diversity and dependency thesis

Ethical Relativism

by James R. Beebe

Dept. of Philosophy

University at Buffalo

Copyright ã 2003

Outline of Essay :

I. Introduction

II. Arguments in Favor of Conventional Ethical Relativism

A. Cultural Diversity

B. Avoiding Ethnocentrism

C. Culturally Conditioned Values

D. Lack of Knowledge

III. Arguments Against Conventional Ethical Relativism

A. A Universal Conscience

B. Conflicting Cultures

C. Create Your Own Culture

D. Reformers

E. Culture vs. Culture

F. Consistency

G. Diversity and Dependency

IV. Arguments in Favor of Subjective Ethical Relativism

A. Immunity to Some Earlier Objections

B. The Importance of Individual Liberty

C. Tolerance

D. Lack of Entitlement

E. Lack of Knowledge

V. Arguments Against Subjective Ethical Relativism

A. Violent Lifestyles

B. Judging Other People

C. Deciding for Yourself

D. The Psychology of Belief

E. The Truth of Relativism

F. Living Together in Peace

G. Moral Absolutes in Relativism

H. You Can’t Live It Out

VI. Conclusion

            Although most people are unfamiliar with the term ‘ethical relativism,’ almost everyone has probably encountered relativist slogans like the following:

(1) What’s right for you may not be what’s right for me.

(2) What’s right for my culture won’t necessarily be what’s right for your culture.

(3) There are no absolute moral truths.

(4) No moral principles are true for all people at all times and in all places.

(5) No ethical principle is any better than any other.

(6) All ethical opinions, lifestyles and worldviews are equally right.

It is possible to be a relativist either about all truths whatsoever or only about truths in certain domains—e.g., truths in ethics or religion.  Though some people claim to be relativists about all truths across the board, it is more common today for people to be relativists about ethics and to be absolutists about truths in other areas, like science and mathematics.  Because ethical relativism is the most common variety of relativism, it will be the focus of our discussion in this essay. 

            There are two basic kinds of ethical relativism: subjective ethical relativism and conventional (or cultural) ethical relativism.  The two kinds of relativism are defined as follows:

Subjective ethical relativism = df the view that:

(i) there are no absolute or universally true moral principles; and

(ii) the truth of moral principles is relative to individuals.

Conventional ethical relativism = df the view that:

(ii) the truth of moral principles is relative to cultures.

Notice that both views deny that there are any absolute or objective ethical truths.  Let’s call the view that there are absolute or universally correct moral principles ‘ moral absolutism .’  The only difference between two relativist views is that they disagree about that to which ethical truths or principles are supposed to be relative.  Each version of ethical relativism, then, is composed of both a negative and a positive element—the negative claim that there are no absolute or objective ethical truths and a positive account of that to which ethical truths are relative. 

            According to subjective ethical relativism (‘subjective relativism,’ for short), whatever you think is right for you really is right for you, and no one can tell you any differently.  What is right for you is completely up to you to decide.  You are sovereign over the principles that can tell you how to live your life. 

            According to conventional ethical relativism (‘conventional relativism,’ for short), what is right for you as an individual depends upon what your culture thinks is right for you.  What your culture says is right for you really is right for you.  The culture or society is the highest authority about what is right for individuals living within that society.  On this view, an individual’s will is subordinated to the will of the cultural majority. 

            In the next two sections we will consider arguments for and against conventional ethical relativism.  We will put off discussing subjective ethical relativism until after we have finished discussing conventional relativism.

            The argument from cultural diversity seeks to support conventional relativism by appealing to empirical facts about the wide variety of cultural practices around the globe.  The argument goes like this: Centuries ago, when cultures were relatively isolated and little information was known about distant lands, it might have been reasonable to think that the traditions and practices of one’s own culture represented the only right way of doing things.  But now we know that each of our conventions and norms reflects only one out of an endless number of possibilities and that other cultures have opted for other ways of doing things.  In this modern age of worldwide communication and information it is no longer reasonable to view the practices of one’s own culture as being the only correct cultural practices.

            Louis Pojman (1999, p. 27) describes some of the facts about cultural diversity that are often cited in support of this position:

For instance, Eskimos allow their elderly to die by starvation, whereas we believe that this is morally wrong.  The Spartans of ancient Greece and the Dobu of New Guinea believe that stealing is morally right; but we believe it is wrong.  Many cultures, past and present, have practiced or still practice infanticide.  A tribe in East Africa once threw deformed infants to the hippopotamus, but our society condemns such acts.  Sexual practices vary over time and clime.  Some cultures permit homosexual behavior, whereas others condemn it.  Some cultures, including Moslem societies, practice polygamy, while Christian cultures view it as immoral.  Anthropologist Ruth Benedict describes a tribe in Melanesia that views cooperation and kindness as vices, and anthropologist Colin Turnbull has documented that the Ik in northern Uganda have no sense of duty toward their children or parents.  There are societies that make it a duty for children to kill their aging parents (sometimes by strangling). 

According to the present argument, to think that your culture’s way of doing things is the only right way or the best way to do things reflects an extreme ignorance of the wide cultural diversity that has always existed in the world.  The anthropological study of foreign lands has (or ought to have) opened our eyes and set us free from any closed-minded adherence to the parochial standards of our own culture.  Faced with the tremendous cultural diversity the world offers, it is simply not reasonable to think that one set of cultural practices can be the one and only right set.  Hence, we must conclude that all cultural practices are equally valid.

            The first argument in favor of conventional relativism uses facts about cultural diversity to argue against moral absolutism.  The second argument claims that there is something intrinsically wrong with the view of moral absolutism itself.  Moral absolutism implies that some cultures are better, ethically speaking, than others.  Conventional relativists, however, argue that such a claim is ethnocentric.  Ethnocentrism , roughly, is judging another culture through the eyes of your own culture and not trying to see things from their perspective.  This almost inevitably leads to thinking that your culture is superior to others.  Most people today agree that ethnocentrism belongs in the same category with racism, sexism and other unacceptable forms of discrimination.  To be racist is to think that, simply because someone belongs to a different ethnic group, that person is inferior.  To be sexist is to think that, simply because someone is a member of the opposite sex, that person is inferior.  All forms of bigotry and prejudice involve judging other people solely on the basis of their group membership.  Ethnocentrism is not any different.  Instead of looking down on other races or sexes, the ethnocentric person looks down on and devalues other cultures. 

            Conventional relativists claim that their position—unlike moral absolutism—is not ethnocentric.  Because conventional relativists maintain that all cultures are equally valid and that no culture is any better than any other, they claim their position avoids any kind of ethnocentrism.  Because ethnocentrism is a pernicious form of discrimination, the apparent fact that conventional relativism avoids it and moral absolutism seems to fall into it is a reason for choosing conventional relativism over moral absolutism. 

 C. Culturally Conditioned Values

            The third argument for conventional relativism begins by focusing on the source of our values and beliefs about morality.  Think about how people have acquired most of their views about what is right and wrong.  From infancy our parents and teachers have sought to instill within us the values of our society.  Friends, books, television, movies, and sometimes priests or preachers have also contributed to helping us internalize the values of the society around us.  Children in America today grow up thinking that women and minorities deserve the same educational and professional opportunities as white men.  One hundred years ago, children in America grew up with different views because those around them impressed upon them a different set of values.  Most women from the nineteenth-century did not believe that women should have the same educational and professional opportunities as men.  It wasn’t just nineteenth-century men who thought that.  Because the women of a century or more ago were surrounded by a culture that did not place a high value on the capacities of women, most of them simply internalized the values of their society and accepted them as true.  It is only because women today grow up in a different cultural milieu that they find themselves with different views about the equality of the sexes.  We are conditioned by our cultures to have the ethical values and beliefs that we do.

            If we were to try to make judgments about the rightness or wrongness of the practices of some other culture, our thoughts would inevitably reflect the beliefs and values of our own culture.  If culture A ’s practices are different from culture B ’s, how is a person from culture A going to assess the practices of culture B ?  If that person were to rely upon the standards of culture A , the practices of culture B would obviously be viewed as wrong because they deviate from what culture A ’s standards say are right.  Of course, if you were to ask someone from culture B about culture A , we would get the same result in the opposite direction. 

            If we were somehow able to throw off all of the cultural baggage we have inherited from our social environments and to break free from all of the cultural conditioning that has shaped our minds, our emotions and our personalities, we might then be able to formulate a completely neutral and objective assessment of some other culture.  However, conventional relativists claim, this is not a realistic human possibility.  We can never get beyond the cultural conditioning that has shaped us and our views of morality.  Therefore, any time we try to sit in judgment on other cultures, we’re going to be just like the hypothetical people from culture A and culture B above: our judgments will simply reflect the values of our own societies.  Since this is the fundamental human condition, there is no reason to think that the judgments any person makes about some other culture will have any objective validity or truth.  Such judgments will always be subjective and culturally conditioned.

            It is as if we all are wearing tinted glasses, the particular tint of your glasses being a function of your upbringing and cultural background.  People from different cultures have glasses that are tinted different colors from ours.  Consequently, their view of reality will be different.  The catch is this: No one can take off their glasses and see reality as it really is in itself.  No one can view truth or reality, except through the distorting lenses of their own cultural biases. 

            Conventional relativists claim that those who believe in moral absolutes are simply blind to the cultural influences that have shaped their ethical opinions.  Absolutists think they can view reality as it is in itself, when in fact they can only see a prejudiced and subjective view of reality. 

            Since we are incapable of freeing ourselves from the cultural influences that have shaped our ethical views, what kinds of judgments should we form about other cultures?  According to conventional relativism, we should stop judging other cultures altogether.  We should stop pretending that our ethical judgments and opinions reflect anything more than the contingent, historical forces that have shaped our lives. 

 D. Lack of Knowledge

            The argument from lack of knowledge makes explicit a theme that is probably implicit in some of the earlier arguments for conventional relativism.  According to this argument, if there is some absolute or objective fact about which cultural practices are the right ones, we simply have no way of discovering what this fact is.  Even if we wanted to believe in moral absolutism, we would be faced with the daunting—and perhaps unanswerable—question, “How can we tell for certain that these cultural practices but not those are morally correct?”  Where would we go to obtain an answer to this question?  Some people suggest that certain sacred texts contain the answers we seek.  But the question that arises for these people is, “How can we tell for certain that this sacred text but not that one contains the absolute truth?”  How do we know that any sacred text will tell us the truth?  Once again the conventional relativist will point out that people raised to believe in one sacred text will think that theirs contains the truth, but people raised to believe in another sacred text will think that theirs is the only true sacred text. 

            For those who do not want to bring religion into the debate, the question is equally challenging.  There are just as many different secular opinions about what is right or wrong as there are religious opinions.  In science we can rely upon experimental methods and empirical observations to resolve differences of opinion.  But what can we use to resolve ethical disagreements?  There do not seem to be any experiments we can run or empirical observations we can make that could show some ethical judgments or principles to be the right ones.  In ethics there seems to be no way to prove some answers right and other answers wrong. 

            Since none of us seems to have any privileged access to the absolute truth about morality, conventional relativists urge us to stop treating our own ethical opinions as infallible or indubitable.  Every culture has its own view of morality.  Since we have no way to prove that some views are better than others, we should simply treat them all as being equally valid or correct.

            Some people respond to the conventional relativist’s arguments—particularly the culturally conditioned values argument—by claiming that all people have an innate ability to know what is right and wrong.  This source of this inborn ability, they say, is your conscience.  Your conscience tells you what the right thing to do is, and it’s the thing that makes you feel bad when you do something you know is wrong.  Because everyone has a conscience, moral absolutists say, we can rise above the cultural conditioning we have received from our social environments.  Contrary to what conventional relativists maintain, we can take off our colored glasses.  Our conscience shows us the truth about morality—not what our culture says is the truth, but the truth itself. 

            Mark Twain’s portrait of Huckleberry Finn provides a compelling counterexample to this line of argument.  Huck is helping his slave friend Jim run away from Miss Watson, Jim’s owner.  The two of them are taking a raft down the Mississippi River to a place where Jim will be legally free.  Huck says:

Jim said it made him all over trembly and feverish to be so close to freedom.  Well I can tell you it made me all over trembly and feverish, too, to hear him, because I begun to get it through my head that he was most free—and who was to blame for it?  Why, me .  I couldn’t get that out of my conscience, no how nor no way.... It hadn’t ever come home to me, before, what this thing was that I was doing.  But now it did; and it stayed with me, and scorched me more and more.  I tried to make out to myself that I warn’t to blame, because I didn’t run Jim off from his rightful owner; but it warn’t no use, conscience up and say, every time: “But you knowed he was running for his freedom, and you could a paddled ashore and told somebody.”  That was so—I couldn’t get around that, no way.  That was where it pinched.  Conscience says to me: “What had poor Miss Watson done to you, that you could see her [racial epithet] go off right under your eyes and never say one single word?  What did that poor old woman do to you, that you could treat her so mean?...”  I got to feeling so mean and miserable I most wished I was dead.  (Quoted in Bennett 1997, pp. 23-24)

Jim tells Huck that he plans to work hard and save enough money to buy his wife and children out of slavery.  He goes on to say that, if he is unable to save enough money to buy them, he will steal them.  Huck is horrified at hearing this:

Thinks I, this is what comes of my not thinking.  Here was this [racial epithet] which I had as good as helped to run away, coming right out flat-flooted and saying he would steal his children—children that belonged to a man I didn’t even know; a man that hadn’t ever done me no harm. 

I was sorry to hear Jim say that, it was such a lowering of him.  My conscience got to stirring me up hotter than ever, until at last I says to it: “Let up on me—it ain’t too late, yet—I’ll paddle ashore at first light, and tell.”  I felt easy, and happy, and light as a feather, right off.  All my troubles was gone.  (Quoted in Bennett 1997, p. 24)

When Huck gets the chance, he sets off toward shore in a canoe, telling Jim he is simply going to look around.  In reality, he plans to turn Jim in. 

As I shoved off, [Jim] says: “Pooty soon I’ll be a-shout’n for joy, en I’ll say, it’s all on accounts o’ Huck I’s a free man...  Jim won’t ever forgit you, Huck; you’s de bes’ fren’ Jim’s ever had; en you’s de only fren’ old Jim’s got now.”

I was paddling off, all in a sweat to tell on him; but when he says this, it seemed to kind of take the tuck all out of me.  I went along slow then, and I warn’t right down certain whether I was glad I started or whether I warn’t.  When I was fifty yards off, Jim says:

“Dah you goes, de ole true Huck; de on’y white genlman dat ever kep’ his promise to ole Jim.”  Well, I just felt sick.  But I says, I got to do it—I can’t get out of it.  (Quoted in Bennett 1997, p. 25)

While ashore, Huck runs into two white men searching for runaway slaves.  They ask Huck whether the man on his raft is white or black.  Huck tells us:

I didn’t answer up prompt.  I tried to, but the words wouldn’t come.  I tried, for a second or two, to brace up and out with it, but I warn’t man enough—hadn’t the spunk of a rabbit.  I see I was weakening; so I just give up trying, and up and says: “He’s white.”  (Quoted in Bennett 1997, p. 25)

            Huck’s conscience was telling him that the right thing to do was to turn Jim in.  Jim was the property of Miss Watson, and his children were the property of someone else.  This “stolen” property needed to be returned to its rightful owner.  In Huck’s mind, he didn’t have the moral fiber, the courage, or the strength of will to do what was right.  He does not turn Jim in, but he does not think that he has done the right thing.  He despises himself for his weakness, and his conscience tells him that he is a morally despicable person. 

            We see that Huck’s conscience is not something that rises above the cultural conditioning he has received throughout his life.  Instead, it merely reflects the values of the slave-owning society he grew up in.  In response to the conventional relativist’s culturally conditioned values argument, some moral absolutists want to argue that only part of us is subject to cultural conditioning.  There is, they claim, another part of us that is immune to cultural conditioning—a part that can really tap into the absolute truth about morality without reflecting the contingent values of our society.  Twain’s very believable description of Huck suggests that cultural conditioning may very well extend so deeply into our psyche that there is no part of us that is left unaffected.  Even our consciences appear to be shaped by the values of our society.  Thus, it seems that this first argument against conventional relativism does not succeed. 

 B. Conflicting Cultures

            Consider the following case: Lacey is both a feminist and a Roman Catholic.  (This case is a modified version of Pojman’s (1997, p. 36) case of Mary, who is a U.S. citizen and a Roman Catholic.)  In her law practice, she specializes in defending the rights of women.  Most of the time, her commitments to feminism and Catholicism do not conflict with each other.  Her religion views women as being created in the image of God and as having an intrinsic value that should not be violated.  Consequently, Catholicism condemns most of the same abuses and injustices suffered by women that feminism condemns.  However, there is one obvious issue where Lacey’s joint commitments conflict: abortion.  According to the feminist movement, abortion is morally permissible.  According to Catholicism, however, abortion is not morally permissible. 

            Conventional relativism claims that what is right for you as an individual is determined by the culture you belong to.  Lacey, however, belongs to more than one culture, and the values of the two cultures conflict.  So, is abortion morally permissible for Lacey or not?  Which culture takes precedence over the other?  Which one should she listen to? 

            Some students think that Lacey should listen to the Catholic church more than to the feminist movement.  But why?  If you find yourself belonging to one religious culture and one secular culture, why should the religious culture take priority?  Simply because it is a religious culture?  That doesn’t seem like much of a reason.  And what do you do if you belong to two secular cultures whose views conflict?  How do you decide which one to follow?

            Before offering an answer to these questions, I want to present another case, due to Pojman (1997, p. 36): John is a college student.  He belongs to a fraternity that is racist in both its creed and its practices.  However, John is also a member of the larger university community, and the university is officially non-racist in its policies and procedures.  The university, it turns out, is located in a town that is overwhelmingly racist.  Because this is John’s sixth year of college (he’s had some trouble with calculus), John has been living, working, shopping, and paying taxes in this town for quite some time.  He is, thus, very much an integral part of his community.  Of course, this town is located in a nation that is officially non-racist in its policies.  So, is racism right or wrong for John?

            Pojman (1997, p. 36) writes,

As a member of a racist university fraternity, KKK, John has no obligation to treat his fellow Black students as an equal, but as a member of the university community (which accepts the principle of equal rights), he does have the obligation; but as a member of the surrounding community (which [rejects] the principle of equal rights), he again has no such obligation; but then again, as a member of the nation at large (which accepts the principle), he is obligated to treat his fellow students with respect. 

Conventional relativism says that what is right for John depends upon what his culture says is right.  But which of the overlapping and conflicting cultures that he belongs to should be listen to? 

            Some students think that the nation is the most important culture John belongs to and that, therefore, he should not be racist.  But what if John identifies more with his fraternity than he does with his country?  Suppose that members of his fraternity believe the nation has gone downhill ever since the Civil Rights Act of 1964, and that it is their sworn duty to combat civil rights for minorities wherever they can.  It is difficult to see how, in such a case, the culture that defines what is right for John should be the nation when he has rejected the beliefs, principles and practices of that nation. 

            Moreover, opting for the nation as the relevant culture for John seems completely arbitrary.  Conventional relativism itself does not say anything about nations, as opposed to smaller cultures.  John could just as well identify with white people worldwide or college students worldwide or people in Western industrialized nations or even with humanity itself.  Why should national affiliation trump all others? 

            The most tempting solution to the dilemmas facing Lacey and John is one that conventional relativists cannot accept.  It is natural to think that the decision about which culture to identify with should be left up to Lacey and John.  They should each decide for themselves which culture’s values will be the ones that determine what is right and wrong for them.  However, this way out of the dilemma is not open to the conventional relativist.  It is subjective relativism that claims that morality is relative to individuals and that individuals can decide what is right and wrong for them.  But conventional relativism says that it is cultures—not individuals—that decide.  A conventional relativist who says that Lacey and John can decide for themselves what is right for them would be giving up on conventional relativism and adopting subjective relativism instead.  Is there a solution to this dilemma that does not require abandoning conventional relativism?  It is difficult to see what such a solution could be. 

            Conventional relativism appears to work fairly well when we are considering a culture other than our own, especially when that culture meets the following conditions:

(a) The culture is non-Western and non-industrialized.

(b) The culture would be described by ethnocentric Westerners as “primitive.” 

(c) The culture is monolithic or homogeneous, meaning that there is (or at least appears to outsiders to be) only one uniform social structure that defines the culture’s social relations.

Condition (c) is simply another way of saying that the culture is not pluralistic.  In other words, it does not contain a variety of overlapping and sometimes conflicting cultures that (as in the Lacey and John cases) cause problems for conventional relativism.  I doubt that “primitive” cultures are ever as uniform or homogeneous on the inside as they appear to Western observers who have only limited and superficial exposure to them.  But even if Western stereotypes of these cultures were accurate, the fact remains that conventional relativism could not be made to work very well when applied to an obviously pluralistic and modern society like our own.  Each of us (like Lacey and John) belongs to many different subcultures, and this sometimes results in conflict.  It seems much more plausible to say that we must each, as individuals, decide what is right for ourselves than to say that “our culture” (whatever that is) decides what is right for us.  In short, subjective relativism seems like it might work better in America than conventional relativism. 

 C. Create Your Own Culture

            Thinking about the cases of Lacey and John, in which people belong to many different overlapping cultures, raises questions about the nature of a culture.  What exactly is a culture anyway?  Cultural anthropologist Richley Crapo (1993, p. 24) defines a culture as “a learned system of beliefs, feelings, and rules for living around which a group of people organize their lives.”  According to this definition, the shared way of life of any group of people counts as that group’s culture.  This seems to imply that you could organize any group of people into some set of shared practices, beliefs and rules and it would count as a culture.  In other words, you could create you own culture.

            David Koresh did.  If you are charismatic enough, you can take any rule—regardless of how lame-brained, idiotic, cruel, ignorant or twisted it may be—and convince at least some people to go along with it.  One of the rules that David Koresh instituted in the Branch Davidian culture was that married couples living in the Branch Davidian compound outside of Waco, TX, were not allowed to have sex with each other.  Only David Koresh was allowed to have sex with the women of the compound, and he could have sex with whomever he wanted.  This included sex with young girls.  According to conventional relativism, David Koresh’s sexual lifestyle was right for him.  Why?  Because the norms of his culture—which was in part something of his own making—said that it was OK. 

            If there is some kind of activity you would like to engage in but that is viewed as immoral by the rest of society, take heart.  According to conventional relativism, all you have to do is to convince a few of your buddies to go along with you, and—Voila!—you will made the previously questionable activity morally correct for you. 

            Surely it is absurd to think that any seemingly evil practice can become morally good simply by convincing a few other people to go along with it.  And yet that is what conventional relativism seems to imply. 

 D. Reformers

            Conventional relativism subordinates the will of the individual to the will of the cultural majority.  What is right for you as an individual is not up to you to decide.  What is right for you is what your culture says is right.  Think about what conventional relativism implies about reformers like Martin Luther King, Jr., or Mahatma Gandhi.  Reformers are people whose beliefs and actions run contrary to those of their surrounding culture and who strive to change the beliefs and actions of their culture for the better.  If MLK’s culture says that African-Americans should not be allowed to eat at the same lunch counters, drink out of the same water fountains, attend the same schools, and sit in the same seats on crowded buses as white people, then it was morally wrong for MLK to praise Rosa Parks for refusing to yield her bus seat to a white person one cold December day in 1955.  If the culture says that African-Americans should be treated as second-class citizens, then according to conventional relativism you are morally obligated to treat African-Americans as second-class citizens.  It is morally wrong for you to buck the system.  The system is always right.  In the eyes of conventional relativism, reformers are—by definition—always in the wrong. 

            It is difficult to stomach the idea that the majority is always right and that the status quo should always be respected.  The people whom we treat as our greatest heroes were people who stood up to the system and fought against the tyranny of the majority.  When was the last time you saw an action movie starring Mel Gibson or Harrison Ford in which they were on the side of the powerful majority?  Our movie heroes are always lone individuals, often misunderstood by those around them, who take courageous stands against the injustices of their society. 

            The fact that conventional relativism implies that reformers are always wrong provides a strong reason for thinking that conventional relativism is false. 

 E. Culture vs. Culture

            Consider the following case: Lothar is a barbarian.  Raping, pillaging and plundering are central to Lothar’s barbarian culture.  One day Lothar and his band of warriors set off for Pleasantville, a quiet little town in sunny, southern California.  The residents of Pleasantville are peace-loving vegetarians and animal rights activists who, when they are not tending to their organic farms or making pottery for burning incense, work as strong advocates for conventional relativism.  When Lothar’s barbarian war party arrives in Pleasantville, the sandal-shod residents of Pleasantville are faced with a dilemma: Should they defend themselves against the barbarian attack or not? 

            You may be thinking, “Dilemma?  What dilemma?  How can the question of whether they should defend themselves against an unwanted attack be difficult to answer?  Of course, they should defend themselves!”  Despite the plausibility of this response, it is important to see that the Pleasantvilleans’ belief in conventional relativism poses a problem for them.  As conventional relativists, they believe that the right thing for Lothar to do is determined by his culture.  But Lothar’s culture says that adult barbarian men should display their bravery and strength by sacking at least one city (preferably one with a Starbucks) per year.  So, according to conventional relativism, it is morally right for Lothar to sack Pleasantville.  Since it is morally correct for him to do so, it doesn’t seem right for the residents of Pleasantville to try and stop the barbarian attack.  They would be keeping Lothar from doing what, according to their own moral standards, is the morally right thing for him to do.  However, if the citizens of Pleasantville do not defend themselves against the barbarian invasion, they will be brutalized and killed. 

            Another problem that would be generated by any attempted defense of Pleasantville concerns ethnocentrism.  One of the main motivations for conventional relativism is that it supposedly allows us to avoid ethnocentrism—the view that our culture is superior to others.  A great deal of harm has been done throughout the ages by people who have forced their way of life upon other cultures.  By claiming that all cultures are morally equal and that no culture is morally better than any other, conventional relativism is supposed to help us lead more tolerant lives.  But killing barbarians doesn’t seem to be a very good way to display tolerance for barbarian culture.  By keeping the barbarians from sacking Pleasantville, the Pleasantvilleans would be ethnocentrically forcing the barbarians to accept what they think is right in opposition to what the barbarians think is right. 

            It is difficult to see how the citizens of Pleasantville could be morally justified in fighting against the barbarians.  Their belief in conventional relativism seems to undermine any moral justification they might have for fighting.  Keep in mind that I am not asking whether they would fight against Lothar and his warring band.  I am asking about the morality of their actions.  I want to know whether conventional relativism can provide a reason for thinking that the defeat of the barbarians would be a morally good thing.  I am unable to see how conventional relativists could provide such a reason.  Since it is absurd to think that it might not be justified for them to defend themselves, the case of Lothar and the barbarians provides a reason for thinking that conventional relativism is false.

            (If my tale of Lothar and the barbarians seems a little too far-fetched to some of you, note that I could have told basically the same story as a clash between the culture of a violent urban gang and the culture of the law-abiding citizens in the city where the gang lives.  Clashes between cultures with opposing viewpoints are more than merely fictional.) 

 F. Consistency

            What should conventional relativists say about a culture that is ethnocentric?  Is it right for them to be ethnocentric?  Or is it wrong?  Remember, according to conventional relativism, what is right for you to do is determined by your culture.  If your culture is ethnocentric, it seems like it should be morally right for you to act in an ethnocentric way.  And yet conventional relativists condemn ethnocentrism as morally wrong.  They use the fact that most people today view ethnocentrism as being wrong as a way to motivate people to become conventional relativists.  But if my culture says that it is OK to look down upon other cultures, then according to conventional relativism it should be OK for me to do so.

            What if my culture is absolutist?  What if a belief in absolute moral truths is a central feature of my culture?  According to conventional relativism, it should be right for me to believe in the existence of moral absolutes.  But, of course, a belief in moral absolutes is incompatible with a belief in conventional relativism because the first component of conventional relativism is the claim that there are no moral absolutes.  So, if my culture is absolutist, conventional relativism says that it is right for me to believe that conventional relativism is false.  Think carefully about this claim before reading further.  Does it make sense for a conventional relativist to believe that conventional relativism is true and yet at the same time to believe that it is OK for me to think that conventional relativism is false?  Conventional relativists think that it does.  It might (just barely) be possible for a conventional relativist to act consistently on their relativist views, but it looks like it will be extremely difficult. 

 G. Diversity and Dependency

            One of the most common ways to argue in favor of conventional relativism involves appealing to facts about cultural diversity.  Conventional relativists say, “How can you believe in moral absolutes?  Just look at all of the moral diversity in the world.  It should be obvious that no moral truths are universal or absolute.”  This line of argument, however, involves a confusion.  To sort out the confusion Pojman (1999, pp. 37-38) distinguishes between the following two claims:

The Diversity Thesis :

What is considered morally right and wrong varies from society to society, so there are no moral principles that all societies accept.

The Dependency Thesis :

What really is morally right and wrong depends upon what societies think is morally right and wrong.

(I have changed the wording of the dependency thesis, but the idea remains the same.)  The diversity thesis makes a claim about people’s moral opinions—about what they think is right or wrong.  It simply says there is a wide diversity of human opinion about morality.  Taken by itself, that claim is pretty harmless and indeed uncontroversial.  The diversity thesis itself does not make any value judgments about this diversity of opinion.  It simply reports the existence of the diversity. 

            Some people try to suggest there really are some moral principles that all societies accept.  It is difficult to know whether this is really true because so many social scientists disagree about this issue.  Some say that the ban on incest is the only universally accepted moral rule, while others claim there are dozens of other such rules.  Many social scientists claim there are no universally accepted moral rules at all.  It’s hard to know who to believe.  We do not, however, need to resolve this issue in order to consider the heart of the controversy concerning conventional relativism.  The most important of the two claims above is the dependency thesis, not the diversity thesis. 

            The dependency thesis says that moral principles depend upon cultural acceptance for their validity or correctness.  In other words, if a culture accepts some principle, then it will be right for the members of that culture.  If they do not accept another principle, that principle will not be right for that culture.  The heart of conventional relativism is the claim that cultural acceptance determines morality. 

            To illustrate the differences between the two theses, think about what a moral absolutist like Socrates would say about the diversity thesis.  Would he think it was true or false?  Many students are initially tempted to think that Socrates would disagree with the diversity thesis.  But think carefully about it.  The diversity thesis simply claims that people disagree about what is right and wrong.  How could Socrates deny that this is obviously true?  A moral absolutist need not (indeed should not) deny that people disagree about morality.  The difference between the conventional relativist and the moral absolutist concerns how the two parties view the diversity of moral opinions.  Conventional relativists think that everybody is equally right, while moral absolutists think that only some moral opinions are right while the rest are all wrong.  According to moral absolutism, the fact that there is an extremely wide diversity of opinions about morality simply shows that there are a lot of very mistaken people in the world. 

            Distinguishing between the diversity thesis and the dependency thesis allows us to see that—contrary to what most conventional relativists think—demonstrating how much diversity of opinion there is in the world does not in any way undermine moral absolutism.  Many relativists think that conventional relativism can be (and has been) proven true simply by doing enough anthropological research on the divergent beliefs and practices of people around the globe.  All of this anthropological research, however, simply supports the diversity thesis.  But it provides no reason for believing the dependency thesis.  Consequently, the argument from cultural diversity fails to show that conventional relativism is true. 

            Conventional relativism seems to face several serious problems.  It is, however, only one of the two main versions of relativism.  Some people claim that subjective relativism is more defensible than conventional relativism.  In this section we examine some of the main arguments offered in favor of subjective relativism. 

 A. Immunity to Some Earlier Objections

            One point in favor of subjective relativism is that it does not fall prey to some of the same objections that were levied against conventional relativism.  For example, recall the cases of Lacey and John.  They each belonged to overlapping cultures with conflicting values, and there did not seem to be any way for conventional relativism to say whether abortion was morally permissible for Lacey or whether racism was morally permissible for John.  Subjective relativism, however, can easily handle this sort of case.  According to subjective relativism, Lacey and John are free to choose how they will live their lives.  If Lacey chooses to make the values of the Catholic church her own personal values, then abortion will not be morally permissible for her.  If, however, she chooses to identify more with the feminist movement and to adopt their values, abortion will be morally permissible for her.  Similar considerations apply in John’s case.  By making moral correctness a function of personal choice, subjective relativism avoids the problem of conflicting cultures. 

            Subjective relativism is also able to provide a seemingly more acceptable verdict in the case of reformers who challenge or reject the values of their culture.  Because conventional relativism subordinates the will of the individual to the will of the culture or society, it seems that little room is left over for individuals to make their own, autonomous decisions about how they ought to live their lives.  Conventional relativism says that anyone who challenges the values of their society will be in the wrong.  Subjective relativism, however, says that what is right for you is up to you.  Regardless of what the majority says or what anyone else in your culture thinks, you are the one who should decide what kind of lifestyle or what kind of values you will adopt.  If you want to be a reformer and you want to challenge the society around you, subjective relativism says you are acting rightly if you are true to yourself. 

            Subjective relativism, then, seems to give answers to the ultimate questions about morality that are more plausible than those given by conventional relativism.  Consequently, some objections that seem to undermine conventional relativism do not harm subjective relativism in any way. 

 B. The Importance of Individual Liberty

            By relativizing ethical truth to individuals rather than cultures, subjective relativism is able to give more consideration to the importance of individual liberty.  In America we place an extremely high value on our freedom, our ability to direct the course of our own lives.  Subjective relativism makes individual liberty and freedom central to morality.  According to subjective relativism, it is not right for anyone to try to force their oppressive morality upon you against your wishes.  The only morality that is right for you is one that you have autonomously chosen. 

            Many subjective relativists believe that the theories of conventional relativism and moral absolutism do not adequately respect individual liberty.  Conventional relativism makes the culture or the majority in control and allows them to trample on the rights of individuals to choose how to live their lives.  Moral absolutism says that you don’t have any choice about what is right for you.  The moral absolutes that define morality are all predetermined ahead of time, and you have no say in the matter.  Because subjective relativism respects individual liberty more than conventional relativism and moral absolutism, subjective relativists claim this is a reason for choosing subjective relativism over these other views. 

 C. Tolerance

            Subjective relativists argue that conventional relativism’s emphasis on tolerance of other cultures is good but that it does not go far enough.  Conventional relativism says that no culture is better than any other and that we should treat them all as being equally valid.  However, conventional relativism does not make tolerance of other people within a society a priority.  Subjective relativists claim that it is not only cultures but individuals as well who deserve to be treated with tolerance.  Subjective relativism claims that no individual’s ethical opinions, values or lifestyle is any better than any other individual’s.  All opinions about morality and lifestyles should be treated as being equally good.  Tolerance of other individuals, then, is an important part of subjective relativism. 

            Think about all of the injustices that have been committed because of intolerance.  In the years following the Protestant Reformation, Protestants and Catholics slaughtered each other by the thousands because each side would not tolerate the religious views of the other side.  Racism, genocide and ethnic cleansing all involve an unwillingness to tolerate other people who are different from us.  Subjective relativists believe that most of the world’s intolerance results from a belief in moral absolutism.  If you believe in moral absolutes, you think there is only one right way to do things.  Moral absolutists think that everybody who disagrees with them is dead wrong.  It is this kind of belief that has led people throughout the centuries to think it is OK to abuse and slaughter other people.  They are the pagans, the barbarians, the heretics.  So, it is only right to rid the world of their corrupting influence. 

            Subjective relativism promotes tolerance and takes its advocacy of tolerance farther than conventional relativism does.  Moral absolutism seems to lead to intolerance.  Subjective relativists think the choice is clear: subjective relativism is preferable to either conventional relativism or moral absolutism. 

 D. Lack of Entitlement

            Subjective relativists ask, “Who’s to judge what is really right and wrong for everybody?  What person has the entitlement or authority to sit in judgment on the rest of us?”  Their answer is: “Nobody.”  When you judge other people, you are placing yourself above them and pretending that you have the authority to decide what is right and wrong for them.  But you do not have any such authority.  So, it is inappropriate for you to judge other people.  If you actually did have the authority to make such judgments, there might not be a problem.  As it is, however, there is nothing that entitles you to pass judgment on anyone else.  You’re no better than the rest of us.  Thus, you have no business judging us. 

            Not only do we not have the authority or entitlement to judge other people, there is something problematic about the act of judging itself.  Judging someone else displays an intolerance for the judged person’s way of life.  To judge another person is to do something unkind to that person.  It is not unlike insulting them because it involves say negative things about them. 

            Subjective relativism urges us to admit that we are not entitled to stand in judgment over other people.  According to subjective relativism, we should view other people’s opinions about morality, their lifestyles, their habits, and their actions as being as equally valid as our own.  It is only by adopting subjective relativism that people can learn to stop judging others. 

 E. Lack of Knowledge

            Subjective relativists have their own lack of knowledge argument that is similar to the one offered by conventional relativists.  None of us, they note, can really tell for sure what is the absolute truth about morality.  What is right for us is something that we each have to make up our own minds about.  Conventional relativists correctly note that none of us has an infallible access to absolute truth, but they go wrong in concluding that what is right for you is something to be determined by your culture.  It is ultimately a personal decision.  You have to decide for yourself what the right way to live is. 

            Moral absolutists think they have everything all figured out.  They think they know what is right for everybody, and they try to force their morality on everyone else.  It is arrogant of them to think they know what the absolute truth is.  No one person has any better access to the truth than the rest of us.  So, none of us should presume to speak on behalf of everyone else.  What is right for you is something for you to decide and no one else. 

            The arguments offered in favor of subjective relativism can sound very convincing to many people.  There are, however, some serious problems for the view lurking just below the surface. 

 A. Violent Lifestyles

            Subjective relativism would have us believe that no opinion about morality is any better than any other and that no lifestyle is any better than any other.  All moral opinions and lifestyles are equally valid, they say.  Consider now the lifestyle of the Baton Rouge serial killer.  If no lifestyles are any better or any worse than any others, that means the lifestyle of the serial killer is not any worse than the lifestyles of Baton Rouge’s law-abiding citizens.  Remember: we are supposed to be tolerant of all lifestyles.  Also, according to subjective relativism, the opinion that rape and murder are morally wrong is not any better than the opinion that rape and murder are fine and dandy.  Both opinions are equally valid.  Each person (including the Baton Rouge serial killer!) gets to decide what is right and wrong for that person.  If a serial killer has decided that rape and murder are right for him, then these horrible crimes really are right for him, and it is inappropriate for us to think any differently. 

            Can anyone seriously believe that the subjective relativist’s assessments of the Baton Rouge serial killer’s lifestyle and ethical views are correct?  I don’t see how they could.  Subjective relativism’s claim that all lifestyles and all opinions about morality are equally valid cannot be true. 

 B. Judging Other People

            Subjective relativism claims that it is wrong to be judgmental, that we should not judge other people.  Something about this claim seems reasonable.  However, subjective relativism’s claim that we should not think that anyone else’s lifestyle or opinion is wrong seems absurd.  But if forming an opinion about someone else is judging them, how can we accept the first relativist claim and reject the second? 

            The key to understanding this issue is realizing there is an ambiguity in the phrase “judging someone else.”  In the most basic sense of this phrase, to judge someone else is to form an opinion about them.  For example, Louisiana voters were recently asked to decide whether Kathleen Blanco or Bobby Jindal should be their next governor.  Voters had to form an opinion about which candidate they think will do the best job.  Regardless of which candidate you may have voted for, no one will condemn you for having “judged other people.”  That’s what you were supposed to do.  In this sense of “judging,” it is impossible not to judge other people.  In fact, it is necessary in order to get along in society.

            The foregoing example reveals that there must be another sense of the phrase “judging someone else” that is reasonably taken to be objectionable.  When we speak of judging someone else in a negative sense, what we sometimes have in mind is an inappropriate rejection of the person being judged.  We have probably all seen an episode of some television show with the following motif: A couple’s estranged son has finally come home.  He has been gone for years, and they have hardly spoken since he left.  The mother weeps tears of joy.  However, the son has brought with him a “special friend,” and he has some important news to share: “Mom, Dad, I’m gay.”  The father disowns the son and will not speak to him.  He says, “You are no longer my son” and orders his son to leave his house.  The father is intolerant, unloving and verbally abusive. 

            When we call the father “judgmental,” we are not merely saying that the father has formed some opinion or other about his son.  We mean there is something about his opinions and the way he is acting upon them that is inappropriate.  To judge other people in this negative sense means that you will shun them, disown them, cut off whatever relationship you had with them, refuse to associate with them, look down upon them, exclude them, or deny them the same privileges as those who have not been so judged.  Subjective relativists claim that judging in this negative sense is wrong.  That seems like a reasonable suggestion.  Responding in a hateful way to another person is usually going to be a bad thing. 

            However, this does not mean that we should stop judging them in the first sense discussed above, but this is what subjective relativists recommend.  Couldn’t the television father continue to believe that his son’s alternative sexual lifestyle was morally wrong but embrace and love his son anyway?  Disagreeing with another person does not (and should not) always lead to shunning, excluding or disowning that person.  In other words, “judging someone” in the first sense of forming an opinion about that person, does not always lead to “judging someone” in the second sense of rejecting or cutting off all social ties with that person.  You can still love someone with whom you disagree. 

            Subjective relativists, however, do not distinguish between these two senses of “judging other people.”  They argue that, since the second sort of judging is inappropriate, so is the first.  But this conclusion does not logically follow.  It is possible to judge in the first sense but not the second.  However bad the second sense of judging may be, this does not mean that the first sense is also bad. 

 C. Deciding for Yourself

            Subjective relativists claim that you should get to decide what is right for you.  Something about this claim seems very true.  As autonomous, rational agents, we have the ability to make our own decisions, and our autonomy and rationality should be respected by others.  However, is seems absurd to think that the Baton Rouge serial killer should be free to decide that rape and murder are right for him.  We need to distinguish two different senses “deciding for yourself.”  Subjective relativists slide from using one sense of “deciding for yourself” to using a completely different sense, without acknowledging this is what they are doing. 

            In one sense, you must decide for yourself what to believe not only in ethics but in every area of your life.  You must evaluate the evidence that is available to you, the arguments for and against various positions, and make up your mind about what you think is true.  For example, if you are unsure about whether to believe in global warming, you can go to the library, check out some books, read some journal articles, and familiarize yourself with the various arguments that have been put forward.  Then, you must decide for yourself whether you think the evidence supports or does not support a belief in global warming.  Talking about “deciding for yourself” in this sense seems unobjectionable. 

            However, subjective relativists also like to talk about “deciding for yourself” in another sense.  In this second sense, subjective relativists claim that the fact that you chose lifestyle X for yourself makes lifestyle X right for you.  To see that this sense of “deciding for yourself” is different from the first, recall the global warming case.  Choosing to believe in global warming does not make global warming a fact.  When it comes to issues like global warming, your choices determine your beliefs, but they do not determine the facts.  You hope that your belief in global warming corresponds to the facts.  But the facts do not depend upon you believing in them in order for them to be the facts.  The facts are the way they are, regardless of what you think about them. 

            By contrast, subjective relativists claim that in ethics your choices determine not only your ethical beliefs but also the ethical facts.  If you decide that cheating on your boyfriend or girlfriend is morally permissible, that makes cheating morally permissible for you.  You never have to wonder or worry whether your ethical beliefs correspond to the facts.  Your beliefs create the ethical facts.  Without your beliefs, there would be no ethical facts about what is right for you.  So, there is never any possibility that you could be wrong about what is right for you.  According to subjective relativism, believing so makes it so. 

            Subjective relativists claim that, because we must obviously decide for ourselves what to believe (in the first sense of “deciding for yourself”) in ethics, we are also able to decide for ourselves (in the second sense of “deciding for yourself”) what the ethical facts are.  In their discussions of “deciding for yourself,” they never distinguish the two senses and use the plausibility of the first sense illicitly to support the second sense.  But the fact that the first sense gives no support to the second sense.  Consequently, subjective relativists cannot appeal to the importance of autonomous decision making to show that their view is true. 

 D. The Psychology of Belief

            The subjective relativism position also seems to misunderstand the psychology of belief.  Subjective relativists believe it is OK for you to think that capital punishment is right but that you should not think the opinions of those who oppose capital punishment are any less correct than your own.  Think about that for a minute.  Is what they recommend even psychologically possible?  How can I believe that position A is true and yet at the same time believe that those who think that position A is false are just as right as I am?  That sounds like nonsense.  If I believe position A is true, I am committed to believing that anyone who thinks position A is false is wrong.  Subjective relativism seems to be asking us to do something that is not humanly possible. 

 E. The Truth of Relativism

            Consider the following simply question: Do subjective relativists believe that subjective relativism is true?  This may seem like an utterly ridiculous question, unless you think carefully about it.  At first glance, the answer seems obvious.  Of course, they believe subjective relativism is true.  That’s what makes them subjective relativists.  However, this seemingly obvious answer causes serious problems for the subjective relativist. 

            Subjective relativists deny that there are any absolute truths in ethics.  No ethical principle, they say, is true for all people at all times and in all places.  The problem, however, is that subjective relativists think subjective relativism is the TRUTH about ethics.  They do not merely think that subjective relativism is true-for-them.  They think it is true for all people at all times and in all places.  They seem to be contradicting themselves.  Subjective relativism also claims that no moral view is any better than any other.  Since subjective relativism is itself a moral view, this means that subjective relativism is not any better than any other moral view, such as moral absolutism.  And yet you will never meet a subjective relativist who does not think that moral absolutism is just plain wrong.  They contradict themselves once again. 

            (By the way, conventional relativism falls prey to this same problem.  Conventional relativists also deny that there are any absolute truths in ethics, but they claim that conventional relativism is the absolute truth about ethics.  They also claim that no culture’s moral viewpoint is any better than any other, and yet they claim that any culture that subscribes to conventional relativism is correct while any culture that subscribes to moral absolutism is objectively wrong.)

            Is there any way for the subjective relativists to keep from contradicting themselves.  There might be one way, but it has some serious drawbacks.  Instead of claiming that subjective relativism is TRUE, subjective relativists could argue that subjective relativism is simply true-for-them and not necessarily true-for-others.  So, if you are a moral absolutist, this kind of subjective relativist would not try to tell you that you are wrong—even if you were intolerant and ethnocentric.  The subjective relativist I am imagining would claim that, while subjective relativism is true-for-them, moral absolutism would still be true-for-you.  This kind of subjective relativist does not fall into self-contradiction. 

            However, the subjective relativist’s position is no longer a very interesting one.  The features that make subjective relativism an attractive position for a lot of people are no longer present: the emphasis on trying to get other people to be more tolerant, the opposition to ethnocentrism, the critique of the injustices done in the name of moral absolutism, etc.  Subjective relativists want other people to become relativists, too.  They want to tell those who believe in moral absolutes that they are really wrong for being absolutists.  Relativists of all stripes are continually criticizing people who believe in absolute moral truths for being closed-minded, intolerant, dogmatic, politically incorrect, and just plain wrong.  But if belief in moral absolutism is merely wrong-for-the-subjective-relativist but not necessarily wrong-for-you, then the relativist is not in a position to criticize you for being an absolutist.  Going with the option under consideration avoids a contradiction, but only by making it impossible for the subjective relativist to disagree with absolutists.

            Consequently, although the position I have just sketched is logically possible, you will never meet a subjective relativist who believes in it.  All of the subjective relativists you will ever come across will believe that subjective relativism is the fundamental, objective and universal truth about morality.  This is not something they can believe in without contradicting themselves.  (For more on contradictions, point your browser to: http://www.geocities.com/beebejames/Contradictions101.)

 F. Living Together in Peace

            Subjective relativists suggest that the only way for us to live together in peace in a pluralistic society like ours is for us to treat everyone else’s opinions and lifestyles as being as equally valid as our own.  They suggest that moral absolutism leads to intolerance and injustice.  If we all stopped thinking that our opinions and ways were superior to those of other people, we would have a more peaceful, more egalitarian society. 

            Subjective relativists, however, misunderstand what democracy is all about.  Living together peacefully in a democracy does not mean having no opinions about what is right and wrong.  It means living together in peace with those with whom you strongly disagree.  It is ridiculous to think that we will never have a peaceful, just and fair society as long as Democrats are convinced that Republicans are wrong (and vice versa), pro-lifers are convinced that pro-choicers are wrong (and vice versa), evangelical Christians are convinced that purveyors of pornography are wrong (and vice versa), proponents of the death penalty are convinced that opponents of it are wrong (and vice versa), and proponents of affirmative action are convinced that opponents of it are wrong (and vice versa).  Subjective relativism makes the absurd suggestion that the only way to live together in harmony is to stop having any convictions about what is right or wrong.  Subjective relativists want us to stop thinking that anybody else’s opinion is wrong.  As long we think that, they say, we will be intolerant, judgmental and unjust.  Such a position completely misunderstands the beauty of democracy. 

            Citizens in a democracy are expected to respect the rights of others and to find peaceful means of resolving their disagreements.  You don’t have to agree with the people on the other side of the aisle, but you are not free to abuse them or deprive them of their rights simply because they disagree with you.  You are free to debate, to persuade, to campaign, to make commercials, and to donate money to political parties or private organizations that promote your interests.  But you are not free to harm those with whom you disagree.  That’s democracy: peaceful disagreement.  It’s not (as subjective relativism suggests) a lack of any real disagreement. 

 G. Moral Absolutes in Relativism

            Subjective relativists contradict themselves not only in claiming that subjective relativism is true but also by believing in the following absolute moral truths:

(1) Every person deserves to be treated with dignity and respect, regardless of race, religion, class, color or creed.

(2) Different lifestyles and cultures should be treated with tolerance.

(3) Intolerance is morally wrong.

(4) The basic human rights of every individual should be defended by a free and just society. 

(5) We should strive to provide the citizens of our nation with as much individual liberty as is compatible with the free exercise of everyone else’s liberty. 

(6) It is wrong to deprive those with whom one disagrees of their right to make their voices heard in an arena of public discourse. 

(7) Racism, sexism and all forms of hateful discrimination are unjust and have no place in an equitable and peaceful society. 

Every relativist I have ever met (or expect to meet) believes in (1) through (7).  And they do not merely believe that (1) through (7) are true-for-them.  They believe these truths apply to all people in all cultures at all times and in all places.  In other words, relativists (subjective and conventional) treat (1) through (7) as moral absolutes.  Thus, contrary to the explicit statements of their own position, they do believe in moral absolutes after all. 

 H. You Can’t Live It Out

            Not only are self-contradictory positions necessarily false, they are also impossible to put into practice.  You couldn’t really live your life in accordance with subjective relativism if you wanted to. 

            Suppose that Boudreaux deliberates about what he should do in the following manner:

(a) One is morally obliged to keep one’s promises. 

(b) I promised my cousin Jethro that I would attend the premiere of his performance art piece. 

(c) I am morally obligated to attend Jethro’s premiere. 

(d) I want to do what is right. 

(e) Therefore, I will attend Jethro’s premiere. 

Statement (a) is an expression of a universal principle about what is morally right or wrong.  (b) is a statement of descriptive fact regarding Boudreaux’s interaction with Jethro.  (c) states a logical consequence of (a) and (b).  (d) expresses one of Boudreaux’s desires, which provides him (let us suppose) with sufficient motivation for caring about (c) and for following through with what he knows to be right.  Finally, (e) expresses Boudreaux’s decision or determination to undertake the specified course of action. 

            Suppose, however, that Boudreaux is a subjective relativist and consider what effect this might have on his practical deliberations.  Since he denies that there are any absolute moral truths, the moral principle in (a) can only be interpreted as being true-for-him, if he has decided to believe in it.  But Boudreaux could just as well have chosen to believe in any of the following, incompatible moral principles:

(f) One is morally obliged to keep one’s promises, unless it is inconvenient to do so. 

(g) One is morally obliged to keep one’s promises only if one has promised to do something pleasurable. 

(h) One is not morally obliged to keep any of one’s promises. 

Which one of these is right-for-Boudreaux?  Whichever one he happens to believe in.  Remember: According to subjective relativism, what is right-for-him is whatever he thinks is right-for-him.  So, none of the above options can be any more accurate or true than the others.  Since, according to subjective relativism, whatever Boudreaux believes to be right really is right-for-him, there is no reason for him to worry that his beliefs might be wrong-for-him.  They are right-for-Boudreaux of necessity.

            How is Boudreaux supposed to choose which one to believe or adopt?  His relativism cannot be of any help in this matter.  If Boudreaux had chosen (f) instead of (a), then (f) would have been right-for-him.  If he had chosen (g), then (g) would have been right-for-him.  And so on.  There is no belief choice he can make which will be ‘wrong-for-him.’ 

            In such circumstances, it is difficult to see how genuine practical deliberation can still be possible.  To deliberate is to weigh one’s options in light of one’s evidence, reasons, consequences and background beliefs.  But no process of weighing is applicable in the relativist’s case because every belief has equal weight or merit.  Regardless of what ethical beliefs Boudreaux may have, each of them is ‘true-for-him.’  Subjective relativism seems to make it impossible for Boudreaux’s practical choices to be anything but arbitrary.

            We have seen that conventional relativism and subjective relativism are both subject to very serious objections.  However, you should not forget that both forms of relativism also put forward some very challenging arguments against moral absolutism.  Relativists seem to be right about the evils of ethnocentrism and the virtue of tolerance.  Both forms of relativism also try to promote a seemingly healthy respect for other ways of life and other people.  Both relativists and absolutists face philosophical objections they must answer if their views of morality are going to be fully adequate. 

Bennett, Jonathan. 1997. “The Conscience of Huckleberry Finn.” In Christina Sommers and Fred Sommers (eds.), Vice and Virtue in Everyday Life: Introductory Readings in Ethics . Ft. Worth, TX: Harcourt Brace College Publishers, pp. 20-34.

Crapo, Richley H. 1993. Cultural Anthropology: Understanding Ourselves & Others , 3rd edn. Guilford, CT: Dushkin Publishing.

Pojman, Louis. 1999. Ethics: Discovering Right and Wrong , 3rd edn. Belmont, CA: Wadsworth.

Differentiation, diversity and dependency in higher education

Research output : Thesis › PhD Thesis - Research UT, graduation UT

• METIS-100006

T1 - Differentiation, diversity and dependency in higher education

AU - Huisman, Jeroen

PY - 1995/10/20

Y1 - 1995/10/20

KW - METIS-100006

M3 - PhD Thesis - Research UT, graduation UT

SN - 90 5189 535 6

CY - Utrecht

Reason and Meaning

Philosophical reflections on life, death, and the meaning of life, summary of cultural relativism.

Philosophical Ethics 

Ethics is that part of philosophy which deals with the good and bad, or right and wrong in human conduct. It asks questions like: What is morality? Is morality objective or subjective? What is the relationship between self-interest and morality? Why should I be moral?

We can divide philosophical ethics into four parts. Meta-ethics analyzes moral concepts, moral justification, and the meaning of moral language. Descriptive ethics describes the moral systems of various cultures. Normative ethics considers moral norms, standards or criteria that underlie moral theories. Applied ethics applies normative theories to moral problems in law, medicine, business, computer science, the environment and more. Over the next few weeks we will discuss normative ethics, or moral theories. We’ll begin with relativism.

Now you might agree that  my previous assault on relativism has been successful, but still claim that while some truths are objective—logical, mathematical, and natural scientific ones for example—other so-called truths are relative—ethical truths for instance. Such considerations lead us to moral relativism, the theory that there are no absolute, objective, and universally binding moral truths . According to the moral relativist, there exist conflicting moral claims that are both true. (X is right, and x is wrong.) In short, the ethical relativist denies that there is any objective truth about right and wrong. Ethical judgments are not true or false because there is no objective moral truth—x is right—for a moral judgment to correspond with. In brief, morality is relative, subjective, and non-universally binding, and disagreements about ethics are like disagreements about what flavor of ice cream is best.

And what specifically might morality be relative to? Usually morality is thought to be relative to a group’s or individual’s: beliefs, emotions, opinions, wants, desires, interests, preferences, feelings, etc. Thus, we distinguish between two kinds of moral relativism: cultural moral relativism and personal moral relativism. (I’ll discuss the first one today, and the second one tomorrow.)

What is Cultural Moral Relativism?  

Cultural moral relativism i s the theory that moral judgments or truths are relative to cultures . Consequently, what is right in one society may be wrong in another and vice versa. (For culture, you may substitute: nation; society; group, sub-culture, etc.) This is another theory with ancient roots. Herodotus, the father of history, describes the Greeks encounter with the Callatians who ate their dead relatives. Naturally, the Greeks found this practice revolting. But the Callatians were equally repelled by the Greek practice of cremation causing Herodotus to conclude that ethics is culturally relative. World literature sounds a recurring theme: different cultures have different moral codes , an insight confirmed by the evidence of cultural differences. The Incas practiced human sacrifice, Eskimos shared their wives with strangers and killed newborns, Japanese samurai tried out his new sword on an innocent passer-by, Europeans enslaved masses of Africans, and female circumcision is performed today in parts of North Africa.

Cultural moral relativism contains two theses: 1) the diversity thesis— moral beliefs, practices, and values are diverse or vary from one culture to another; and 2) the dependency thesis —moral obligations   depends upon cultures, since they are the final arbiters of moral truth. In short, cultural relativism implies that no cultural values have any objective, universal validity, and it would be arrogant for one culture to make moral judgments about other cultures.

The thesis of diversity is descriptive; it describes the way things are. Moral beliefs, rules, and practices, in fact, depend upon facets of culture like social, political, religious, and economic institutions. By contrast the thesis of dependency is prescriptive ; it describes how things ought to be. Morality should depend on culture because there is nothing else upon which it is based. Now we might argue for cultural relativism as follows:

Argument 1 – (from the diversity thesis)

• Different cultures have different moral codes;
• Thus, there is no morality independent of culture.

The weakness of this argument is that  the conclusion doesn’t follow from the premise . The fact that cultures disagree about morality doesn’t show that morality is relative. After all, cultures disagree about whether abortion is moral or immoral, but their disagreement doesn’t mean there is no truth about the matter. It might be that one culture is just mistaken. Consider how cultures might disagree as to whether the earth or sun is at the center of our solar system. Their disagreement doesn’t mean there is no truth about the matter. Similarly, societies might disagree about whether they should put their young to death, but that disagreement proves nothing, other than societies disagree. So cultural disagreements are not enough to prove cultural relativism. Consider another argument:

Argument 2 – (from the dependency thesis)

• What is often regarded as the moral truths depends on cultural beliefs;
• Thus, there is no moral truth independent of culture.

The argument commits the fallacy that logicians call “begging the question.” This occurs when you assume the truth of what you are trying to prove. (For example, if you ask me why I think abortion is wrong and I say, because it’s bad, I’ve begged the question.) In argument 2, one is trying to show that right and wrong depend on culture. It begs the question to say that right and wrong depend on culture because they depend on culture.

Now might we make a stronger case for the relativist if we put the two theses together?

Premise 1 – Right and wrong vary between cultures (diversity). Premise 2 – Right and wrong depend upon a cultural context (dependency). Conclusion – Thus, right and wrong are relative to culture.  

Critique of Cultural Moral Relativism – Premise 1

This seems better; at least the conclusion follows from the premises. But are these premises true? Let’s consider the first premise (diversity). Nothing seems more obvious than the fact of cultural differences. Eskimos believed in infanticide; most Americans do not. Most Americans believe executing criminals is morally justifiable; most French find the practice barbaric. Clearly, there are different cultural mores. But maybe the differences between cultural values are not as great as they seem.

Consider that Eskimos live in harsh climates where food is in short supply and mothers nurse their babies for years. There simply isn’t enough food for all their children, nor enough backs upon which nomadic people can carry their children. So Eskimos want their children to live just like we do, and it is the harsh and unusual condition that force them to make difficult choices. Sometimes they kill a weaker child so that both the stronger and weaker children won’t die. We may disagree with the practice, but we can imagine doing the same in similar circumstances. Thus, the underlying principle—life is valuable—has been applied differently in different contexts. Maybe cultures aren’t so different after all.

Consider that there is more crime in America than in France. Most Americans seem to believe that criminals deserve to be punished for their crimes, that severe punishment brings peace to the victim’s family, that capital punishment is a deterrent to crime, etc. The French are more likely to renounce retribution or doubt that capital punishment brings victim’s families peace or deters crime. But notice again. Both cultures are steered by a principle—act justly—even though they apply the principle differently. So upon closer inspection, there doesn’t seem to be as much disagreement as it first appeared. So the differences in cultural values might be more apparent than real.

Now suppose we could show that there are moral principles that all cultures share? Wouldn’t that show that morality was not relative to culture? Many scientists claim that there are moral principles common to all cultures. 1 For instance, all cultures share: regulations on sexual behavior; prohibitions against unjust killing; requirements of familial obligations and child care; emphasis on truth-telling; and reward for reciprocity and cooperation. If we take these two ideas together—cultural moral differences aren’t as great as they appear, and all cultures share some moral values—then the diversity thesis is false . And if the first premise is false, then the conclusion of the cultural relativist’s argument doesn’t follow.

However notice that even if the first premise is false , that doesn’t prove that moral objectivism is true. Cultures that share the same moral values could all be wrong! So the empirical evidence concerning similarities and differences between moral codes isn’t relevant to the question of whether morality is absolute or relative. And that means that while we haven’t proven the truth of cultural absolutism, we have undermined the cultural relativist. For the evidence about diversity of culture is irrelevant, then we have undermined the relativist’s first premise, and with it the conclusion of his/her argument.

Critique of Cultural Moral Relativism – Premise 2

While undermining the first premise sufficiently undermines cultural relativism, let’s turn to the second premise (dependency) to see if it fares any better. Now it does appear true that some moral “truths” depend on culture—for example, regulations on sexual behaviors or funeral practices. But it is not self-evident that all moral truth depends on culture. Moral truth may be independent of culture in the same way that other truths are independent of culture. Ethics may be objectively grounded in reason, the god’s commands, the most happiness for the most people, human nature, or something else.

But rather than trying to contradict all the relativist’s arguments for the second premise, consider the implications of taking cultural relativism seriously. If cultural relativism is true then all of the following (counter-intuitive) are true. 1) We cannot make cross-cultural judgments . We could not consistently criticize a culture for killing all those over forty, exterminating ethnic groups, or banishing children to the Antarctic. 2 ) We cannot make intra-cultural judgments . We cannot say, even within our culture, whether we should send children to their death or to school, whether we should torture our criminals or reward them. 3) The idea of moral progress is incoherent . All you can say is that cultures change, not that one is better than another. The old culture practiced slavery; we do not, and that’s the end of it. The appearance of moral progress is illusory.

But all of this is counter-intuitive. We might think that cultures can do what they want regarding funeral practices, but what about human sacrifice? Aren’t there some things that are just plain wrong, in both other cultures and our own? Don’t you believe that society is better now because it has outlawed slavery? Cultural relativism answers no to both questions. But can such a strongly counterintuitive theory be correct?

Summary and Transition

Thus cultural relativism is as incoherent and unsubstantiated as epistemological relativism, as I argued yesterday. The logical arguments for cultural relativism fail, and we have good reasons to doubt the truth of the premises of cultural relativism. Finally, cultural relativism contradicts our moral intuition. While we can’t prove that cultural relativism is mistaken—you can only really do this in logic or mathematics—we have shown that there are many reasons to doubt the theory, and few reasons to accept it.

Share this:

3 thoughts on “ summary of cultural relativism ”.

I’d like to offer an odd point of view on this question, although I hasten to point out that I have no expertise, and my analysis is nowhere near as careful or thorough as your own. Treat this idea as a curious stimulus for thought, not a clearly established thesis.

I look at reality and I see no morality. It’s not in this tree; I don’t find it when I dig a hole, nor does it seem to be under the sea or in the stars. It is a strictly human concept; in fact, it is a human. To my narrow physicist’s point of view, this means that it is not objectively real; it’s merely a human perception.

Of course, there are plenty of human perceptions that *are* real in some sense. English is a language; so is Urdu; and they are certainly real in the sense that they are used in the real world. In fact, I think that languages provide us with a useful analogy. Moral systems are arbitrary in the same sense that languages are arbitrary. That is, there is absolutely nothing in the physical world that dictates that the sound “cat” intrinsically refers to our pets. “Gato” and “feles” are just as arbitrary. Nevertheless, language is constrained to follow an innate “deep structure” that is universal to all humans.

In the same fashion, we could argue that morality is arbitrary yet constrained by certain universal principles. I here argue that morality has been subjected to exactly the same evolutionary pressures that applied to humans as individuals. Cultures with stupid moral systems were superseded by cultures with more pragmatic moral systems. The prohibition against murder, for example, is obviously of great pragmatic value for any society. Similar principles elicit prohibitions against many other behaviors.

As you point out, applications of the deep principles can manifest themselves in surprisingly different fashions in different physical environments. Yet there remains one fundamental principle: societies evolve moral systems that optimize their ability to survive and prosper in the physical environment in which they find themselves.

Anyway, for what it’s worth, that’s how I perceive the existence of moral systems. I understand how many people feel a need for some objective moral system to exist, something that we could find graven in stone that would give us peace of mind in knowing what truly is right and wrong. I maintain that no such stone can ever exist.

I’m sure that, with your vastly greater experience wrestling with these issues, you can pick out some blunders in my thinking, so I’d love to see your reactions.

Most of these issues are addressed in my ethics book or at good sites like Wikipedia, Stanford Enc. of Phil, or the internet enc. of phil. Everything you say is spot on and you could have a new career as an ethicist. Ethics is so important, we so need more moral and intelligent people. Basically you have adopted an anti-realist position in meta-ethics in your comments.

Moral Anti-Realism (or Moral Irrealism) is the meta-ethical doctrine that there are no objective moral values. It is usually defined in opposition to Moral Realism, which holds that there are indeed objective moral values, that evaluative statements are factual claims which are either true or false, and that their truth or falsity is independent of our perception of them or our beliefs, feelings or other attitudes towards them. Thus, Moral Anti-Realism can involve either a denial that moral properties exist at all, or the acceptance that they do exist, but that their existence is mind-dependent and not objective or independent.

You do hedge your bets slightly with this quote “In the same fashion, we could argue that morality is arbitrary yet constrained by certain universal principles.” This position was defended by another non-professional philosopher Michael Shermer in his wonderful and readable book “The Science of Good & Evil.” I think you would like it.

The position you adopt is popular but not the most popular position. Here are the stats from the site “what phils believe” Meta-ethics: moral realism 56.4%; moral anti-realism 27.7%; other 15.9%.

Finally you mention evolution and we are of one mind here. I am an evolutionary ethicist and you can read all about that too. I think game design might have to go on hold while you become an ethicist.

CRAWFORD INVENTS WHEEL!! SCIENTISTS AND ENGINEERS AGHAST!! “We never thought of that!” Seen as “the death of distance”!!

Leave a Reply

Your email address will not be published. Required fields are marked *

Notify me of new posts by email.

This site uses Akismet to reduce spam. Learn how your comment data is processed .

Louis Pojman destroys relativism

October 6, 2012.

Louis Pojman: Against Relativism and For Objectivism

source:  http://www2.drury.edu/cpanza/relativism.pdf

I. Philosophical arguments aspire to the form of deductive validity.

This means that philosophers strive to make their arguments deductively valid. A deductively valid argument is one where, if the premises are true, the conclusion can’t possibly be false (it has the right logical form such that this will be the case). So what a philosopher tries to do is construct a valid argument form, and then make sure that the premises ARE true. In such a case, the conclusion cannot be false. If so, no better argument for that conclusion can be given.

II. Pojman’s Strategy Against the Argument for relativism:

P1. Beliefs about what is right and wrong differ across cultures (the Diversity thesis). P2. What is right and wrong is dependent upon, or relative to, culture (the Dependency thesis). — C1. Thus, there is no objective right and wrong.

Pojman recognizes that this is a valid argument. As such, IF the premises are true, the conclusion (which denies moral objectivism) must be true. If moral objectivism must be false, then moral relativism must be true. Pojman tries to attack this argument. The attack has two stages.

STAGE ONE: show that one or more of the premises (of the relativist’s argument) are NOT true. If Pojman can do this, then even if the argument is valid, the truth of the conclusion will not necessarily follow (because it would only follow IF the premises were true). *Note: Even if Pojman is successful, showing that the conclusion is not necessarily true does not entail that it is false. It simply shows that the conclusion could be false.

STAGE TWO: show that the conclusion is false by showing that the reverse of the conclusion must be true. In other words, Pojman’s project here will be to show that moral objectivism must be true. If it must be true, then the claim that there is no objective right and wrong cannot be true. If he succeeds in both stages, the argument for relativism is defeated.

III. Pojman’s Stage One Argument

Pojman must show that one or more of the premises in the argument for relativism is (or are) false. Now Pojman realizes that the first premise (called P1 in the argument for relativism) is not objectionable. What this means is that there is no reason why Pojman would need to find it false. As a matter of fact, Pojman thinks it is true. If you go to various cultures, you will find various different definitions of right and wrong. Is this harmful to moral objectivism?

If Pojman acknowledges that P1 is true, does this harm moral objectivism? No. Recall why Ruth Benedict’s argument (which is really just P1) doesn’t work to get you relativism on its own. The reason is this: one cannot validly move from a statement about beliefs to a statement about fact. If you could, then this argument would be conclusive: P1. Beliefs about what is right and wrong differ across cultures — C1. Thus, there is no objective right and wrong

The conclusion here is NOT necessarily true, even if the premise P1 is true. That means thatthe argument is not valid. We know it is not valid because it is possible for the premises to be true and the conclusion to be false. To see how, just construct another argument with a similar form (this is called an argument from analogy):

P1. Beliefs about the shape of the Earth differ across cultures — C1. Thus, there is no objective right and wrong about the shape of the Earth.

Clearly, this argument is not valid. It is possible for people to disagree about the shape of the Earth, but this does not entail that there is no objective answer about the shape of the Earth. So Pojman reasons that if this is so, and the argument for relativism using just P1 as a premise has the same form, then the claim that people differ about moral beliefs does not entail that there is no objective answer about what is right and wrong.

So Pojman allows for P1 to be true, since it does not harm objectivism about morality. Clearly, then, the worrisome premise is P2, called the dependency thesis. The dependency thesis is the claim that what is right and wrong is itself relative to culture (this differs from P1 – it is not a claim about beliefs, it is a claim about the nature of right and wrong itself). Clearly P2 entails relativism about morality. If P2 is true, then C1 cannot be false.

Pojman recognizes, however, that P2 – the dependency thesis – has two forms, what he calls ‘weak’ and ‘strong’ dependency. Before he attacks P2 he must be sure that he is attacking the right version of P2.

Here’s an example.

In America and in Europe we share the belief that one ought to be polite to strangers. Let’s call this belief X. Now this does not mean that the way in which politeness is expressed in America and in Europe is the same. As a matter of fact, they differ.

In Europe, politeness to a stranger might mean kissing the stranger on both cheeks. In America, this would be disturbing. Rather, a handshake would be polite, one which would be rude in Europe. From this we can ascertain that the right way to express politeness is relative to the country you are in.

Does moral objectivism need to be worried about ‘weak’ dependency? No. It can be true that moral belief X (politeness in this case) is objective and not relative to culture, even if the ways in which politeness is ‘rightly’ expressed is relative to culture.

What about strong dependence? This is the form of P2 Pojman wants to attack. It says not just that the ways in which moral beliefs are expressed is relative to culture, but that the moral beliefs themselves are relative to culture.

IV. The Case Against P2 or the Strong Dependency Thesis

The Strong Dependency Thesis is used by two camps of relativism to support why their positions are right. The two camps are:

1. Subjectivism: morality is dependent on individuals, not culture

2. Conventionalism: morality is dependent on culture

If Pojman’s argument against P2 is going to work, then it will have to turn out that both subjectivism and conventionalism are false. If they are both false, then P2 does not lead to any true theories (and thus P2 must be false). If either of the two is not false, then it will turn out that the strong dependency thesis could in fact be true, since it does lead to a theory that could be true.

So Pojman must attack both camps and show that they cannot work.

The Argument Against Subjectivism

Pojman’s Objection: The purpose of morality is to settle interpersonal conflicts. By definition, however, a subjectivist does not share a moral language with another person.-4- Moreover, as we see in chapter 3 of Rachels, subjectivism states that moral statements are just claims about feelings. And two people cannot be in disagreement about their feelings.

Thus subjectivism cannot settle interpersonal conflicts, because no interpersonal conflicts can exist. Since interpersonal conflicts on morality DO exist, and because we DO think morality is used to settle those disputes, subjectivism is false.

Against Conventionalism

Pojman recognizes that he cannot use the same argument that he used against subjectivism and lodge it against conventionalism. The reason is evident: if conventionalism is true, then people do in fact share moral language. So they can in fact settle interpersonal conflicts. So Pojman needs a different argument.

Pojman’s Objection: Conventionalism entails tolerance.  So there is at least one absolute value at the ehart of relativism – tolerance of others’ opinions.

V. Stage Two: Establishing the Truth of Moral Objectivism

Pojman’s argument for moral objectivism requires reducing morality to biology. What this means is this: Pojman thinks that there are certain moral rules that are entailed if we, as a species, wish to stay alive and flourish.

Example: if the human species is to survive, then a moral rule would be ‘one ought not to kill another’. If it were permissible to kill others, then the chances of species survival would be lower than if it were not permissible.

Here is Pojman’s argument: P1. Objective moral principles are those adherence to which meet the needs and promote the most significant needs of persons. P2. Some principles are such that adherence to them meets and promotes the significant needs of persons. -5- — C1. Thus, there are some objective moral rules.

Note that Pojman thinks the argument is valid. So if the premises are true, the conclusion cannot be false. If the conclusion in this case (there are some objective moral rules) cannot be false, then it must be true. If so, then the conclusion of the relativist=s argument (which denies objective moral rules) must be false. If so, the Pojman has succeeded in completely dismantling the relativist’s argument.

Study with us

Practise Questions 2020

Religious Studies Guides – 2020

Check out our great books in the Shop

Thank you so mush. I appreciate the information.

Leave a Reply Cancel

This site uses Akismet to reduce spam. Learn how your comment data is processed .

Strengths and Weaknesses of the Dependency Essay

The dependency thesis states that what is good or bad depends on what the people of that particular society, regard as wrong or right. Another definition is that; the moral principles that have been accepted by a certain cultural are the correct ones, irrespective of what other cultures practice. If a certain community accepts that upon meeting someone older than yourself you should bow your head, then not bowing your head to an elder person is morally wrong (1).

The dependency thesis has some strengths and weaknesses. One of the strengths is that; if by any chance you get entangled in another community, you become unaffected by some of the earlier obligations.

For example, in a Kenyan society, abortion is wrong. If a Kenyan girl is married to a Chinese, whose culture condones abortion, she will be free from the no abortion rule. When this Kenyan girl is married to a Chinese, she will have the choice of aborting if wished. According to the thesis, it is not right for one community to force its rule unto another person from a different community.

This means that a foreigner may live amidst another community, and continue practicing what he thinks is right. For example, a reporter from BBC will be able to report the lifestyle of the Fulani people, without himself being forced to abide to the traditions of the Fulani (3).

If somebody is supposed to adhere to his culture’s principles, it means that there is no supreme person who is supposed to judge everybody. This means there is nothing that entitles one human being to judge others. The thesis also advocates that we should see other people’s culture as important as ours.

This has enabled people to tolerate other people’s culture. Being able to tolerate other people’s culture has enabled people from different cultures to live together. For example, a Muslim and a Christian can be very good roommates even if they have different believes. Both cultures teach to respect other people’s culture (4).

One of the major draw back of this thesis is that, it gives room for one community rising over the other with catastrophic results. From the thesis, Hitler can be viewed to be equally moral as Gandhi. Both Hitler and Gandhi did and preached what they believed is right for their people.

If a certain community has a moral principle which allows one to kill another person, the results can be catastrophic. During the crusade mission in the 10 th century, Muslims and Christians slaughtered each other. Both of them were thinking that they were doing the right thing.

One irony in this case is that they were all killing, each other to please the same person. During the Rwandan genocide, the Hutus were made to believe that they needed to defend their country from Tutsis. Even though they were the majority, they went on to kill approximately 800,000 Tutsis (5).

The other flaw of this thesis is that, people who advocate for change are considered to be wrong. For example, can we say that Jesus was morally wrong sine he was not accepted by his community? People who may come up with ways that are supposed to make the living condition of a community better, maybe shut down since they may be going against what is accepted as morally right. Another fault is that a person can fail to be held responsible for doing something wrong, simply because he does not belong to any community (7).

Beebe, James. “Ethical Relativism.” ACSU . ACSU, 15 January 2003. Web.

• Chicago (A-D)
• Chicago (N-B)

IvyPanda. (2018, August 16). Strengths and Weaknesses of the Dependency. https://ivypanda.com/essays/strengths-and-weaknesses-of-the-dependency-thesis/

"Strengths and Weaknesses of the Dependency." IvyPanda , 16 Aug. 2018, ivypanda.com/essays/strengths-and-weaknesses-of-the-dependency-thesis/.

IvyPanda . (2018) 'Strengths and Weaknesses of the Dependency'. 16 August.

IvyPanda . 2018. "Strengths and Weaknesses of the Dependency." August 16, 2018. https://ivypanda.com/essays/strengths-and-weaknesses-of-the-dependency-thesis/.

1. IvyPanda . "Strengths and Weaknesses of the Dependency." August 16, 2018. https://ivypanda.com/essays/strengths-and-weaknesses-of-the-dependency-thesis/.

Bibliography

IvyPanda . "Strengths and Weaknesses of the Dependency." August 16, 2018. https://ivypanda.com/essays/strengths-and-weaknesses-of-the-dependency-thesis/.

• Achondroplasia Genetic Disorder: Pedigree
• Law Ethics: The Use of Force During Emergencies
• Summary Atlas Shrugged Part One and Two
• My father's black pride
• Stoning in the Twenty-First Century: The Dangers of Cultural Relativism
• Social and Cultural Diversity and Stereotypes
• Cultural Competence Among Professionals
• What is Different to us, is the Same to Others

• For Current Majors and Minors
• For Faculty & Graduate Students
• For Community Members and Alumni
• Affiliated Faculty
• Visiting Scholars
• Graduate Students
• Ancient History
• Comparative Race and Ethnicity
• Comparative Gender + Sexualities History
• Medieval & Early Modern Europe
• History of Public Policy
• History of Science
• Latin America
• Medieval Studies
• Middle East
• Modern Europe
• Public History
• United States History
• World History
• Empires in Question
• Gender + Sexualities
• History and Political Economy
• Public History and Theory
• Race, Blackness, & Abolition
• Science, Technology, and Society
• Dissertation Titles
• Undergraduate Journal
• Affiliated Centers, Programs, and Groups
• Undergraduate Prospective Students
• Undergraduate Academic Advising
• History, B.A.
• History of Public Policy and Law, B.A.
• Minor in History
• Minor in Labor Studies
• Minor in Poverty, Inequality and Social Justice
• American History & Institutions Requirement | Exam Information
• Careers for History Majors
• Phi Alpha Theta (History Majors’ Club)
• Senior Honors Seminar (History 194AH/194BH)
• History of Public Policy and Law Senior Thesis Seminar (HIST 195IA/IB)
• Undergraduate Journal of History
• Why Study History?
• Undergraduate Awards & Fellowships
• Undergraduate Student Resources
• Graduate Student Resources
• Prospective Students
• History Graduate Students Association (HGSA)
• Central Continuing Fellowships
• Departmental Dissertation (and Diversity) Fellowships
• Research and Conference Travel Grants
• Student Self-Nominated Awards and Prizes
• Faculty Nominated Awards and Prizes
• Teaching Assistantships
• Teaching Opportunities
• Fields of Study
• Living in Santa Barbara, CA
• Winter 2024
• Spring 2024
• Summer 2024
• “The Archive” Newsletters
• Latest News
• History Associates
• Diversity, Inclusion, and Equity Statement
• UCSB History Department Statement on Floyd Uprising
• Faculty Resources
• HASC (Staff Support Center)
• Campus Maps & Buildings
• UCSB Library
• Connect Email
• Department of History
• History Associates Graduate Fellowship
• History Graduate Student Support

Nora Kassner awarded John D’Emilio LGBTQ History Dissertation Award

The Organization of American Historians (OAH) has awarded Nora M. Kassner , who recently completed her PhD in our department, the 2024 John D’Emilio LGBTQ History Dissertation Award, which recognizes the best PhD dissertation in U.S. LBGTQ history. The Award was presented during the OAH’s 2024 Conference on American History .

Kassner’s, “Hard to Place: Gay and Lesbian Foster Families and the Remaking of U.S. Family Policy” (completed under the direction of Alice O’Connor), with an incisive and personable voice, makes a stunning intervention in scholarship on queer family formation and the historiography of U.S. family policy in the twentieth century. Spanning the 1970s through the 1990s, with a regional focus on foster care systems in Florida, New Jersey, and California, Kassner’s dissertation argues that foster parenting was an essential and understudied catalyst for securing state recognition and protection for gay and lesbian parents. Kassner deftly braids together extensive archival research and oral history to craft a granular study of the day-to-day decisions of foster parents, social workers, and activists. In doing so, she accounts for the hyperlocal, idiosyncratic practices that gradually amounted to more inclusive parent licensing nationally. Kassner also captures the racial politics that fueled the selective liberalization of U.S. family policy in the late twentieth century. The committee was especially impressed by Kassner’s application of Black feminist theory to critique the sinister co-dependency between state divestment in Black families, which produced an influx of “hard to place” children in U.S. foster care, and state investment in the white gay and lesbian foster families who fostered and adopted them.

Congratulations, Nora!

Site Administration

Department of History University of California, Santa Barbara Santa Barbara, California 93106-9410

Directory Fax: 805.893.7671

Spot a problem? Contact the Webmaster

• Undergraduate Program
• Graduate Program
• Current Courses
• Department Chair
• Entries feed
• Comments feed
• WordPress.org

7 Princeton Traditions in My Last Semester

May 16, 2024, amélie lemay.

As a follow-up to my sophomore blog post about 7 traditions in my first on-campus semester , I now present to you 7 traditions from my final semester. 

1. Taking 3 courses + thesis

In the final semester, seniors generally take a lighter course load to have additional time to focus on the thesis. This spring I only took 3 courses plus the thesis (which counts as a course), giving me more time to focus on my project than when I have a typical 4-5 course load. This also gave me time for graduate school interviews, student visit days, and other tasks associated with planning for life post-Princeton.

2. Choosing a grad school program

Come March, I was notified of my acceptances to the different graduate school programs I'd applied to. In the fall, I'll be starting a doctoral program in Civil and Environmental Engineering at MIT working with Dr. Desirée Plata! Being able to share this news with my professors and letter of recommendation writers was exciting and rewarding.

3. Printing and binding my thesis

In mid-April, my thesis was wrapping up, and it was time for official printing and binding. Printing your thesis is optional, but it's traditional to present a leather-bound copy to your advisor. I chose to print my thesis and was incredibly proud to present the culmination of my project to Dr. Bourg.

4. Stepping into the Fountain of Freedom post-thesis submission

Following submission of the thesis, seniors will step into the Fountain of Freedom to officially mark the beginning of the mythical "PTL" (post-thesis life). The water wasn't very warm on the day after my department's thesis submission date (April 15), but I still honored the tradition by stepping into the water.

5. Wearing my class jacket

Formerly known as a " beer jacket ," to be worn by seniors at the Nassau Inn to protect their day clothes, the class jacket is now the de facto uniform for Reunions. The jacket prominently displays your class year, making it easy to spot your classmates among the masses of orange and black that flock to campus for Reunions each May. Our class voted on the design in the fall, and I'm really pleased with the final design.

6. Taking photos by the bronze tigers

Our class government offered free sessions with a pro photographer by the bronze tigers, and I also took photos of my friends myself. We brought numerous graduation props (thesis, class jacket, cap) to the session.

7. Walking through FitzRandolph Gate

At Commencement, I'll walk through FitzRandolph Gate for the first time since the class of 2024 Pre-Rade in my first on-campus semester. Legend has it that students who walk through the gates between the Pre-Rade and Commencement won't graduate in four years. All appears to be on track for me to officially receive my diploma on May 28, but I certainly won't be taking any chances between now and then.

And with that, my undergraduate experience at Princeton has come to a close! I've truly loved my time here, and I'll forever be grateful to Old Nassau.

Related Articles

Commencement commences, amor fati: embracing my path through princeton, plasa’s inaugural latine history series.

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

• View all journals
• My Account Login
• Explore content
• About the journal
• Publish with us
• Sign up for alerts
• Open access
• Published: 17 May 2024

Recruitment of plasma cells from IL-21-dependent and IL-21-independent immune reactions to the bone marrow

• Marta Ferreira-Gomes   ORCID: orcid.org/0000-0002-7171-7952 1   na1 ,
• Yidan Chen   ORCID: orcid.org/0000-0003-3232-6403 1 , 2   na1 ,
• Pawel Durek 1   na1 ,
• Hector Rincon-Arevalo   ORCID: orcid.org/0000-0001-5614-7894 1 , 2 , 3 , 4 ,
• Frederik Heinrich   ORCID: orcid.org/0000-0001-6097-5422 1 ,
• Laura Bauer   ORCID: orcid.org/0000-0002-1435-8121 5 ,
• Franziska Szelinski   ORCID: orcid.org/0000-0001-7850-5971 1 , 2 ,
• Gabriela Maria Guerra 1 ,
• Ana-Luisa Stefanski   ORCID: orcid.org/0000-0001-7689-6454 1 , 2 ,
• Antonia Niedobitek 1 ,
• Annika Wiedemann 1 , 2 ,
• Marina Bondareva 1 ,
• Jacob Ritter   ORCID: orcid.org/0000-0001-8962-1564 1 , 2 ,
• Katrin Lehmann 1 ,
• Sebastian Hardt 6 ,
• Christian Hipfl 6 ,
• Sascha Hein   ORCID: orcid.org/0000-0001-7257-4538 7 ,
• Eberhard Hildt   ORCID: orcid.org/0000-0002-3020-9564 7 ,
• Mareen Matz 8 ,
• Henrik E. Mei   ORCID: orcid.org/0000-0003-0697-7755 1 ,
• Qingyu Cheng 1 , 2 ,
• Van Duc Dang 1 , 2 ,
• Mario Witkowski   ORCID: orcid.org/0000-0002-8791-5558 1 , 9 ,
• Andreia C. Lino   ORCID: orcid.org/0000-0002-3791-8232 1 , 2 ,
• Andrey Kruglov 1 ,
• Fritz Melchers 1 ,
• Carsten Perka 6 ,
• Eva V. Schrezenmeier 1 , 4 , 8 ,
• Andreas Hutloff   ORCID: orcid.org/0000-0002-0572-8151 5 ,
• Andreas Radbruch   ORCID: orcid.org/0000-0001-5753-0000 1 , 2   na2 ,
• Thomas Dörner 1 , 2   na2 &
• Mir-Farzin Mashreghi   ORCID: orcid.org/0000-0002-8015-6907 1   na2  

Nature Communications volume  15 , Article number:  4182 ( 2024 ) Cite this article

8 Altmetric

Metrics details

• Gene regulation in immune cells
• Immunological memory
• Plasma cells
• RNA vaccines

Bone marrow plasma cells (BMPC) are the correlate of humoral immunity, consistently releasing antibodies into the bloodstream. It remains unclear if BMPC reflect different activation environments or maturation of their precursors. Here we define human BMPC heterogeneity and track the recruitment of antibody-secreting cells (ASC) from SARS-CoV-2 vaccine immune reactions to the bone marrow (BM). Trajectories based on single-cell transcriptomes and repertoires of peripheral and BM ASC reveal sequential colonisation of BMPC compartments. In activated B cells, IL-21 suppresses CD19 expression, indicating that CD19 low -BMPC are derived from follicular, while CD19 high -BMPC originate from extrafollicular immune reactions. In primary immune reactions, both CD19 low - and CD19 high -BMPC compartments are populated. In secondary immune reactions, most BMPC are recruited to CD19 high -BMPC compartments, reflecting their origin from extrafollicular reactivations of memory B cells. A pattern also observable in vaccinated-convalescent individuals and upon diphtheria/tetanus/pertussis recall-vaccination. Thus, BMPC diversity reflects the evolution of a given humoral immune response.

Introduction

Long-lived plasma cells (PC) are rare cells that reside and rest in dedicated survival niches of the bone marrow (BM), which continuously secrete antibodies against previously encountered pathogens 1 . These cells have been considered to originate mostly from T cell-dependent B-cell activation in secondary lymphoid organs. The concept of long-lasting humoral immunity provided by long-lived (memory) plasma cells 2 , 3 has gained acceptance and attention during the COVID-19 pandemic, when the presence of spike-specific memory plasma cells in the BM was considered a correlate of long-lasting protection against severe disease and death 4 , 5 . This protection, conferred by stable systemic antibody titres, can last for decades, if not a life time 6 . Despite their relevance, the recruitment of plasma cells from secondary lymphoid organs into the BM and their establishment there as long-lived plasma cells is not well understood. Plasmablasts generated in primary and secondary immune reactions might differ in their competence to enter the BM and survive there as long-lived plasma cells 1 . BM plasma cells (BMPC) are heterogeneous with respect to expression of e.g., CD19 7 , 8 , CD38 7 , PD-1 8 , CD39 and CD326 9 , but whether this reflects different environments encountered during their generation, their maturation in the BM or different qualities as BMPC, e.g., life-spans, has remained unclear. It also is unclear whether BMPC are constantly recruited to the BM during an immune reaction or only at the end, when affinity maturation is finished 10 , 11 , 12 .

Here, we address these conceptual questions by performing a global analysis of the transcriptional heterogeneity and antigen-receptor repertoire of human BMPC, and we follow the recruitment of plasma cells (PC) to the BM in primary and secondary immune reactions to SARS-CoV-2 vaccines. We find 10 “clans” of BMPC, compartments differing in and reflecting the instructive signals they received as activated B cells. By comparing the transcriptomic signatures of newly generated circulating antibody-secreting cells (ASC) from peripheral blood to the transcriptomes of established BMPC, we track the recruitment of PC to the bone marrow in the primary and secondary immune reaction. We identify interleukin-21 (IL-21) as the signal to downregulate expression of CD19 on activated B cells. CD19 low PC are thus generated in IL-21-dependent follicular germinal centre reactions, and CD19 high PC are generated in IL-21-independent extrafollicular (re)activations of (memory) B cells. In the immune reactions analysed here, PC generated in primary reactions are recruited to distinct CD19 low and CD19 high clans, while PC generated in secondary immune responses are nearly exclusively recruited to clan 0 (CD19 high ). Most ASC exiting the immune response early on express IgG1—an isotype induced by IL-21 13 , 14 . However, as the immune response progresses, ASC measured at later time points also express IgA1 and IgA2, indicative of TGF-β instruction at the time of activation and class switching 15 . ASC expressing IgG2, reflecting instruction by interferons 16 , were not as frequently induced by the vaccines. Taken together, PC recruited to the bone marrow in secondary immune reactions are derived from non-follicular reactivation of memory B cells, while CD19 low BMPC reflect the direct IL-21-dependent output of primary germinal centre reactions. Upon repeated vaccination, spike-specific BMPC, but also tetanus-specific BMPC are present in most clans, reflecting the evolution of the respective immune response, the continued recruitment of PC to the BM during the immune reaction, and their lasting maintenance there.

Transcriptional and phenotypic heterogeneity of BMPC

Single-cell transcriptomes of 49,347 BMPC were obtained from eight patients who underwent hip joint-replacement surgery (Supplementary Table  1 ). BMPC were enriched as viable CD38 high CD138 high CD3 − CD10 − CD14 − or CD38 high CD27 high CD3 − CD14 − cells (Supplementary Fig.  1a ), incubated in addition with DNA-barcoded antibodies for Cellular Indexing of Transcriptomes and Epitopes by Sequencing (CITE-seq), and subjected to single cell transcriptome as well as full-length B-cell receptor (BCR) sequencing. Cells were clustered according to their transcriptomes and visualised by uniform manifold approximation and projection for dimension reduction (UMAP) 17 . From the 15 defined subpopulations, clusters 2, 3, and 10 were excluded from further consideration, as they contained low quality cells expressing 16.4% (median, MAD = 6.6%) mitochondrial genes and/or a significantly low number of transcripts (a median of 453, 842, and 662, respectively; MAD = 133, 266, 152) (Supplementary Fig.  1b ). Clusters 7 and 14 represented CD20-expressing B cells and pre-B cells, respectively, and thus were not considered further as BMPC. The remaining 10 clusters (Fig.  1a ), amounting to 38235 cells and present in all eight donors (Fig.  1b ), were classified as BMPC according to their expression of the signature genes PRDM1 , IRF4 , XBP1 , and SDC1 (Fig.  1c ), and surface proteins, including CD27, CD38 and CD138 (Supplementary Fig.  1c, d ). 38% of the BMPC analysed resided within cluster 0, with 34% of them expressing CD19 transcripts and being of CD19 high phenotype (Fig.  1b–d , Supplementary Fig.  1e ). Most of these cells expressed IgA1 and IgG1 antibodies (Fig.  1e, f ). Clusters 1 and 4 contained predominantly CD19 low cells, 93% and 84%, respectively, not expressing CD19 transcripts (Fig.  1c, d , Supplementary Fig.  1e ). While cluster 1 was enriched for IgG1-expressing BMPC, cluster 4 contained mostly IgM and IgA-expressing BMPC (Fig.  1e, f ). Cells from cluster 5 are a population of XBP1 low IRF4 high IgA1 BMPC, with 27% expressing CD19 transcripts. Cluster 4 is enriched in IgA2 and IgM BMPC expressing IgJ , CCR10 and ITGB7 , indicative of a mucosal origin and TGF-β instruction 18 (Fig.  1c–f ). Cluster 6 consists of BMPC expressing CD9 and IgG2, hallmarks of a type II interferon (IFN)-driven immune reaction (Fig.  1d–f ). Cells expressing elevated levels of STAT1 and IFITM1 (Fig.  1d ) were contained in cluster 8, i.e. BMPC generated in a type I IFN response. This observation is supported by gene set enrichment analysis (GSEA), where the relative expression of genes associated with defined pathways is shown in a density plot (Fig.  1g ). Clusters 9 and 13 consist of BMPC expressing HLA-DR, a hallmark of recent generation from activated B cells 19 (Fig.  1d , Supplementary Fig.  1c, d ). They represent proliferating plasmablasts (cluster 13) and newly generated, still HLA-DR + plasma cells (cluster 9), with PD-1 signalling GSEA indicating their recent interaction with PD-1 expressing T cells (Fig.  1g ). BMPC of these two clusters also show a GSEA characteristic of high translational activity and oxidative phosphorylation (Fig.  1g ). Finally, clusters 11 and 12 are BMPC expressing heat shock genes and NR4A1 (Nur77), respectively, (Fig.  1d ) indicative of cellular stress. Nur77 has been described as an antagonist of B-cell lymphoma (Bcl)-family members and inducer of apoptosis in myeloma cells 20 , as well as being linked to B and T-cell self-reactivity 21 , 22 .

a Bone marrow plasma cells (BMPC; CD138 high CD38 high or CD38 high CD27 high ) from 8 patients undergoing total hip replacement surgery were isolated and sorted by FACS for single-cell sequencing (gating strategy in Supplementary Fig.  1a ). Amplified area: UMAP representation of remaining 38235 plasma cells after exclusion of contaminant and poor quality cells (see Supplementary Fig.  1b ). Clusters of transcriptionally similar cells were identified using shared nearest neighbour (SNN) modularity optimisation. b Percentage of BMPC found in each cluster per donor’s total cells analysed. Horizontal lines indicate the median. n  = 8 independent donors. c UMAP representation of the expression levels of selected PC signature genes across BMPC clusters. d Bubble plots of expression levels of the top five marker genes for each cluster (left) and of additional selected genes (right). Colour scale shows the z scores of the average expression of a gene within the indicated cluster. Bubble sizes correspond to the fraction of cells expressing a defined gene within the indicated cluster. e Density plots of immunoglobulin isotype expression within the BMPC compartment. f Frequency of plasma cells expressing a defined immunoglobulin isotype, displayed according to the clusters in a , per donor’s total cells where a full BCR could be identified. For better readability, each isotype was plotted separately, even though the percentages are related to the total BCRs from each donor. Horizontal lines indicate the median. n  = 8 independent donors. g Density plots of BMPC significantly enriched in gene sets from different biological pathways as calculated by Gene Set Enrichment Analysis (GSEA). Source data are provided as a Source Data file.

Overall, the increased FAS (CD95) and/or lower BCL2 expression in CD19 high clusters (Supplementary Fig.  1d, f ) suggest that they might be transcriptionally more prone to succumb to extrinsic and intrinsic apoptosis inducers, while CD19 low clusters may be more resilient. In support of this hypothesis, GSEA indicates that cells of clusters 1 and 6 show STAT3, STAT5, and TNF receptor family signal transduction signatures. They also express IL5R as well as the TNF-family receptors TNFRSF13B (TACI) and TNFRSF17 (BCMA) (Fig.  1g , Supplementary Fig.  1f ), the latter ones protecting plasma cells from anabolic stress 23 , 24 , 25 . Clusters 1 and 6 are also enriched in plasma cells expressing genes associated with hypoxia, a condition known to favour plasma cell survival in BM niches 26 . Finally, at the border between cluster 0 and 1, there is an enrichment of cells expressing genes associated with glycolysis, a metabolic condition crucial for plasma cell survival in BM niches 27 (Fig.  1g ). In summary, analysis of the phenotype and transcriptional profile of individual BMPC reveals a remarkable heterogeneity, even within the 10 clusters identified, which thus define clans of BMPC, rather than homogeneous clusters.

BMPC clans express exclusive antibody repertoires

Consistent with their origin from distinct types of immune reactions, the various BMPC clans also expressed different repertoires of antigen receptors. Overall, the different BMPC clans were similar in diversity and we did not find significantly expanded clones (Fig.  2a ). The repertoires of the different clans, defined by the germline sequences of heavy and light chains of the BCR and CDR3 similarity of their individual cells, did not overlap beyond what would be expected stochastically (Supplementary Fig.  2a ). Also, pseudotime analysis did not identify clear relationships and developmental paths between the BMPC clans (Supplementary Fig.  2b ). Mutations in both the framework and the CDR sequences were more frequent in clans 0, 8 and 11, i.e., the CD19 high BMPC, as compared to the CD19 low BMPC clans 1, 4 and 6 (Fig.  2b–d , Supplementary Fig.  2c ). This is in line with our previous findings 28 .

a  Number of clonal families of BCRs found within the different BMPC clusters (diversity) and probability of finding different clonal families by random selection of cells (Simpson diversity index). A clonal family was defined by V and J gene composition and a CDR3 region with <20% Hamming distance in both the heavy and light chains originating from one donor. b Mutation rates in the framework regions FR1-3 (left) and in the CDR1-3 regions (right) of the heavy and light chain rearrangements across BMPC clusters. c Bubble plot of the mutation rates in the framework regions FR1-3 (left) and in the CDR1-3 regions (right) of BCRs per isotype and cluster. Colour scale indicates median mutation rates. Values below or above the scale limits are shown in blue or red, respectively. Bubble sizes correspond to the percentage of cells expressing a defined isotype within the indicated cluster. d Violin plot depicting the mutation rates in the V gene of the BCRs of BMPC per cluster. Statistical significance between clusters is shown in Supplementary Data  1 (two-tailed Mann–Whitney U test). Horizontal lines represent the median mutation rate. Violins are coloured by the z score of CD19 gene expression in each cluster. e Identification of SARS-CoV-2 spike-specific and tetanus toxoid-specific public clones (in black) among analysed BMPC. Public clones were defined by exhibiting over 80% CDR3 sequence identity in both heavy and light chains when compared to the BCR of sequenced peripheral blood and bone marrow spike- and tetanus-specific cells from vaccinated individuals (see Supplementary Fig.  1a and Supplementary Data  1 ). f Relative distribution of spike-specific (red) and tetanus-specific (blue) BMPC (depicted in e ) per cluster. g Comparison of mutation rates within the framework regions FR1-3 (left) and the CDR1-3 regions (right) of spike-specific (red) and tetanus-specific (blue) BMPC per isotype. Horizontal lines represent the median mutation rate. Statistics were performed using a two-tailed Mann–Whitney U test. Source data are provided as a Source Data file.

In order to locate antigen-specific cells in the various plasma cell clans, we analysed BMPC secreting antibodies specific for the receptor-binding domain (RBD) or full spike protein of SARS-CoV-2, or the tetanus toxoid (TT) C fragment. All BM samples were processed during the COVID-19 pandemic, with patients having either had contact with SARS-CoV-2 or been vaccinated between 56 and 312 days prior to BM sampling (Supplementary Table  1 ). In all cases where serum could be analysed (Supplementary Table  1 ), patients had RBD- and spike-specific serum antibodies, as well as TT-specific serum antibodies (Supplementary Fig.  2d ). Since most BMPC do not express antigen-receptors on their cell surface 29 , 30 , direct isolation of viable antigen-specific BMPC for single-cell sequencing was not possible. Rather, to obtain a pool of antigen-specific BCR sequences we sorted and sequenced individual RBD/spike- and TT-binding memory B cells and plasmablasts/plasma cells either from the peripheral blood or the BM of vaccinated individuals (Supplementary Fig.  1a , gating strategy 2). Based on the CDR3 regions in heavy and light chains, we designated as “public” RBD/spike- and TT-specific clones, those BMPC clones with more than 80% similarity (modelled optimum for CDR3 specificity 31 ) shared between individuals. Of the 38235 BMPC analysed, 84 expressed spike-specific and 120 tetanus-specific public clonotypes (Fig.  2e ). These public BMPC clones were present in several clans in all individuals analysed, with the exception of clan 12 and clans 11 and 13 for tetanus specificity (Fig.  2f , Supplementary Fig.  2e ). Their percentage among total BMPC is highly individual-dependent being up to 2.03% in the CD19 high compartment (0.05 to 0.32% for spike- and 0 to 2.03% for tetanus-specific clones) and up to 1.25% in the CD19 low compartment (0.04 to 0.24% for spike- and 0 to 1.25% for tetanus-specific clones). Mutation rates in the framework were similar between spike- and tetanus-specific BMPC irrespective of the isotype they expressed, except for spike-specific IgA1 BMPC (Fig.  2g ). These were significantly less mutated than tetanus-specific IgA1 BMPC. Mutation rates in the CDR regions show that tetanus-specific IgM, IgG2 and IgA2 BMPC accumulate significantly more mutations than their spike-specific counterparts (Fig.  2g ). In summary, tetanus- and SARS-CoV-2 spike-specific BMPC expressed somatically mutated antibodies and were located in several clans in the donors analysed.

BMPC heterogeneity reflects the different timings/stages of immune reactions

To follow the recruitment dynamics of PC to BM in repeated antigenic challenges of a given immune response, we compared single-cell transcriptomes of ASC exiting the immune reaction into the blood at various time points after primary and secondary vaccination to the transcriptomes of BMPC. This longitudinal clinical study included 36 healthy individuals who had received different vaccines (Supplementary Table  2 ), namely the mRNA vaccine Comirnaty (SARS-CoV-2 spike, BioNTech/Pfizer, BNT), the viral vector-based vaccine Vaxzevria (SARS-CoV-2 spike, Oxford/AstraZeneca, AZ), and/or the mixed protein vaccine Boostrix (diphtheria toxoid, tetanus toxoid and pertussis toxoid, GlaxoSmithKline, DTP). To obtain single-cell transcriptomes and BCR repertoires, viable CD3 − CD14 − CD27 high CD38 high cells (Supplementary Fig.  3a ) were isolated from peripheral blood 7 and 14 days after primary immunisation (BNT, AZ), 7 days and 7 months after secondary BNT immunisation, and 7 days after third BNT vaccination (Fig.  3a ). Circulating ASC from 7 days after primary vaccination of convalescent COVID-19 individuals, as well as 7 days and 6 months after boost with DTP vaccine were also isolated. Altogether, we sequenced 55071 peripheral blood ASC, based on which a new UMAP was generated. A uniform distribution of the cells within the UMAP with respect to the vaccine protocol and the time post vaccination was observed (Supplementary Fig.  3b ). Based on gene expression of CD38, HLA-DMA and MKI67 (Fig.  3b ) we classified the peripheral ASC into newly generated plasmablasts ( CD38   high HLA-DMA high ), proliferating plasmablasts ( MKI67   + ), and plasma cells ( CD38   low HLA-DMA low ) 19 . Cells from each stage preferentially expressed isotypes reflecting the cytokine milieu that they had been exposed to during their activation leading to antibody class switch recombination (Supplementary Fig.  3c ). While the plasmablasts expressed mostly IgM, IgG1, IgG2, IgA1 and IgA2, the plasma cells expressed mainly IgA1.

a Schematic representation of vaccination, blood collection and sample analysis. Arrows indicate time points when transcriptome and full-length B-cell receptor repertoire sequencing was performed. b UMAP representation of the expression levels of selected genes in 55071 CD27 high CD38 high sorted peripheral blood ASC from 36 healthy individuals after COVID-19 or diphtheria, tetanus, pertussis (DTP) vaccination (see Supplementary Table  2 for information on participants and different vaccination schemes). Colour scale represents the expression level of the indicated genes. c Identification of SARS-CoV-2 spike-specific (red) public clones among analysed ASC. Public clones were defined by displaying >80% CDR3 sequence identity when compared to the BCR of sequenced peripheral blood and bone marrow spike- and tetanus-specific cells from vaccinated individuals (see Supplementary Fig.  1a and Supplementary Data  1 ). The number of public clones found is shown on the UMAP. d Bubble plot of mutation rates in the framework regions (FR1-3, left) and in the CDR regions (CDR1-3, right) of identified spike-specific public clones per isotype identified in time point after COVID-19 vaccination. Colour scale indicates median mutation rates. Values above the scale limits are shown in red. Bubble sizes correspond to the percentage of spike-specific cells expressing a defined isotype within the indicated group. e Density plots of BMPC significantly enriched in gene signatures from peripheral blood ASC isolated at different time points after vaccination as identified by Gene Set Enrichment Analysis (GSEA). Violin plots of the normalised enrichment score (NES) per BMPC cluster are depicted in Supplementary Fig.  4a . Statistical significance between NES scores is shown in Supplementary Data  1 (two-tailed Mann–Whitney U test). Source data are provided as a Source Data file.

Using the same pool of antigen-specific BCR sequences and criteria for similarity used for the analysis of spike-specific BMPC, we tracked public putatively spike-specific clonotypes among all peripheral ASC. Spanning the different time points analysed, a total of 749 ASC that expressed such clones were identified (Fig.  3c, d ). 14 days after primary vaccination, when spike-specific serum antibodies became detectable (Supplementary Fig.  3d ), the identified public ASC of the BNT and AZ vaccinees predominantly expressed IgG1 and IgA1 (Fig.  3d ). However, spike-specific public ASC from AZ vaccinees showed higher somatic mutation rates. In both groups after the second immunisation (3 and 12 weeks after the first, respectively), almost all identified spike-specific clones were IgG1. Public ASC from the AZ/BNT vaccinees showed even higher mutation rates. At 7 months after BNT/BNT vaccination, public ASC expressed a variety of antibody classes and an increase in mutation rates. 7 days after a third dose of BNT, public spike-specific ASC expressed IgG1, IgG2 and IgA1, with increased mutation rates as compared to 14 days after primary and 7 days after secondary vaccination. Interestingly, convalescent individuals who had received just one dose of BNT 7 days earlier had predominantly IgG1 spike-specific BCRs, with similar mutation rates as individuals 7 days after a third BNT vaccination (Fig.  3d ). The integration of both BMPC and blood ASC data sets (batch corrected for sequencing depth, effects among individuals and origin of the cells) into a new UMAP projection show that BMPC cluster with blood ASC (Supplementary Fig.  3e ), an exception being the plasmablasts found in the BM (cluster 13), which cluster together with blood proliferating plasmablast (MKI67+, Fig.  3b ).

We then defined gene signatures characteristic for the circulating ASC at the different time points (Supplementary Data  1 ), and from the public antigen-specific ASC (Supplementary Data  1 ), and projected them onto the transcriptomes of the BMPC by GSEA (Fig.  3e , Supplementary Fig.  4a–d ). The gene signatures obtained from each individual vaccinee at a given time point after vaccination showed only marginal inter-individual variation (Supplementary Fig.  5a ). 7 and 14 days following primary immunisation with BNT or AZ vaccines, the signatures of circulating ASC marked clans 0, 1 and 4 of BMPC, both in terms of frequency and enrichment score (Fig.  3e , Supplementary Fig.  4a, b ), with BNT delegating more PC into clans 1 and 4 than AZ, and AZ more into clan 9 than BNT. This difference is likely due to the fact that AZ also elicits an immune response against its adenovirus components (Supplementary Fig.  5b ), as well as T-REx HEK293 proteins from virus production 32 . The resemblance of ASC to BMPC clan 1 and particularly clan 4 also held true 7 days following secondary vaccination with BNT (when the primary dose was given 28-35 days previously). In stark contrast, those primed with AZ, or infected with SARS-CoV-2 and boosted later with BNT, developed circulating ASC resembling BMPC of clans 0, 9, and 13. Moreover, clans 0, 9, and 13 expressed the signatures of circulating ASC egressing into the blood 7 days after vaccination with DTP — a boost to already established long-term memory. In those vaccinated with a third dose of BNT, 7 days later, the circulating ASC also shared signature gene expression with clan 0 of BMPC, but also clans 4 and 5, i.e. IgA1 and IgA2 expressing CD19 low BMPC.

In order to obtain gene signatures from prolonged germinal centre output, we also analysed the circulating ASC 6/7 months after vaccination in individuals immunised twice with BNT or boosted with DTP 33 . At this time, we were still able to detect newly generated spike-specific ASC (Fig.  3d ). Regarding ASC from BNT vaccinees, their signatures were mainly shared with BMPC of clans 0, 1, 5, 6 and 9 (Fig.  3e , Supplementary Fig.  4a, b ). 6 months after DTP boost, circulating ASC resembled the ones obtained 7 months after BNT/BNT, with the exception of clan 1. This showed that while BNT prolonged response gives rise to CD19 high but also CD19 low BMPC, the prolonged response to a bona-fide DTP recall response gives rise predominantly to CD19 high BMPC. In summary, a prominent recruitment of PC to CD19 low clans is only observed upon primary and closely-followed secondary vaccination with BNT and primary vaccination with AZ. Later on, most PC are recruited to CD19 high clans.

Vaccination-induced ASC reflect cytokine instructions

To identify the signals responsible for the differential transcriptional signatures of ASC and BMPC generated by vaccination, we compared these signatures to transcriptomes of B cells activated ex vivo under different conditions. The B cells were stimulated with anti-CD40 (to mimic T-cell help) and various combinations of cytokines for different durations 34 , 35 . Obtained gene signatures were projected by GSEA onto the UMAP of peripheral ASC collected at different times post vaccination (Fig.  4a ) and onto the UMAP of BMPC (Fig.  4b , Supplementary Fig.  6a ). While IL-6 and IL-21 gene signatures at 12 and 24 h highlight the proliferating peripheral ASC (Fig.  4a ), they were mainly enriched in clans 5 and 13 of BMPC (Fig.  4b , Supplementary Fig.  6a ). Signatures of B cells stimulated with IFN-α for 12 h marked BMPC of clan 8 (see also Fig.  1d, g ), whereas interestingly, 24 h of IFN-α stimulation resulted in the marking of BMPC clans 4 and 6 (Fig.  4b , Supplementary Fig.  6a ). B cells activated in the presence of IFN-α, TGF-β, IL-6 and IL-21 for 12 h showed signatures comparable to BMPC of clans 1 and 4 and mainly the HLA-DMA high cells of the peripheral ASC (Figs.  4 a, b and 3b ). After 24 h the signatures resembled those of clan 0 (Fig.  4b ). Accordingly, the transcriptomes of ASC generated at defined time points after primary, secondary and tertiary vaccination do differ (Supplementary Fig.  6b ), indicating the dynamic evolution of the immune response, i.e. the cytokines involved, and confirming the assignment of BMPC clans to its distinct phases.

a , b Gene Set Enrichment Analysis (GSEA) based on gene signatures from ex vivo-differentiated plasmablasts in presence of different cytokine combinations (data sets from Stephenson et al. 35 ). Briefly, naive B cells were cultured in differentiating conditions for 6 days after which different cytokines were added to the culture. After 12 or 24 h, cells were harvested and sequenced. Gene signatures were identified by comparing the different conditions and time points to 0 h. a Density plots of ASC (time points and vaccine protocols combined) with significant enrichment identified by GSEA of gene signatures from ex vivo-differentiated plasmablasts. b Density plots of BMPC with significant enrichment identified by GSEA of gene signatures from ex vivo-differentiated plasmablasts. Violin plots of the normalised enrichment score (NES) per BMPC cluster are depicted in Supplementary Fig.  6a . Statistical significance between NES scores is shown in Supplementary Data  1 (two-tailed Mann–Whitney U test). c , d T follicular helper cells (Tfh, CD19 − CD4 + CD45RA − CXCR5 ++ ) from tonsils or T peripheral helper cells (Tph, CD19 − CD4 + CD45RA − CXCR5 − ) from BAL of sarcoidosis patients were co-cultured 1:1 with tonsillar memory B cells (CD19 + CD4 − IgD − CD38 − ) for 7 days in presence of staphylococcal enterotoxin B (SEB). Where indicated, T-cell help was blocked with an anti-CD40L antibody and/or recombinant soluble IL-21R. c Histograms of CD19 expression by differentiated plasmablasts (see Supplementary Fig. 7). Two Tfh and two Tph donors were analysed. d Frequencies of CD19 hi plasmablasts. Statistics were performed using an RM parametric one-way ANOVA followed by multiple testing according to Fischer’s LSD test. Source data are provided as a Source Data file.

IL-21 downregulates expression of CD19 on activated B cells

Notably, B cells activated ex vivo with signals mimicking T-cell help, i.e. CD40L and IL-21, developed transcriptional signatures similar to BMPC from clans 1 and 4, i.e. CD19 low BMPC. To test the hypothesis that CD19 low ASC and BMPC are indeed generated in T cell-dependent follicular immune reactions, we sorted either tonsillar follicular T helper cells (CD45RA − CXCR5 high ) or peripheral T helper cells (CD45RA − CXCR5 − ) from bronchoalveolar lavage (BAL) and cultured them with tonsillar memory B cells (IgD − CD38 − ) in the presence of staphylococcal enterotoxin B (SEB) 36 . To block CD40/CD40L interactions and IL-21 receptor-induced signalling of the activated B cells, anti-CD40L and/or soluble IL-21 receptor (sIL-21R) were also added to the cultures. After 7 days of culture, we analysed the expression of CD19 on the CD27/CD38-expressing ASC generated (Fig.  4c, d , Supplementary Fig.  7a, b ). Whether or not CD40-CD40L interaction was blocked, the resulting ASC were CD19 low . In contrast, blocking IL-21 signalling inhibited CD19 downregulation in the ASC, with a 2- to 15-fold increase in the frequency of CD19 high cells, for the 4 donors analysed (Fig.  4c, d ). The IL-21-mediated downregulation of CD19 expression on activated B cells implies that CD19 low BMPC are derived from follicular immune reactions, while CD19 high BMPC are generated in extrafollicular immune reactions in the absence of IL-21 expressing follicular T helper cells. This is in line with the fact that the CD19 low compartment is enriched in cells exhibiting hallmarks of STAT3 signalling (Fig.  1g ), known to be induced by IL-21 in germinal centres 37 , 38 .

Sequential recruitment of BMPC to the CD19 low and CD19 high compartments in the tertiary immune reaction to Comirnaty

We also analysed SARS-CoV-2 spike-specific and TT-specific BMPC by multiparametric flow cytometry. For a total of 20 BM samples (Supplementary Table  1 ) we identified both RBD of spike- and TT-specific plasma cells by intracellular staining with the respective antigen, using two fluorescent conjugates each, to ensure specificity of the staining (Fig.  5a , Supplementary Fig.  8a ). The frequencies of RBD- and TT-specific BMPC did not significantly differ, with medians of 0.092% and 0.16%, respectively (Fig.  5b ). These numbers are consistent with previous observations 19 , 39 . However, while the majority of RBD-specific plasma cells (67%) were CD19 high , the majority of TT-specific plasma cells (63%) were CD19 low (Fig.  5c and Supplementary Fig.  8b ). 75% of both RBD- and TT-specific BMPC expressed IgG, while 22% of RBD- and only 5% of TT-specific cells expressed IgA (Fig.  5d , Supplementary Fig.  8c ). The remaining cells expressed IgM. Interestingly, with time elapsed after the third COVID-19 vaccination, frequencies of CD19 low RBD-specific did increase, from about 20% one month after the third vaccination to more than 40% two months later (Fig.  5e ). This increase is in line with the notion that secondary (and tertiary) immune reactions start with the extrafollicular reactivation of memory B cells, followed by generation of new and adapted ASC in a subsequent follicular immune reaction 40 .

a Representative pseudocolour plots of intracellular double-positive SARS-CoV-2 RBD (left) or tetanus toxoid (TT, right) staining in CD38 high CD138 + CD14 − CD3 − live singlet BMPC (gating strategy in Supplementary Fig.  8a ). b – e Each symbol represents one donor/sample (see Supplementary Table  1 ). Filled symbols represent BMPC samples which were also analysed by single-cell sequencing (Fig.  1a ). b Frequencies of RBD-specific and TT-specific BMPC within total BMPC. Horizontal lines indicate the median. Statistics were performed using the one-tailed Mann–Whitney U test. n  = 20 BM samples. c Frequencies of CD19 low cells within RBD-specific BMPC (red), TT-specific BMPC (blue) and total BMPC (black). Horizontal lines indicate the median. Statistics were performed using the Kruskal–Wallis tests with Dunn’s correction for multiple comparisons. n  = 20 BM samples. d Frequencies of IgG+ (left) and IgA+ (right) cells within RBD-specific BMPC (red), TT-specific BMPC (blue) and total BMPC (black). Horizontal lines indicate the median. Statistics were performed using the Kruskal–Wallis tests with Dunn’s correction for multiple comparisons. n  = 20 BM samples. e Correlation between the frequency of CD19 low RBD-specific BMPC and days after 3rd vaccination against SARS-CoV-2. Statistics were performed using one-tailed Spearman’s correlations. n  = 11 BM samples. Source data are provided as a Source Data file.

Here we provide a comprehensive single-cell gene expression analysis of human bone marrow plasma cells (BMPC). We find a remarkable similarity between 8 individuals, each with 10 clans of BMPC, characterised by transcriptomic signatures reflecting their imprinting in different types of immune reactions. We track the recruitment of PC precursors to the BM by comparing transcriptional signatures of ASC, newly generated in immune reactions to defined vaccines against SARS-CoV-2 spike protein or diphtheria/tetanus/pertussis, to transcriptional signatures of BMPC and human B cells activated ex vivo with defined stimuli.

ASC released from secondary immune reactions are detected in the blood, peaking after about 7 days, while those of primary immune reactions are detected a few days later 19 , 41 , 42 . Signatures of ASC released from primary and secondary immune reactions differ significantly and indicate sequential recruitment of BMPC to different clans. In primary and secondary immune reactions, which were induced closely following the primary response, BMPC are recruited to CD19 low clans, while in true secondary and tertiary reactions, BMPC are recruited nearly exclusively to CD19 high clans. We show that CD19 is downregulated by IL-21, an essential signal of follicular helper T cells in follicular immune reactions. Thus, while in the initial immune reaction BMPC are mostly derived from follicular immune responses, in subsequent immune reactions, they are mostly derived from extrafollicular immune responses. The incremental increase in CD19 low spike-specific BMPC over time after the third vaccination with BNT suggests that after the extrafollicular reactivation of memory B cells, there is a continued follicular immune reaction that follows. Additionally, the transcriptional signatures reveal instruction by interferon and later also by TGF-β in response to vaccination. In the end, the recruitment of BMPC in vaccinations to SARS-CoV-2 spike or tetanus toxoid generates BMPC of most clans in all individuals, reflecting the evolution of a given humoral immune response to sequential challenges. This ensures a broad immunity in terms of antibody function, output of mature cells from follicular reactions, and a fast extrafollicular recall response.

The SARS-CoV-2 pandemic has revealed a remarkable illiteracy on immunological memory, the molecular and cellular basis of adaptive immunity. The persistence of humoral immunity, i.e. specific serum antibodies, has been demonstrated 3 , 6 as well as the decades-long persistence of the cells secreting them: the plasma cells 43 , 44 . The bone marrow is known to maintain long-lived plasma cells 2 , 3 . Bone marrow-resident plasma cells secreting spike protein-specific antibodies have been demonstrated in SARS-CoV-2 convalescent and vaccinated persons 4 , 5 , 45 at frequencies about equal to those secreting tetanus/diphtheria-specific antibodies. Although it is already evident that BMPC are derived from different types of immune reactions from the class of the antibodies they secrete, little is known about their heterogeneity. With the advent of single-cell transcriptomics, different studies on the transcriptional heterogeneity of murine and human BMPC have been published 46 , 47 . Here we show that distinct immune reactions, exemplified for SARS-CoV-2 spike and tetanus, are diverse and dynamic, and that specific BMPC are found in most vaccinees in several, if not all, compartments of BMPC. Moreover, in each subsequent challenge with the vaccine, PC are delegated to different compartments. This not only reflects the heterogeneity of instruction in each immune reaction, but also a sequential dominance of extrafollicular over follicular immune reactions.

Of particular interest is our observation that in primary immune reactions, and the closely-followed secondary vaccination with BNT, ASC exiting the immune reaction 7 days after vaccination show a transcriptional gene expression signature resembling that of clans 1 and 4 BMPC, including downregulated CD19 expression. After the original description of CD19 low and CD19 high BMPC 7 , came studies showing that CD19 low BMPC are refractory to B-cell depletion by rituximab 28 , secrete antibodies against unique childhood experiences like measles and mumps, and are resistant to CD19-specific CAR T cells 48 , defining CD19 low BMPC as long-lived 49 . The role of CD19 high BMPC was, however, less clear. Here we demonstrate that ASC downregulate CD19 upon instruction by IL-21 in follicular immune reactions. This defines CD19 high BMPC clusters as being of extrafollicular origin, while CD19 low BMPC are of follicular origin. CD19 has been implied in supporting ERK signalling and AKT phosphorylation in human plasma cells 50 and also CXCR4 signalling in IgD-deficient B cells 51 . We have previously described, that CXCR4 can support the survival of BMPC ex vivo 52 and in vivo 53 . Moreover, we have demonstrated the pivotal role of the PI3K/AKT/Foxo1/3a signalling axis for survival of BMPC, preventing activation of caspases 3 and 7 by metabolic stress 23 . On the level of single cell transcriptomes, both CD19 low and CD19 high BMPC show genes associated with resilience to apoptosis, namely, MCL1 54 . CD19 high BMPC also thus qualify for being long-lived, but they in all likelihood originate from repeated antigenic challenge, such as influenza, as already demonstrated by Halliley and colleagues 49 . Using a systemic and innovative approach we demonstrate the recruitment of ASC generated upon primary and secondary vaccination with SARS-CoV-2 vaccines to CD19 low and CD19 high BMPC clans. In primary immune reactions to BNT, and the closely-followed boost, and to AZ, most ASC are recruited to CD19 low clans 1 and 4, while in true secondary immune reactions, most ASC generated at early time points are recruited to CD19 high clans, in particular clan 0. At later time points, ASC may also be recruited from follicular immune reactions, as is indicated by an increase in antigen-specific CD19 low BMPC over a timespan of several months.

IL-21 is a signature cytokine of follicular T helper cells, which orchestrate B cell activation in germinal centre reactions 55 , 56 , 57 , 58 . It impacts proliferation, affinity maturation and memory B-cell generation in a paracrine, synapse-independent way, and at high concentrations, also affects the differentiation of activated B cells into plasmablasts 38 , 57 , 59 . This regulation is B cell intrinsic 56 , although the precise gene regulatory mechanisms are not fully understood. As we show here, IL-21 also (down)regulates expression of CD19 on B cells activated to differentiate into ASC. Accordingly, we observe recruitment of ASC generated in primary immune responses induced by SARS-CoV-2 vaccines to CD19 low BMPC clans. Also, IL-21’s influence in germinal centre plasma cell differentiation has been shown to occur via a robust and prolonged STAT3 activation 37 , 38 , which is consistent with the respective GSEA highlighting BMPC belonging to the CD19 low compartment. In primary responses, plasma cells are expected to be generated from naïve B cells activated in germinal centres, whereas in secondary immune reactions, extrafollicular reactivation of memory B cells generated in previous immune reactions has been postulated as a way of delivering an immediate burst of specific antibody production, as imprinted in the previous immune reaction 60 . While reports of both IL-21-dependent and -independent extrafollicular reactions have been described 61 , 62 , 63 , in the immune responses here analysed extrafollicular reactivation was IL-21-independent, since the ASC generated maintained CD19 expression and their canonical transcriptional signature allocates to CD19 high BMPC. Indeed, in the donors analysed, CD19 high BMPC made up as much as 20.2 to 90.1% of all BMPC. There is no evidence that these BMPC are not as long-lived as CD19 low BMPC, and do not use the same molecular mechanisms to persist 23 . CD19 could even be expected to support the antigen-receptor-independent, stromal cell contact-dependent survival of these cells. Indeed, we found substantial populations of tetanus-specific CD19 high BMPC in the bone marrow of the donors analysed.

The derivation of BMPC from follicular and extrafollicular immune reactions has fundamental implications for understanding imprinting and regulation of (humoral) immunological memory. Feedback-regulation by pre-existing specific antibodies was first demonstrated by T. Smith in 1909 64 , and more recently by the group of Nussenzweig 65 , who showed that vaccinees treated with SARS-CoV-2-specific therapeutic antibodies before, upon vaccination, did not respond to the epitopes recognised by these antibodies. The rapid extrafollicular reactivation of memory B cells generated in previous immune reactions thus shapes secondary immune responses in providing immediate, imprinted immunity to the original antigen (original antigenic sin) 66 and blocking reactions to epitopes recognised by them 67 , 68 . As we show here, ASC of these imprinted reactions are also recruited to the BM, providing long-lasting enhanced immunity. Adaptation to variants of the original antigen may occur later, as is suggested here by the increasing frequency of CD19 low SARS-CoV-2-specific BMPC months after the tertiary vaccination with BNT. It remains to be shown to what extent this tertiary germinal centre reaction is regulated by the antibodies produced in the fast extrafollicular reaction, but also, to what extent the extrafollicular reaction is regulated by the pre-existing antibodies of already established BMPC. Understanding the interplay of follicular and extrafollicular immune reactions in shaping the repertoire of BMPC will be key to develop vaccines and vaccination protocols for the establishment of sustainable and lasting immunity, and the adaptation of immunity to newly arising variants.

Human donors

The recruitment of study participants was conducted in accordance with the Ethics Committee of the Charité Universitätsmedizin Berlin in compliance with the Declaration of Helsinki (EA1/261/09, EA1/009/17 and EA2/010/21). Informed consent was obtained from all bone marrow/peripheral blood donors included in the study. No compensation was provided to participants. Bone marrow samples were obtained from 25 patients undergoing total hip arthroplasty without any underlying malignant or inflammatory disease (12 females and 13 males with a median age of 62 years, see Supplementary Table  1 ). Peripheral blood was obtained from 36 healthy volunteers at different time points after Comirnaty, Vaxzevria or Boostrix vaccination (16 females and 19 males with a median age of 32 years, see Supplementary Table  2 ). Due to the small number of analysed samples, no disaggregated sex or gender analysis was performed.

Bone marrow plasma cell (BMPC) isolation

Bone marrow samples were fragmented and transferred to a 50 mL tube where they were vortexed to separate cells from bone fragments. Samples were subsequently rinsed through a 70 µM filter with PBS/1% BSA/5 mM EDTA/2 µg/mL actinomycin D to obtain a cell suspension. Plasma cells were enriched from bone marrow using StraightFrom Whole Blood and Bone Marrow CD138 MicroBeads and StraightFrom Whole Blood CD19 MicroBeads (Miltenyi Biotec) according to manufacturer’s instructions. Enriched cells were incubated with Fc Blocking Reagent (Miltenyi Biotec) following manufacturer’s instructions and subsequently stained for 30 min at 4 °C with the following anti-human antibodies: CD3-VioBlue (BW264/56, Miltenyi Biotec, Cat. 130-113-133, 1:400), CD10-VioBlue (97C5, Miltenyi Biotec, Cat. 130-099-670, 1:11); CD14-VioBlue (TÜK4, Miltenyi Biotec, Cat. 130-113-152, 1:200); CD38-APC (HIT2, BioLegend, Cat. 303510, 1:25) and CD138-PE (44F9, Miltenyi Biotec, Cat. 130-119-840, 1:50) or CD3-VioBlue (BW264/56, Miltenyi Biotec, Cat. 130-113-133, 1:400), CD14-VioBlue (TÜK4, Miltenyi Biotec, Cat. 130-113-133, 1:200), CD27-APC-Cy7 (O323, BioLegend, Cat. 302816, 1:25), CD38-PerCP-Cy5.5 (HIT2, BD Biosciences, Cat. 551400, 1:100) and tetanus toxoid (AJ vaccines) coupled with Alexa Fluor 647 or Alexa Fluor 488 and SARS-Cov2 Spike Protein (Biotin, Miltenyi Biotec, Cat. 130-127-682) pre-incubated with streptavidin PE or streptavidin PE-Cy7 according to manufacturer’s instructions. Simultaneously, cells were incubated with DNA barcoded antibodies for Cellular Indexing of Transcriptomes and Epitopes by Sequencing (CITE-seq, see antibody list). DAPI was added before sorting to allow dead cell exclusion. Two different gating strategies were used for sorting (CD138 + CD38 high or CD27 high CD38 high , see Supplementary Fig.  1a ), depending on the need to sort either bulk plasma cells or also antigen-specific memory B cells. Memory B cells were also sorted but not included in this analysis. All sortings were performed using a MA900 Multi-Application Cell Sorter (Sony Biotechnology). Cell counting was performed using a MACSQuant flow cytometer (Miltenyi Biotec). The sorted cells were further processed for single-cell RNA sequencing.

Peripheral blood antibody-secreting cell (ASC) isolation

Cells were enriched by positive selection from peripheral blood using a combination of StraightFrom Whole Blood CD19 and CD3 MicroBeads and StraightFrom Whole Blood and BM CD138 MicroBeads (Miltenyi Biotec) according to manufacturer’s instructions. 2 µg/mL actinomycin D was added to the buffer used during the first centrifugation. Enriched cells were incubated with Fc Blocking Reagent (Miltenyi Biotec) following manufacturer’s instructions and subsequently stained for 30 min at 4 °C with the following anti-human antibodies: CD3-FITC (UCHT1, DRFZ in-house, 1:10), CD14-VioBlue (TÜK4, Miltenyi Biotec, Cat. 130-113-152, 1:200), CD27-PE (MT271, Miltenyi Biotec, Cat. 130-113-630, 1:100) and CD38-APC (HIT2, BioLegend, Cat. 303510, 1:25). Simultaneously, cells were incubated with DNA barcoded antibodies for Cellular Indexing of Transcriptomes and Epitopes by Sequencing (CITE-seq), which allowed identification of samples from different donors (see antibody list, hashtags). DAPI was added before sorting to allow dead cell exclusion. See Supplementary Fig.  3a for gating strategy. While B cells and T cells were also isolated, they were not included into the analysis. All sortings were performed using a MA900 Multi-Application Cell Sorter (Sony Biotechnology). Cell counting was performed using a MACSQuant flow cytometer (Miltenyi Biotec). The sorted cells were further processed for single-cell RNA sequencing.

Single-cell RNA-library preparation and sequencing

Single-cell suspensions were obtained by cell sorting and applied to the 10x Genomics workflow for cell capturing and scRNA gene expression (GEX), BCR and CITE-Seq library preparation using the Chromium Single Cell 5’ Library & Gel Bead Kit version 2 for BMPC or version 1.1 for ASC, as well as the Single Cell 5’ Feature Barcode Library Kit (10x Genomics). After cDNA amplification, the CITE-Seq libraries were prepared separately using the Dual Index Kit TN Set A for BMPC or the Single Index Kit N Set A for ASC. BCR target enrichment was performed using the Chromium Single Cell V(D)J Enrichment Kit for Human B cells. Final GEX and BCR libraries were obtained after fragmentation, adapter ligation and final Index PCR using the Dual Index Kit TT Set A for BMPC or the Single Index Kit T Set A for ASC. Qubit HS DNA assay kit (Life Technologies) was used for library quantification and fragment sizes were determined using the Fragment Analyzer with the HS NGS Fragment Kit (1-6000 bp) (Agilent).

Sequencing was performed on a NextSeq2000 device (Illumina) applying the sequencing conditions recommended by 10x Genomics for libraries prepared with Next GEM Reagent Kits. NEXTSeq 1000/2000 P3 reagent kits (200 Cycles, Illumina) were used for 5’ GEX and CITE-Seq libraries (for version 2, read1: 26nt, read2: 90nt, index1: 10nt, index2: 10nt; for version 1.1, read1: 26nt, read2: 98nt, index1: 8nt, index2: 0nt) and NEXTSeq 1000/2000 P3 reagent kits (300 Cycles, Illumina) were used for BCR libraries (for version 2, read1: 151nt, read2: 151nt, index1: 10nt, index2: 10nt., 2% PhiX spike-in, for version 1.1, read1: 151nt, read2: 151nt, index1: 8nt, index2: 0nt., 2% PhiX spike-in).

Single-cell transcriptome analysis

Raw sequence reads were processed using cellranger (version 5.0.0). Demultiplexing, mapping, detection of intact cells as well as quantification of gene expression was performed using cellranger’s count pipeline in default parameter settings with refdata-cellranger-hg19-1.2.0 as reference and expected number of 3000 cells per sample. Noteworthy, the used reference does not contain immunoglobulin genes as defined by the respective biotype, which would otherwise strongly influence the downstream analysis. An average of 48000 mean reads per cell was obtained, which is more than double of the 20000 reads per cell recommended by the manufacturer for 10x genomics 5’ version 2. ( https://kb.10xgenomics.com/hc/en-us/articles/115002022743-What-is-the-recommended-sequencing-depth-for-Single-Cell-3-and-5-Gene-Expression-libraries -). This led to 20378, 15322, 10818, 10707, 8876, 8394, 9209 and 6206 intact cells for 8 bone marrow samples. Cellranger’s aggr was used to merge the libraries without size normalisation and to perform a Uniform Manifold Approximation and Projection (UMAP). Loupe Browser (version 5, 10x Genomics) was used to identify and define bone marrow plasma cells (BMPC) by manual gating. Plasma cells defined clear regions with cells expressing PRDM1 , SDC1 , XBP1 and IRF4 genes. This led to 11052, 10772, 5505, 5661, 2222, 5528, 4556, 4051 plasma cells from each of the 8 BM samples. The BMPC were further analysed in R (version 4.1.2) using the Seurat package (version 4.0.5) 69 and the cellranger’s aggr output and the respective cellular barcodes. In particular, the transcriptome profiles of the BM samples were read and plasma cells were extracted using Read10x, CreateSeuratObject and subset. To identify cells with similar transcriptional profiles among different sequencing libraries, sample specific batch effects were removed as described in FindIntegrationAnchors (Seurat) R Documentation. In particular, samples were analysed individually using SplitObject by LibraryID, NormalizeData with LogNormalization as normalisation.method and scale.factor of 10,000, FindVariableFeatures with 2000 variable genes and vst as selection method, ScaleData and finally RunPCA to compute 50 principle components for each sample. Next, common anchors were identified by FindIntegrationAnchors usind rpca as reduction, 2000 anchor.features and 1:30 dimensions and finally merged using IntegrateData. Based on the integrated data, a uniform manifold approximation and projection (UMAP) was computed using ScaleData, RunPCA to compute 50 principle components and RunUMAP using 1:30 dimensions. Transcriptionally similar clusters were identified by shared nearest neighbour (SNN) modularity optimisation using FindNeighbors with pca as reduction and 1:30 dimensions as well as FindClusters with resolutions ranging from 0.1 to 1.0 in 0.1 increments using FindCluster. Further analyses were based on non-integrated, log normalised values represented as ln (10,000 × UMIsGene)/UMIsTotal +1) and the above integrations-based clusters and UMAP. By visual inspection of the percentage of mitochondrial genes, UMI counts, number of identified genes as well as expression of typical marker genes projected on the UMAP, clustering with a resolution of 0.5 was judged to best reflect the transcriptional community structure. Clusters comprising low quality cells as well as clusters comprising contaminations were not considered in further analyses. For the pseudotype trajectory analysis the considered BMPC clusters were reintegrated as described above. Pseudotime trajectory analysis was performed by applying the default pipeline of the R package monocle3 70 , with the exception of using the integrated data as input matrix 71 .

The single-cell transcriptome analysis of blood ASC was performed in accordance to the BMPC analysis. In particular, libraries of pooled samples from 36 individuals at different time points (see Supplementary Table  2 ) were demultiplexed and mapped, intact cells were detected, and gene expression was quantified by cellranger’s count pipeline and merged by cellranger aggr. This led to 32162 and 32506 intact cells at d7 and d14 after Comirnaty 1st dose of (BNT d7, d14), respectively; 42047 and 26339 cells at d7 and 7 months after Comirnaty 2nd dose (BNT/BNT d7, 7mo), respectively; 32860 cells at d7 after Comirnaty 3rd dose (BNT/BNT/BNT d7); 28282 and 24622 cells at d7 and d14 after Vaxzevria 1st dose (AZ d7, d14), respectively; 16567 cells at d7 after Comirnaty 2nd dose (1st dose Vaxzevria, AZ/BNT d7); 21524 cells at d7 after 1st Comirnaty dose of donors recovered from a SARS-CoV-2 infection (COVID/BNT d7); and 29875 and 22811 cells at d7 and 6mo after Boostrix boost (DTP d7, 6mo), respectively. Libraries were merged by cell ranger’s aggr. Loupe Browser (version 5, 10x Genomics) was used to identify and define ASC by manual gating. ASC defined clear regions with cells expressing higher levels of PRDM1 , CD27 and CD38 genes. This led to 4462 and 5762 ASC at d7 and d14 after Comirnaty 1st dose of (BNT d7, d14), respectively; 5983 and 4169 ASC at d7 and 7 months after Comirnaty 2nd dose (BNT/BNT d7, 7mo), respectively; 6036 ASC at d7 after Comirnaty 3rd dose (BNT/BNT/BNT d7); 8540 and 2143 ASC at d7 and d14 after Vaxzevria 1st dose (AZ d7, d14), respectively; 2969 ASC at d7 after Comirnaty 2nd dose (1st dose Vaxzevria, AZ/BNT d7); 5586 ASC at d7 after 1st Comirnaty dose of donors recovered from a SARS-CoV-2 infection (COVID/BNT d7); and 5679 and 3742 ASC at d7 and 6mo after Boostrix boost (DTP d7, 6mo), respectively. ASC were further analysed with the Seurat package R package using the cellranger’s aggr output and the respective cellular barcodes. UMAP was performed by removal of library and donor specific batch effects using the Seurat’s integration as described above for BMPC. Visualised is the log normalised gene expression of non-integrated data.

The Integration of the filtered BMPC clusters and blood ASC (Supplementary Fig.  3e ) was performed as describe above, considering LibraryID and Donor.

Single-cell immune profiling

Raw sequence reads were processed using cellranger (version 5.0.0). Vdj was used in default parameter settings for demultiplexing and assembly of the B cell receptor sequences using refdata-cellranger-vdj-GRCh38-alts-ensembl-2.0.0 as reference. The cellranger output was further analysed in R (version 4.1.2) using the Seurat package (version 4.0.5) 69 .

B cell receptor isotypes and receptor sequences were assigned to the corresponding cells in the single-cell transcriptome analysis by identical cellular barcodes. In case of multiple contigs, the most abundant, productive and fully sequenced contig for the heavy and light BCR chain was used. This led to the annotation of 95% (3856), 90% (4954), 90% (5097), 94% (2081), 87% (9551), 85% (9105), 91% (5032) and 93% (4237) plasma cells for the 8 BM samples; 69% (3060) and 67% (3860) ASC at d7 and d14 after Comirnaty 1st dose of (BNT d7, d14), respectively; 65% (3893) and 66% (2762) ASC at d7 and 7 months after Comirnaty 2nd dose (BNT/BNT d7, 7mo), respectively; 70% (4248) ASC at d7 after Comirnaty 3rd dose (BNT/BNT/BNT d7); 53% (4525) and 64% (1364) ASC at d7 and d14 after Vaxzevria 1st dose (AZ d7, d14), respectively; 65% (1931) ASC at d7 after Comirnaty 2nd dose (1st dose Vaxzevria, AZ/BNT d7); 61% (3405) ASC at d7 after 1st Comirnaty dose of donors recovered from a SARS-CoV-2 infection (COVID/BNT d7); and 69% (3938) ASC at d7 after Boostrix boost (DTP d7). The high-confidence contig sequences with an associated transcriptional profile were reanalysed using the HighV-QUEST at IMGT web portal for immunoglobulin (IMGT) to retrieve the germline sequence between of the FR1-CDR1-FR2-CDR2-FR3, the V-, J- and D-gene information as well as the nucleotide and amino acid CDR3 sequence. The HighV-QUEST output, in particular the IMGT-gapped-nt-sequences and V-REGION-mutation-and-AA-change-table were used to reverse engineer the gapped germline FR1-CDR1-FR2-CDR2-FR3 sequence. A clonal family of a BCR receptor was defined by the germline FR1-CDR1-FR2-CDR2-FR3 sequence, the used VJ-genes, the length of the CDR3 sequence and CDR3 identity greater than 80% (modelled optimum for CDR3 specificity 31 ) between all members of a clonal family considering both the heavy and light chain. The clonal family annotation was used to compute the diversity, Simpson Diversity Index as well as the overlap table. Significance of an overlap was evaluated by 1000 permutation of the clonal family annotation of the cells (see Supplementary Data  1 for statistics). Mutation counts in framework regions (FR1, FR2, FR3) were taken from V-REGION-nt-mutation-statistics of the HighV-QUEST output. Mutation rates were defined as the sum of the estimated mutation counts in the heavy and light chain normalised to the length of the corresponding nt sequence length.

Spike- and tetanus-specific clones were derived from BCR-sequencing of spike- and tetanus-specific B cells isolated with either fluorophore-coupled RBD/Spike protein of SARS-CoV-2 or tetanus toxoid after vaccination of healthy individuals with Comirnaty or Boostrix vaccines. In particular, raw sequence reads from BCR-sequencing were processed using cellranger vdj pipeline as described above. Solely contig pairs for the heavy and light chain were considered. Unpaired contigs, that is contigs without a corresponding contig with the same cellular barcode, were removed from further analyses. In case of multiple contigs for the same cellular barcode, the most abundant, productive and fully sequenced contig for the heavy and light BCR chain were used. Putative spike- and tetanus-specific clones in BMPC and ASC samples were identified by comparing them with the experimentally validated spike- and tetanus-specific B cell clones. To meet the selection criteria, clones were required to exhibit consistent lengths in both heavy and light chains within the CDR3 region. Additionally, samples were defined based on a minimum CDR3 nucleotide identity threshold, calculated as the sum of identical nucleotides normalised to the total length of these regions, with a minimum requirement of 80% identity. This stringent approach ensured that only clones meeting both criteria were considered putative spike- or tetanus-specific clones for further analysis (see Supplementary Data  1 ).

Gene set enrichment analysis (GSEA)

GSEA was performed for each cell based on the difference to the mean of log normalised expression values of all cells in the analysed set as pre-ranked list and 1000 randomisations (PMID: 16199517, PMID: 12808457). Significant up- or downregulation was defined by a FDR ≤ 0.50 and normalised p value < 0.05 43. For visualisation, NES for significant cells were plotted. The GSEA was performed for indicated cells using hallmark gene sets (PMID: 26771021), REACTOME (PMID : 29145629), KEGG (PMID: 10592173), ex vivo-differentiated plasmablast gene signatures (defined as described before 34 , 35 ), as well as ASC time-specific gene sets and spike-specific ASC time-specific gene sets from the ASC analysis as defined by marker genes for different time points after vaccination (Supplementary Data  1 ). Hallmark gene sets, REACTOME and KEGG were obtained from the MSigDB Collections (PMID: 26771021).

For the ASC signature gene set samples at day 7 and day 14 after first Comirnaty vaccination, day 7 and 7 months after second Comirnaty vaccination, day 7 after third Comirnaty vaccination, day 7 and day 14 after Vaxzevria vaccination, day 7 after second Comirnaty vaccination (heterologous), day 7 after Comirnaty vaccination from donors recovered from a SARS-CoV-2 infection, as well as samples at day 7 and 6 months after vaccination with Boostrix, genes with an Area under the ROC Curve greater than 0.6 and an adjusted p value ≤0.05 (Mann–Whitney U Test) were defined as marker genes. For the spike-specific ASC signature gene set samples at day 14 after first Comirnaty vaccination, day 7 and 7 months after second Comirnaty vaccination, day 7 after third Comirnaty vaccination and day 7 after Comirnaty vaccination from donors recovered from a SARS-CoV-2 infection, genes with an Area under the ROC Curve greater than 0.6 and an adjusted p value ≤0.05 (Mann–Whitney U Test) were defined as marker genes. No significant signature genes were found for spike-specific ASC obtained at day 14 after first Comirnaty vaccination.

The GSEA results were visualised by density plot on UMAPs and when appropriate also on violin plots of the NES score of significant enriched cells with a positive NES score. Differences in positive NES scores were evaluated using the Mann–Whitney U Test (see Supplementary Data  1 for statistics).

BM mononuclear cells flow cytometry analysis

BM mononuclear cells were enriched by density gradient centrifugation over Ficoll-Paque PLUS (GE Healthcare BioSciences) 28 . Briefly, samples were fragmented, rinsed with PBS/0.5%BSA/EDTA (PBE) (Miltenyi Biotech). The collected BM mononuclear cells were filtered with a 70 µm cell strainer (BD Biosciences), and then washed twice with PBE for staining.

All flow cytometry analyses were performed using a BD FACS Fortessa (BD Biosciences). To ensure comparable mean fluorescence intensities over time of the analyses, Cytometer Setup and Tracking beads (BD Biosciences) and Rainbow Calibration Particles (BD Biosciences) were used. For staining, LIVE/DEAD Fixable Blue Dead Cell Stain Kit (ThermoFisher Scientific) was used to exclude dead cells according to the manufacturer’s protocol. BM cells were surface-stained for 30 min at 4 °C with the following anti-human antibodies: CD138-BUV737 (MI15, BD Biosciences, Cat. 564393, 1:20), CD14-BUV395 (M5E2, BD Biosciences, Cat. 740286, 1:50), CD3-BUV395 (UCHT1, BD Biosciences, Cat. 563546, 1:50), CD27-BV786 (L128, BD Biosciences, Cat. 563328, 1:50), CD19-BV711 (SJ25C1, BD Biosciences, Cat. 563038, 1:50), CD20-BV510 (2H7, BioLegend, Cat. 302340, 1:50), IgD-PE/Dazzle594 (IA6-2, BioLegend, Cat. 348240, 1:500), CD38-APC-Cy7 (HIT2, BioLegend, Cat. 303534, 1:500), HLA-DR- PE (Tü36, BD Biosciences, Cat. 555561, 1:10), CD56-BV421 (HCD56, BioLegend, Cat. 318328, 1:25) diluted in Brilliant Stain buffer (BD Horizon). Cells were washed twice with PBE, fixed for 20 min at 4 °C using Fixation/Permeabilization Solution Kit (BD Cytofix/Cytoperm Plus) and washed twice with perm/wash buffer. Cells were then stained intracellularly for 30 min 4 °C with recombinant purified RBD (DAGC149, Creative Diagnostics, New York, USA) and TT (AJ vaccines), which were coupled with either Alexa Fluor 647 or Alexa Fluor 488 to identify antigen-specific cells as described above and in ref. 72 , 73 , and with anti-human antibodies to detect expressed isotypes: IgA-biotin (G20-359, BD Biosciences, Cat. 555884, 1:50), IgG-PE-Cy7 (G18-145, BD Biosciences, Cat. 561298, 1:500), IgM-BV421 (G20-127, BD Biosciences, Cat. 562618, 1:100). Double-positive cells were considered as antigen-specific cells (See Fig.  5a ). Flow cytometric data were analysed by FlowJo software 10.7.1 (TreeStar).

One-tail spearman’s correlation coefficients were estimated to assess the relationship between the frequencies of CD19 − frequency of antigen-specific BMPCs and the time since the third SARS-CoV-2 vaccination. Mann–Whitney U test was used for comparison of two groups and Kruskal-Wallis with Dunn’s post-test was used for multiple comparisons. All statistical analyses were conducted using Prism version 9 (GraphPad), and P values of <0.05 were considered significant.

Enzyme-linked immunosorbent assay for the detection of serum-specific antibody titres on patients undergoing hip replacement surgery

To determine the tetanus toxoid and SARS-CoV-2 RBD-specific antibody titres, 96-well plates were coated overnight with 0.5 µg/ml of either tetanus toxoid (AJ vaccines) or SARS-CoV-2 (2019-nCoV) Spike RBD-His recombinant protein (Sino biological, Cat. 40592-V08B-100). Coated plates were washed, blocked for 1 h with PBS 5% BSA and incubated overnight at 4 °C with serial dilutions of sera. Specific IgA antibodies were detected using anti-human IgA-Biotin (Southern Biotech, Cat. 2050-08) followed by streptavidin-HRP (Invitrogen, Cat. N100) and specific IgG antibodies were detected using anti-human IgG-HRP (Southern Biotech, Cat. 2040-05). Detection antibody incubation was performed at room temperature for 1 h. After washing 5 times with PBS-T, Tetramethylbenzidine (TMB) Substrate (Invitrogen, Cat. 88-7324-88) was added. The reaction was stopped by the addition of 2 N H2SO4 (Sigma-Aldrich: Cat. 84736). Optical densities were measured on Spectramax (Molecular devices). Optical densities were measured on Spectramax plus 384(Molecular devices). OD values were further plotted against respective sample dilutions, and areas under the curve (AUC) were quantified using Graphpad Prism 9.3.1.

Flow cytometric assay for the detection of serum-specific antibody titres on patients undergoing hip replacement surgery

HEK293T cells (ATCC CRL-3216) were transfected with a plasmid expressing wild-type SARS-CoV-2 S protein. Next day, the proportion of transfected cells was determined by staining with anti-SARS-CoV-2 Spike Glycoprotein S1 antibody (clone: CR3022, Abcam, Cat. ab273073) for 30 min, wash cells once with PBS/0.2 % BSA and subsequent staining with goat anti-human IgG-Alexa647 (Southern Biotech, Cat. 2014-31). Further transfected cells were collected and incubated with sera for 30 min, washed twice with PBS/BSA and stained with goat anti-human IgG-Alexa647 (Southern Biotech, Cat. 2014-31) and anti-human IgA FITC (Sothern Biotech, Cat. 2052-02). Cells were washed with PBS/0.2 % BSA and either measured directly, dead cell exclusion by DAPI or stained for dead cells with Zombie Violet (Biolegend, Cat. 423113) in PBS for 5 min at room temperature and fixed in 4 % paraformaldehyde solution overnight at 4 °C. Samples were acquired on a FACSCanto (BD Biosciences) or a MACSQuant 16 (Miltenyi) and analysed using FlowJo v10 (Tree Star Inc.) analysis software. In the respective fluorescent channels, the geometric mean of fluorescent intensity (MFI) of Spike expressing and non-expressing cells was quantified, and ΔMFI = MFI (S+)-MFI (S−) for IgG and IgA was determined. ΔMFI values were further plotted against respective serum dilutions, and AUC were quantified using Graphpad Prism 9.3.1.

Enzyme-linked immunosorbent assay for the detection of serum-specific antibody titres on vaccinated individuals

The amount of SARS-CoV-2 spike RBD-specific antibodies was quantified using an in-house ELISA described previously 74 . Briefly, purified RBD protein was used for coating at a concentration of 5 µg/ml and 50 µl per well in a 96-well microtitre plate (Costar 3590, Corning Incorporated, Kennebunk, USA). For the ChAd-Y25 titre, the same ELISA approach was used, and 5 × 10 8 viral particles/well of the Vaxzevria vaccine (AstraZeneca, Oxford) were used for coating. After overnight coating at 4 °C, 230 µl of 10% FCS in PBS per well was used for blocking. Blocking was performed for 1 h at RT. Plates were washed four times with PBS-T (PBS containing 0.05% Tween). 50 µl of in blocking buffer 1:100 diluted sera were incubated in the wells for 1.5 h. An HRP-linked anti-human IgG antibody (Cytiva, Cat. NA933-1ML, Dassel, Germany) at a dilution of 1:3000 was used as a secondary antibody and incubated for 1.5 h on the plates. After washing five times, plates were developed for 5 min with 100 µl TMB solution (eBioscience, San Diego, USA) and stopped with the same volume of 1 N sulphuric acid. Absorbance was measured directly at 450 m on an Infinite M1000 reader (Tecan Group, Männedorf, Switzerland).

Co-culture of helper T cells with memory B cells

Tonsillar follicular helper T cells (Tfh, CD19 − CD4 + CD45RA − CXCR5 high ) from patients who underwent routine tonsillectomy or peripheral memory T helper cells from bronchoalveolar lavage (BAL) of sarcoidosis patients (mostly peripheral helper T cells, Tph, CD19 − CD4 + CD45RA − ) were sorted on an ARIA II flow cytometry sorter (Becton Dickinson). Patient samples were obtained from the Unfallkrankenhaus Marzahn (tonsils) or the Charité Universitätsmedizin Berlin (BAL). Sorted T cells were co-cultured for 7 days with heterologous tonsillar memory B cells (CD19 + CD4 −I gD − CD38 − ) at a 1:1 ratio in the presence of 4 ng/ml staphylococcal enterotoxin B (Toxin Technology) as described previously 36 . To block T cell help, 20 μg/ml anti-CD40L antibody (clone TRAP1) and/or 10 μg/ml recombinant soluble IL-21 receptor (R&D Systems, Cat. 9249-R2) were added to the culture. Cells were acquired on an LSR II Fortessa flow cytometer (Becton Dickinson) and analysed using FlowJo version 10 software (Tree Star Inc.).

All antibodies utilised in the study are listed in Supplementary Table  3 for reference.

Reporting summary

Further information on research design is available in the  Nature Portfolio Reporting Summary linked to this article.

Data availability

Next Generation Sequencing data sets generated in this study are available in the Gene Expression Omnibus (GEO) repository under accession number GSE253862 . Data was mapped using the human genome reference hg19 [ https://www.10xgenomics.com/support/software/cell-ranger/downloads/cr-ref-build-steps ]. The published data sets used for GSEA are available in the Molecular Signatures Database (MSigDB) [ https://www.gsea-msigdb.org/gsea/msigdb/ ]: Hallmark (PMID: 10592173), Reactome (PMID : 29145629) and KEGG (PMID: 10592173); and in the GEO repository under accession number GSE120369 (from Stephenson et al . 35 . PMID: 30642980). Flow cytometry data files for the analysis of human bone marrow plasma cells are available in the Flow Repository under accession ID FR-FCM-Z7A5 , while those for the analysis of the co-culture of helper T cells with memory B cells can be found under accession ID FR-FCM-Z7CB . All other data are available in the article and its Supplementary files or from the corresponding author upon request. Additional support for further analysis of the study findings is available upon request from the corresponding author or the special correspondence for bioinformatics [[email protected]].  Source data are provided with this paper.

Code availability

The software used in this study is open source. No custom code was used. Cellranger from 10x genomics: https://support.10xgenomics.com/single-cell-gene-expression/software/downloads/latest . Seurat packages 4.1.1: https://cloud.r-project.org/web/packages/Seurat/index.html . Monocle3 package: https://cole-trapnell-lab.github.io/monocle3/docs/installation/

Radbruch, A. et al. Competence and competition: the challenge of becoming a long-lived plasma cell. Nat. Rev. Immunol. 6 , 741–750 (2006).

Article   CAS   PubMed   Google Scholar  

Manz, R. A., Thiel, A. & Radbruch, A. Lifetime of plasma cells in the bone marrow. Nature 388 , 133–134 (1997).

Article   ADS   CAS   PubMed   Google Scholar  

Slifka, M. K., Antia, R., Whitmire, J. K. & Ahmed, R. Humoral immunity due to long-lived plasma cells. Immunity 8 , 363–372 (1998).

Turner, J. S. et al. SARS-CoV-2 infection induces long-lived bone marrow plasma cells in humans. Nature 595 , 421–425 (2021).

Kim, W. et al. Germinal centre-driven maturation of B cell response to mRNA vaccination. Nature 604 , 141–145 (2022).

Article   ADS   CAS   PubMed   PubMed Central   Google Scholar  

Amanna, I. J., Carlson, N. E. & Slifka, M. K. Duration of humoral immunity to common viral and vaccine antigens. N. Engl. J. Med 357 , 1903–1915 (2007).

Arce, S. et al. CD38 low IgG-secreting cells are precursors of various CD38 high-expressing plasma cell populations. J. Leukoc. Biol. 75 , 1022–1028 (2004).

Wiedemann, A. et al. Human IgA-expressing bone marrow plasma cells characteristically upregulate programmed cell death protein-1 upon B cell receptor stimulation. Front Immunol. 11 , 628923 (2020).

Dang, V. D. et al. CD39 and CD326 are bona fide markers of murine and human plasma cells and identify a bone marrow specific plasma cell subpopulation in lupus. Front Immunol. 13 , 873217 (2022).

Article   CAS   PubMed   PubMed Central   Google Scholar  

Bortnick, A. et al. Long-lived bone marrow plasma cells are induced early in response to T cell-independent or T cell-dependent antigens. J. Immunol. 188 , 5389–5396 (2012).

Weisel, F. J., Zuccarino-Catania, G. V., Chikina, M. & Shlomchik, M. J. A temporal switch in the germinal center determines differential output of memory B and plasma cells. Immunity 44 , 116–130 (2016).

Robinson, M. J. et al. Long-lived plasma cells accumulate in the bone marrow at a constant rate from early in an immune response. Sci. Immunol. 7 , eabm8389 (2022).

Article   PubMed   Google Scholar  

Fujieda, S., Saxon, A. & Zhang, K. Direct evidence that gamma 1 and gamma 3 switching in human B cells is interleukin-10 dependent. Mol. Immunol. 33 , 1335–1343 (1996).

Pene, J. et al. Cutting edge: IL-21 is a switch factor for the production of IgG1 and IgG3 by human B cells. J. Immunol. 172 , 5154–5157 (2004).

Islam, K. B., Nilsson, L., Sideras, P., Hammarstrom, L. & Smith, C. I. TGF-beta 1 induces germ-line transcripts of both IgA subclasses in human B lymphocytes. Int. Immunol. 3 , 1099–1106 (1991).

Kitani, A. & Strober, W. Regulation of C gamma subclass germ-line transcripts in human peripheral blood B cells. J. Immunol. 151 , 3478–3488 (1993).

Becht, E. et al. Dimensionality reduction for visualizing single-cell data using UMAP. Nat. Biotechnol. 37 , 38–44 (2018).

Article   Google Scholar  

Mei, H. E. et al. Blood-borne human plasma cells in steady state are derived from mucosal immune responses. Blood 113 , 2461–2469 (2009).

Odendahl, M. et al. Generation of migratory antigen-specific plasma blasts and mobilization of resident plasma cells in a secondary immune response. Blood 105 , 1614–1621 (2005).

Luciano, F. et al. Nur77 converts phenotype of Bcl-B, an antiapoptotic protein expressed in plasma cells and myeloma. Blood 109 , 3849–3855 (2007).

Liebmann, M. et al. Nur77 serves as a molecular brake of the metabolic switch during T cell activation to restrict autoimmunity. Proc. Natl Acad. Sci. USA 115 , E8017–E8026 (2018).

Tan, C. et al. Nur77 links chronic antigen stimulation to B cell tolerance by restricting the survival of self-reactive B cells in the periphery. J. Immunol. 202 , 2907–2923 (2019).

Cornelis, R. et al. Stromal cell-contact dependent PI3K and APRIL induced NF-kappaB signaling prevent mitochondrial- and ER stress induced death of memory plasma cells. Cell Rep. 32 , 107982 (2020).

Cornelis, R., Chang, H. D. & Radbruch, A. Keeping up with the stress of antibody production: BAFF and APRIL maintain memory plasma cells. Curr. Opin. Immunol. 71 , 97–102 (2021).

Burt, P. et al. Data-driven mathematical model of apoptosis regulation in memory plasma cells. Cells 11 . https://doi.org/10.3390/cells11091547 (2022)

Nguyen, D. C. et al. Factors of the bone marrow microniche that support human plasma cell survival and immunoglobulin secretion. Nat. Commun. 9 , 3698 (2018).

Article   ADS   PubMed   PubMed Central   Google Scholar  

Lam, W. Y. et al. Mitochondrial pyruvate import promotes long-term survival of antibody-secreting plasma cells. Immunity 45 , 60–73 (2016).

Mei, H. E. et al. A unique population of IgG-expressing plasma cells lacking CD19 is enriched in human bone marrow. Blood 125 , 1739–1748 (2015).

Pinto, D. et al. A functional BCR in human IgA and IgM plasma cells. Blood 121 , 4110–4114 (2013).

Hibi, T. & Dosch, H. M. Limiting dilution analysis of the B cell compartment in human bone marrow. Eur. J. Immunol. 16 , 139–145 (1986).

Burckert, J. P. et al. Functionally convergent B cell receptor sequences in transgenic rats expressing a human B cell repertoire in response to tetanus toxoid and measles antigens. Front. Immunol. 8 , 1834 (2017).

Article   PubMed   PubMed Central   Google Scholar  

Greinacher, A. et al. Insights in ChAdOx1 nCoV-19 vaccine-induced immune thrombotic thrombocytopenia. Blood 138 , 2256–2268 (2021).

Turner, J. S. et al. SARS-CoV-2 mRNA vaccines induce persistent human germinal centre responses. Nature 596 , 109–113 (2021).

Ferreira-Gomes, M. et al. SARS-CoV-2 in severe COVID-19 induces a TGF-beta-dominated chronic immune response that does not target itself. Nat. Commun. 12 , 1961 (2021).

Stephenson, S. et al. Growth factor-like gene regulation is separable from survival and maturation in antibody-secreting cells. J. Immunol. 202 , 1287–1300 (2019).

Bauer, L. et al. Follicular helper-like T cells in the lung highlight a novel role of B cells in sarcoidosis. Am. J. Respir. Crit. Care Med. 204 , 1403–1417 (2021).

Diehl, S. A. et al. STAT3-mediated up-regulation of BLIMP1 Is coordinated with BCL6 down-regulation to control human plasma cell differentiation. J. Immunol. 180 , 4805–4815 (2008).

Luo, W. et al. IL-21R signal reprogramming cooperates with CD40 and BCR signals to select and differentiate germinal center B cells. Sci. Immunol. 8 , eadd1823 (2023).

Giesecke, C. et al. Tissue distribution and dependence of responsiveness of human antigen-specific memory B cells. J. Immunol. 192 , 3091–3100 (2014).

Alsoussi, W. B. et al. SARS-CoV-2 Omicron boosting induces de novo B cell response in humans. Nature 617 , 592–598 (2023).

Blanchard-Rohner, G., Pulickal, A. S., Jol-van der Zijde, C. M., Snape, M. D. & Pollard, A. J. Appearance of peripheral blood plasma cells and memory B cells in a primary and secondary immune response in humans. Blood 114 , 4998–5002 (2009).

Bernasconi, N. L., Traggiai, E. & Lanzavecchia, A. Maintenance of serological memory by polyclonal activation of human memory B cells. Science 298 , 2199–2202 (2002).

Landsverk, O. J. et al. Antibody-secreting plasma cells persist for decades in human intestine. J. Exp. Med. 214 , 309–317 (2017).

Eberhardt, C. S. et al . Persistence of varicella-zoster virus-specific plasma cells in adult human bone marrow following childhood vaccination. J. Virol. 94 . https://doi.org/10.1128/JVI.02127-19 (2020).

Mudd, P. A. et al. SARS-CoV-2 mRNA vaccination elicits a robust and persistent T follicular helper cell response in humans. Cell 185 , 603–613 e615 (2022).

Delaloy, C., Schuh, W., Jack, H. M., Bonaud, A. & Espeli, M. Single-cell resolution of plasma cell fate programming in health and disease. Eur. J. Immunol. 52 , 10–23 (2022).

Duan, M. et al. Understanding heterogeneity of human bone marrow plasma cell maturation and survival pathways by single-cell analyses. Cell Rep. 42 , 112682 (2023).

Bhoj, V. G. et al. Persistence of long-lived plasma cells and humoral immunity in individuals responding to CD19-directed CAR T-cell therapy. Blood 128 , 360–370 (2016).

Halliley, J. L. et al. Long-lived plasma cells are contained within the CD19(-)CD38(hi)CD138(+) subset in human bone marrow. Immunity 43 , 132–145 (2015).

Walker, K. et al. Inherited CD19 deficiency impairs signaling in plasma cells. Res. Square . https://doi.org/10.21203/rs.3.rs-1498757/v1 (2022).

Becker, M., Hobeika, E., Jumaa, H., Reth, M. & Maity, P. C. CXCR4 signaling and function require the expression of the IgD-class B-cell antigen receptor. Proc. Natl Acad. Sci. USA 114 , 5231–5236 (2017).

Cassese, G. et al. Plasma cell survival is mediated by synergistic effects of cytokines and adhesion-dependent signals. J. Immunol. 171 , 1684–1690 (2003).

Cheng, Q. et al. CXCR4-CXCL12 interaction is important for plasma cell homing and survival in NZB/W mice. Eur. J. Immunol. 48 , 1020–1029 (2018).

Peperzak, V. et al. Mcl-1 is essential for the survival of plasma cells. Nat. Immunol. 14 , 290–297 (2013).

Dvorscek, A. R. et al. IL-21 has a critical role in establishing germinal centers by amplifying early B cell proliferation. EMBO Rep. 23 , e54677 (2022).

Zotos, D. et al. IL-21 regulates germinal center B cell differentiation and proliferation through a B cell-intrinsic mechanism. J. Exp. Med 207 , 365–378 (2010).

Quast, I. et al. Interleukin-21, acting beyond the immunological synapse, independently controls T follicular helper and germinal center B cells. Immunity 55 , 1414–1430 e1415 (2022).

Zotos, D. et al. The concerted change in the distribution of cell cycle phases and zone composition in germinal centers is regulated by IL-21. Nat. Commun. 12 , 7160 (2021).

Ozaki, K. et al. Regulation of B cell differentiation and plasma cell generation by IL-21, a novel inducer of Blimp-1 and Bcl-6. J. Immunol. 173 , 5361–5371 (2004).

Elsner, R. A. & Shlomchik, M. J. Germinal center and extrafollicular B cell responses in vaccination, immunity, and autoimmunity. Immunity 53 , 1136–1150 (2020).

Ettinger, R. et al. IL-21 and BAFF/BLyS synergize in stimulating plasma cell differentiation from a unique population of human splenic memory B cells. J. Immunol. 178 , 2872–2882 (2007).

Linterman, M. A. et al. IL-21 acts directly on B cells to regulate Bcl-6 expression and germinal center responses. J. Exp. Med. 207 , 353–363 (2010).

Wang, S. et al. IL-21 drives expansion and plasma cell differentiation of autoreactive CD11c(hi)T-bet(+) B cells in SLE. Nat. Commun. 9 , 1758 (2018).

Smith, T. Active immunity produced by so called balanced or neutral mixtures of diphtheria toxin and antitoxin. J. Exp. Med. 11 , 241–256 (1909).

Schaefer-Babajew, D. et al. Antibody feedback regulates immune memory after SARS-CoV-2 mRNA vaccination. Nature 613 , 735–742 (2023).

Francis, T. On the doctrine of original antigenic sin. Proc. Am. Phil. Soc. 104 , 572–578 (1960).

Arevalo, C. P. et al. Original antigenic sin priming of influenza virus hemagglutinin stalk antibodies. Proc. Natl Acad. Sci. USA 117 , 17221–17227 (2020).

Schiepers, A. et al. Molecular fate-mapping of serum antibody responses to repeat immunization. Nature 615 , 482–489 (2023).

Butler, A., Hoffman, P., Smibert, P., Papalexi, E. & Satija, R. Integrating single-cell transcriptomic data across different conditions, technologies, and species. Nat. Biotechnol. 36 , 411–420 (2018).

Cao, J. et al. The single-cell transcriptional landscape of mammalian organogenesis. Nature 566 , 496–502 (2019).

Hao, Y. et al. Integrated analysis of multimodal single-cell data. Cell 184 , 3573–3587 e3529 (2021).

Frolich, D. et al. Secondary immunization generates clonally related antigen-specific plasma cells and memory B cells. J. Immunol. 185 , 3103–3110 (2010).

Rincon-Arevalo, H. et al . Impaired humoral immunity to SARS-CoV-2 BNT162b2 vaccine in kidney transplant recipients and dialysis patients. Sci. Immunol. 6 . https://doi.org/10.1126/sciimmunol.abj1031 (2021).

Hein, S. et al. Quantitative and qualitative difference in antibody response against omicron and ancestral SARS-CoV-2 after third and fourth vaccination. Vaccines (Basel) 10 . https://doi.org/10.3390/vaccines10050796 (2022).

Download references

Acknowledgements

The authors are most grateful to the patients for their consent to participate in this study. We would like to thank Antje Blankenstein, Johanna Penzlin and Simon Reinke from the BCRT Cell Harvesting Laboratory and the Charité Orthopedics Surgery Department for the provision of samples. We also thank Jenny Kirsch and Toralf Kaiser from the DRFZ Flow Cytometry Core Facility for support in cell sorting and Mairi McGrath for text revision. This work was kindly supported by the following entities: the German Research Foundation (DFG) through the grants LI3540/1-1 to A.C.L., SCHR1658/1-1 to E.V.S., Do491/8-1/2 (SPP Immunobone) and Do491/7–5, 10-1, 11-1 to T.D., TRR130/TP24 to H.E.M. and T.D, TRR130/TP16 to A.R., HU1294/8-1/2 to A.H., and the Clinical Research Unit KFO 5023 ‘BecauseY’/Project number 504745852 to A.K.; the Federal Ministry of Education and Research (BMBF) with financing of the projects TReAT and CONAN to M.F.M., and the grant BCOVIT, 01KI20161 to E.V.S.; the state of Berlin and the “European Regional Development Fund” through the grant ERDF 2014-2020, EFRE 1.8/11, to M.F.M.; the Leibniz Association through the Leibniz Collaborative Excellence projects TargArt to M.F.M., ImpACT to A.K. and M.F.M., and CHROQ to F.M.; the Berlin senate through the financing of the project: “Modulation of the mucosal immune response to prevent severe COVID-19 disease progression by commensal bacteria or vaccination” to M.F.M.; the Berlin Institute of Health through the Starting Grant Multi-Omics Characterisation of SARS-CoV-2 infection, Project 6 “Identifying immunological targets in COVID-19” to M.F.M.; COLCIENCIAS scholarship call 727-2015 to H.R-A.; and the Charité-Universitätsmedizin Berlin through the Rahel-Hirsch-Stipendium to A.L.S.

Open Access funding enabled and organized by Projekt DEAL.

Author information

These authors contributed equally: Marta Ferreira-Gomes, Yidan Chen, Pawel Durek.

These authors jointly supervised this work: Andreas Radbruch, Thomas Dörner, Mir-Farzin Mashreghi.

Authors and Affiliations

Deutsches Rheuma-Forschungszentrum Berlin, ein Institut der Leibniz Gemeinschaft, Berlin, Germany

Marta Ferreira-Gomes, Yidan Chen, Pawel Durek, Hector Rincon-Arevalo, Frederik Heinrich, Franziska Szelinski, Gabriela Maria Guerra, Ana-Luisa Stefanski, Antonia Niedobitek, Annika Wiedemann, Marina Bondareva, Jacob Ritter, Katrin Lehmann, Henrik E. Mei, Qingyu Cheng, Van Duc Dang, Mario Witkowski, Andreia C. Lino, Andrey Kruglov, Fritz Melchers, Eva V. Schrezenmeier, Andreas Radbruch, Thomas Dörner & Mir-Farzin Mashreghi

Department of Rheumatology and Clinical Immunology, Charité-Universitätsmedizin Berlin, Berlin, Germany

Yidan Chen, Hector Rincon-Arevalo, Franziska Szelinski, Ana-Luisa Stefanski, Annika Wiedemann, Jacob Ritter, Qingyu Cheng, Van Duc Dang, Andreia C. Lino, Andreas Radbruch & Thomas Dörner

Grupo de Inmunología Celular e Inmunogenética, Facultad de Medicina, Instituto de Investigaciones Médicas, Universidad de Antioquia UdeA, Medellín, Colombia

Hector Rincon-Arevalo

Department of Nephrology and Medical Intensive Care, Charité-Universitätsmedizin Berlin, corporate member of Freie Universität Berlin, Humboldt-Universität zu Berlin, and Berlin Institute of Health, Berlin, Germany

Hector Rincon-Arevalo & Eva V. Schrezenmeier

Institute of Immunology, University Hospital Schleswig-Holstein, Kiel, Germany

Laura Bauer & Andreas Hutloff

Department of Orthopedic Surgery, Charité-Universitätsmedizin Berlin, Berlin, Germany

Sebastian Hardt, Christian Hipfl & Carsten Perka

Paul-Ehrlich-Institut, Bundesinstitut für Impfstoffe und biomedizinische Arzneimittel, Langen, Germany

Sascha Hein & Eberhard Hildt

Berlin Institute of Health at Charité-Universitätsmedizin Berlin, Berlin, Germany

Mareen Matz & Eva V. Schrezenmeier

Department of Microbiology and Infection Immunology, Charité-Universitätsmedizin Berlin, Berlin, Germany

Mario Witkowski

You can also search for this author in PubMed   Google Scholar

Contributions

Conceptualisation: A.R., T.D., M.F.M.; methodology: M.F.G., Y.C., P.D., H.R.A., L.B., F.S., G.M.G., A.C.L.; software: P.D., F.H., F.S.; validation: M.F.G., A.C.L., M.F.M.; formal analysis: P.D., F.H., F.S.; investigation: M.F.G., Y.C., H.R.A., L.B., G.M.G., A.L.S., A.N., A.W., M.B., J.C.R., K.L., S.H., V.D.D.; resources: S.H., C.H., Q.C., M.W., H.E.M., E.V.S.; data curation: M.F.G.; P.D., M.F.M.; writing–original draft: M.F.G., M.F.M.; writing–review & editing: M.F.G., Y.C., F.M., A.C.L., A.R., T.D.; visualisation: M.F.G., Y.C., P.D., F.H., F.S.; supervision: E.H., M.M., A.C.L., A.K., C.P., A.H., A.R., T.D., M.F.M.; project administration: T.D., M.F.M.; funding acquisition: A.R., T.D., M.F.M.

Corresponding author

Correspondence to Mir-Farzin Mashreghi .

Ethics declarations

Competing interests.

The authors declare no competing interests.

Peer review

Peer review information.

Nature Communications thanks the anonymous reviewer(s) for their contribution to the peer review of this work. A peer review file is available.

Additional information

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary information

Supplementary information, peer review file, description of additional supplementary files, supplementary data 1, reporting summary, source data, source data, rights and permissions.

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/ .

Reprints and permissions

About this article

Cite this article.

Ferreira-Gomes, M., Chen, Y., Durek, P. et al. Recruitment of plasma cells from IL-21-dependent and IL-21-independent immune reactions to the bone marrow. Nat Commun 15 , 4182 (2024). https://doi.org/10.1038/s41467-024-48570-0

Download citation

Received : 09 June 2023

Accepted : 07 May 2024

Published : 17 May 2024

DOI : https://doi.org/10.1038/s41467-024-48570-0

Share this article

Anyone you share the following link with will be able to read this content:

Sorry, a shareable link is not currently available for this article.

Provided by the Springer Nature SharedIt content-sharing initiative

By submitting a comment you agree to abide by our Terms and Community Guidelines . If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.

Quick links

• Explore articles by subject
• Guide to authors
• Editorial policies

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

• Open access
• Published: 08 May 2024

Integrating depth-dependent protist dynamics and microbial interactions in spring succession of a freshwater reservoir

• Indranil Mukherjee 1 ,
• Vesna Grujčić 2 ,
• Michaela M. Salcher 1 ,
• Petr Znachor 1 , 3 ,
• Jaromír Seďa 1 ,
• Miloslav Devetter 1 , 4 ,
• Pavel Rychtecký 1 ,
• Karel Šimek 1 , 3 &
• Tanja Shabarova 1  

Environmental Microbiome volume  19 , Article number:  31 ( 2024 ) Cite this article

273 Accesses

5 Altmetric

Metrics details

Protists are essential contributors to eukaryotic diversity and exert profound influence on carbon fluxes and energy transfer in freshwaters. Despite their significance, there is a notable gap in research on protistan dynamics, particularly in the deeper strata of temperate lakes. This study aimed to address this gap by integrating protists into the well-described spring dynamics of Římov reservoir, Czech Republic. Over a 2-month period covering transition from mixing to established stratification, we collected water samples from three reservoir depths (0.5, 10 and 30 m) with a frequency of up to three times per week. Microbial eukaryotic and prokaryotic communities were analysed using SSU rRNA gene amplicon sequencing and dominant protistan groups were enumerated by Catalysed Reporter Deposition-Fluorescence in situ Hybridization (CARD-FISH). Additionally, we collected samples for water chemistry, phyto- and zooplankton composition analyses.

Following the rapid changes in environmental and biotic parameters during spring, protistan and bacterial communities displayed swift transitions from a homogeneous community to distinct strata-specific communities. A prevalence of auto- and mixotrophic protists dominated by cryptophytes was associated with spring algal bloom-specialized bacteria in the epilimnion. In contrast, the meta- and hypolimnion showcased a development of a protist community dominated by putative parasitic Perkinsozoa, detritus or particle-associated ciliates, cercozoans, telonemids and excavate protists (Kinetoplastida), co-occurring with bacteria associated with lake snow.

Conclusions

Our high-resolution sampling matching the typical doubling time of microbes along with the combined microscopic and molecular approach and inclusion of all main components of the microbial food web allowed us to unveil depth-specific populations’ successions and interactions in a deep lentic ecosystem.

Microbial eukaryotes, the main contributors of eukaryotic diversity on Earth, include a remarkable diversity of single-celled planktonic protists that are omnipresent across all aquatic environments. Their significance in ecosystems stems from their diverse functions in carbon fluxes and energy transfer in aquatic food webs [ 1 , 2 ], encompassing a spectrum of ecological and biochemical roles and nutrition modes, from autotrophy/mixotrophy to heterotrophy (including predation, decomposition, parasitism, and osmotrophy) [ 3 , 4 , 5 , 6 , 7 ].

For a long time, research on protists faced limitations due to labour-intensive microscopic analyses and the often low morphological resolution of these organisms, particularly evident in the case of numerically dominant but taxonomically diverse small cells of heterotrophic nanoflagellates (HNF) [ 8 , 9 , 10 ]. However, the situation changed with the advent of high-throughput sequencing and the accessibility of 18S rRNA gene amplicon approaches. This transformation propelled protist research into a prominent focus within microbial ecology. Subsequently, there has been a rapid increase in studies from the early 2000s reporting an unprecedented diversity of nano-sized protists in marine [ 11 , 12 , 13 , 14 , 15 ] and freshwater [ 16 , 17 , 18 , 19 , 20 , 21 , 22 ] environments.

Most studies conducted in freshwaters have focused on the epilimnion, the upper water layer of highest productivity, while protist communities of deeper strata have been largely neglected, with only a few exceptions [ 21 , 23 , 24 , 25 , 26 , 27 ]. Additionally, the temporal resolution of most datasets is limited to low sampling frequencies (weeks to months) that is insufficient for capturing the rapid dynamics of fast-growing protists (replicating in hours to days) [ 7 , 28 , 29 ]. This considerably hinders the understanding of dynamic environmental events such as phytoplankton spring blooms [ 30 , 31 ] or impacts of dramatic environmental disturbances [ 32 ]. In temperate freshwater lakes, the onset of spring is marked by a physical mixing event after ice melt, which uniformly distributes microbial populations in the water column. The increase of light intensity and air temperature leads to a thermal stratification of the water column and rapid growth of phototrophic organisms in the epilimnion that serve as a base for the aquatic food web. The succession of phytoplankton and zooplankton during this phase was well described in the original and revised Plankton Ecology Group (PEG) model [ 33 , 34 ]. Recently, an attempt was made to expand the PEG model to encompass seasonal dynamics of prokaryotes [ 35 ]. The detailed resolution of bacterial spring dynamics showed a dominance of fast-growing phytoplankton bloom-associated Bacteroidota and Gammaproteobacteria at the beginning of spring bloom [ 30 , 31 , 36 , 37 ], succeeded by small-sized Actinobacteriota that are generally more resistant to protistan grazing [ 30 , 38 ]. However, our understanding of bacterial dynamics in the hypolimnion during this seasonal event remains limited [ 31 ]. While the revised PEG model includes protistan abundance [ 34 ], and protists have been documented in various prokaryotic studies to estimate top-down control, the broader scope of bacterial-protist interactions beyond prey-grazer dynamics remains unexplored. Additionally, there is still a lack of information regarding the spring succession of individual protistan populations, their ecological associations, and their functions within the aquatic food web across distinct lake strata [ 39 ].

Shotgun metagenomic analyses which successfully resolve bacterial populations by providing metagenome assembled genomes of high quality, have an unfortunate constraint for research on protist communities. This is due to a small number of sequenced protist genomes available in public databases and the increase in computational power and necessary depth of sequencing which grows exponentially with the increase in cell and genome size of sequenced organisms [ 31 ]. Therefore, many studies introduce arbitrary cutoffs of 20 or 5 µm for filtration and thus do not provide information on larger protists as well as symbiotic interactions, which might be crucial during the springtime [ 33 , 34 ].

In this study conducted in the temperate freshwater Římov reservoir (Czech Republic), we used a cutoff of 200 µm for biomass collection and hybrid approach combining 18S rRNA gene amplicon sequencing with the fluorescent labelling technique Catalysed Reporter Deposition-Fluorescence in situ Hybridization (CARD–FISH) [ 6 , 22 , 24 , 40 , 41 ]. This allowed us to visualise and enumerate specific protistan lineages, which dominated the sequencing data [ 39 , 42 ]. Additionally, we analyzed 16S rRNA gene amplicons of the prokaryotic community, phyto- and zooplankton, viruses, and chemical parameters, aiming at identifying dominant protist populations and their major interactions to refine our comprehension of the spring plankton succession in different strata of the reservoir. We hypothesised that protist communities in the hypolimnion significantly differ from those found in upper water layers. Specifically, we expected that variations in prey availability and the prevalence of attached lifestyle will strongly influence protist dynamics, ultimately leading to distinct microbial community interactions within the hypolimnion.

Study site and sampling procedure

The Římov reservoir, situated in South Bohemia, Czech Republic, is a dimictic, meso-eutrophic canyon-shaped reservoir which serves as an important drinking water supply. It covers an area of 2.06 km 2 with a volume of 34.5 × 10 6  m 3 and an average summer retention time of 77 days. It has been studied since 1979 at well–established stations [ 43 ], one of them located in the lacustrine zone near the dam (48.8475817N, 14.4902242E, max. depth 43 m) was used for sampling in our study.

Sampling was conducted during the period 31st March to 25th May 2016, with the high-frequency sampling, i.e. three times per week, conducted between 7 April to 12 May over the most intensive part of the spring bloom phase (for the detailed description of the sampling dates and hydrological parameters see Additional file 1 ). We sampled 3 depths (0.5, 10 and 30 m), which corresponded to the epi-, meta-, and hypolimnion, respectively. The sampling started during the mixing period (at homogeneous temperature distribution in the water column) covered the establishment of stratification and was terminated with the end of clear-water phase recognized by increased chlorophyll a concentrations. The samples were taken with a Friedinger sampler (Šramhauser; spol.s.r.o., Dolní Bukovsko, Czech Republic). For each depth ten litres of water were prefiltered through a 200 µm mesh plankton net into a plastic barrel which was precleaned with household bleach and rinsed with Milli-Q and sample water. Physical and chemical parameters, i.e., water temperature, pH, dissolved oxygen, and oxygen saturation were measured with a multiparametric probe YSI EXO2 (Yellow Springs Instruments, Yellow Springs, OH, USA). A submersible fluorescence probe (FluoroProbe; bbe-Moldaence, Kiel, Germany) was employed to measure chlorophyll a (Chl- a ) concentrations at 0.2 m intervals down till 20 m depth. Water transparency was measured using a Secchi disc. Samples for chemical analysis were collected in separate bottles. Phytoplankton samples were collected from 0.5 m depth and preserved with a Lugol’s solution for further processing. Crustacean zooplankton was sampled once a week by vertical hauls using an Apstein plankton net (200-um mesh). Net hauling provided an integrated sample for the upper 5 m water column representing the epilimnetic layer. Two hauls were combined into one sample and preserved with formaldehyde (4% final concentration) for subsequent processing in the laboratory. Small-sized rotifers were sampled analogically once a week as an integrated sample from the upper 5 m water column using a plastic tube of the appropriate length. Subsequently, a volume of 40 l of collected water was quantitatively filtered using a 35 µm thickening net. The collected material was preserved with formaldehyde (4% final concentration).

Chemical analysis

Samples were analysed for pH, dissolved organic carbon (DOC), dissolved nitrogen (DN), dissolved silica (DSi), total phosphorus (TP), dissolved phosphorus (DP), dissolved reactive phosphorus (DRP), NH 4 -N, NO3-N and absorbance was measured at wavelengths 254, 300, 350 and 400 nm (Additional file 2 ) using methods summarized in Znachor et al. [ 43 ].

Enumeration of microbial cells, viruses, phytoplankton and zooplankton

For each depth, subsamples were fixed with the Lugol-formaldehyde-thiosulfate decolourization technique (2% final concentration of formaldehyde) to minimize ejection of protistan food vacuole contents [ 44 ]. These samples were used for the enumeration of bacteria on black 0.2 μm pore-size filters (Osmonics, Inc., Livermore, CA, USA) and eukaryotes (flagellates and ciliates) on black 1 μm pore-size filters. All samples were stained with DAPI (4′, 6-diamidino-2-phenylindole, 1 μg ml −1 final concentration) and microbes were counted via epifluorescence microscopy (Olympus BX 53; Optical Co., Tokyo, Japan). For enumeration of virus-like particles (VLP), subsamples were fixed with glutaraldehyde (1% final concentration) for 10 min, flush-frozen with liquid nitrogen and stored at -80 °C until further processing. VLP were counted with an inFlux V-GS cell sorter (Becton Dickinson, Franklin Lakes, NJ. USA) as previously described [ 45 ].

Phytoplankton species were enumerated employing the Utermöhl method with an inverted microscope (Olympus IX 71) [ 46 ]. The mean dimension of algal cells were obtained for biovolume calculation using the approximation of cell morphology to regular geometric shapes [ 47 ].

For the analysis of zooplankton, formaldehyde from the preserved material was removed and partially replaced by tap water. Further processing was performed by a classical microscopical counting of different species with a series of species determination keys [ 48 ]. Rotifer abundance was analysed in exact subsamples in counting chamber using dissecting microscope Leica DM 2500 under magnification of 25–40 ×. Species determination was done according to Koste 1978 [ 49 ] in light of more recent literature in particular families and recent taxonomy.

DNA extraction and sequencing

Prokaryotic and eukaryotic biomass was collected on 0.2 µm pore-size filters (47 mm diameter; Osmonics, Minnetonka, MN, USA) from 1500 to 2000 ml of water. DNA was extracted with the Power Water DNA isolation kit (MO BIO Laboratories, Inc, Carlsbad, CA, USA). Prokaryotic 16S rRNA fragments (V4 region) were amplified using primer pair 515F and 926R [ 50 ] and sequenced on an Illumina MiSeq platform (PE300) with V3 chemistry at Genome Research Core of the University of Illinois (Chicago, USA). Eukaryotic amplicons (V9 region) were prepared using Euk_1391F and EukBr-7R primers ( https://earthmicrobiome.org/protocols-and-standards/18s/ ) and sequenced on an Illumina MiSeq platform (PE250) with V2 chemistry at SEQme company (Dobříš, Czech Republic).

Sequence analysis

Primers were cut from the demultiplexed reads using Cutadapt software v2.8 [ 51 ]. Trimmed sequences were processed using DADA2 pipeline v1.16.0 [ 52 ] with standard parameters ( https://benjjneb.github.io/dada2/tutorial.html ) in R (R Core Team 2020). For taxonomic identification at ASV (Amplicon Sequence Variant) level, SILVA v138 [ 53 , 54 ] and PR 2 v4.14.0 [ 55 ] were used for prokaryotes and eukaryotes, respectively. All irrelevant reads (mitochondria and plastids for prokaryotes and metazoa and fungi for eukaryotes as the study was focussed on protists) and singletons were excluded, and both datasets were rarefied to the smallest read number prior diversity estimation and statistical analysis. In order to access relatedness of microbial communities of different lake strata and their temporal dynamics, a Bray–Curtis dissimilarity distance matrices were calculated for protists and prokaryotes. Based on the obtained matrices, we performed nonparametric multidimensional scaling (nMDS) analysis using XLSTAT14 (Addinsoft, USA). Diversity estimators and indexes were calculated using vegan package in R [ 56 ]. A co-occurrence network was used to find associations between common protistan and prokaryotic ASVs (relative abundances > 0.5% in at least one sample). Subsequently, all possible pairwise Spearman’s rank correlations were calculated with the R script https://github.com/RichieJu520/Co-occurrence_Network_Analysis [ 57 ]. Only robust (|r|> 0.7) and statistically significant ( p  < 0.05) correlations were visualized in Gephi v0.9.2 [ 58 ] with subsequent modular analysis. The sequence data generated from amplicon sequencing were submitted to the European Nucleotide Archive (ENA) and are available under the BioProject: PRJEB66298.

Phylogenetic tree reconstruction, design of novel eukaryotic probes, and catalysed reporter deposition fluorescence in situ hybridization (CARD-FISH)

Representative amplicons of the 30 most abundant protistan ASVs were aligned with the SINA aligner [ 59 ] and imported into ARB [ 60 ] using the SILVA database SSURef_NR99_123 [ 61 ]. Alignments were manually refined and a maximum likelihood tree (1000 bootstraps) including their closest relatives was constructed on a dedicated web server [ 62 ] (Additional files 3 – 5 ). Oligonucleotide probes targeting a small, monophyletic lineage of katablepharids (Kat2-651), Telonema (Telo-1250) and Novel Clade 10 of Cercozoa (NC10–1290) were designed in ARB using the tools probe_design and probe_check and evaluated with the web tool math-FISH [ 63 ]. The formamide percentages were optimized on environmental samples (Table  1 , Additional file 6 ).

CARD-FISH was carried out for eight protistan lineages (Table  1 ) following published protocols [ 39 ]. Hybridized cells were visualized with an Olympus BX 53 epifluorescence microscope under 1000 × magnification at blue/UV excitations.

Estimation of bacterivory rates of HNF and ciliates

Flagellate and ciliate bacterivory rates in the epilimnion were estimated using fluorescently labelled bacteria (FLB) [ 64 ], prepared from a mixture of strains from the genus Limnohabitans and Polynucleobacter [ 65 ]. Briefly, the FLB tracers were added to constitute 8–20% of total bacteria. Samples were incubated at in situ temperature with FLB tracers for 5 and 30 min for ciliate and flagellate grazing rates, respectively. Incubation was terminated by fixation with Lugol-formaldehyde-thiosulfate and DAPI stained subsamples were prepared as described above for microscopical analysis [ 65 , 66 ]. A minimum of 100 ciliates and 200 HNF were inspected for FLB ingestion in each sample. To estimate total protistan grazing, average bacterial uptake rates of ciliates and HNF were multiplied by their in situ abundances.

Physical and chemical parameters and abundance of microbes

The temperature profile from the first sampling date (31st March 2016) indicated an almost homogeneously mixed water column with only two degrees difference between the surface and the bottom (Fig.  1 a). Similarly, the concentration of dissolved oxygen was almost uniform between 10 to 12 mg l −1 in the entire water column (Fig.  1 b). At the end of the sampling campaign (25th May), when the water column was stratified, the epilimnion temperature reached 16.6 °C, with a thermocline established between 5–10 m depth. Soon after the beginning of the sampling campaign, hypoxia started to develop in the bottom layers, which became anoxic towards the end, however, the sampling depth of hypolimnion (30 m) was always oxygenated (> 6.9 mg l −1 ; Fig.  1 b). A Chl- a maximum (19.6 μg l −1 ) was observed in the epilimnion in the first week of sampling after which the concentration dropped and remained relatively low till the end of the study (Fig.  1 c).

Main physical and chemical parameters and abundances of microorganisms observed in the Římov reservoir during the study. a Thermal structure of the water column (Temp). b Vertical distribution of oxygen (O 2 ). c Chlorophyll a profile (0–20 m). d Abundances of ciliates in the epilimnion and Secchi depths. e Abundances of heterotrophic nanoflagellates (HNF) at three depths. f Bacterial abundances at three depths. g Abundances of virus like particles (VLP) at three depths

Ciliates increased in the first 2 weeks to maximum abundance of 60 ind. ml −1 (Fig.  1 d), and were dominated by prostomes i.e., Urotricha spp. and Balanion planctonicum, representing efficient hunters of small algae and flagellates [ 67 ]. Ciliate abundances dropped sharply during the clear water phase and recovered simultaneously with Chl- a concentrations at the study end (Fig. 1 c). Low counts of HNF, prokaryotes and VLP, were recorded at the start of the sampling (Fig.  1 e–g). During the first 2 weeks, the numbers of HNF increased in all water strata, with a maximum of 7.7 × 10 3 cells ml −1 in the epilimnion and lower peaks in deeper layers (4.2 × 10 3 and 2.9 × 10 3 cells ml −1 at 10 m and 30 m, respectively; Fig.  1 e). Thereafter, the abundances gradually decreased in all layers with some occasional peaks. Bacterial abundances reached maxima in the epilimnion in mid-April (5.3 × 10 6 cells ml −1 ), whereas numbers remained low in the metalimnion and hypolimnion (averaging 2.6 × 10 6 cells ml −1 and 2.0 × 10 6 cells ml −1 , respectively) (Fig.  1 f). VLP steadily increased at 0.5 m to 5.6 × 10 7 VLP ml −1 and plateaued towards the end, while abundances at 10 and 30 m remained relatively stable with maxima of 3.8 × 10 7 VLP ml −1 at 10 m and 3.9 × 10 7 VLP ml −1 at 30 m (Fig.  1 g).

Phytoplankton biovolume in the epilimnion peaked with 3.6 mm −3  l −1 in the first week of April, showed a sharp decline within the following two weeks and remained low until the end (Fig.  2 a). Cryptophytes ( Cryptomonas reflexa, Rhodomonas minuta ) dominated the phytoplankton throughout the sampling period together with diatoms ( Cyclotella sp., Fragilaria sp. ) and chrysophytes ( Chrysococcus sp.). Rotifers and cladocerans followed the phytoplankton dynamics and showed maxima in the second week of sampling (Fig.  2 b). Copepod numbers increased slowly, but their populations remained stable in the second part of the study when densities of rotifers and cladocerans decreased.

Phytoplankton biovolume ( a ) and zooplankton abundance ( b ) in the epilimnion of Římov reservoir during the study

Grazing impact of HNF and ciliates on bacteria

On average, HNF and ciliates grazed 0.32 × 10 6 bacteria day −1 , corresponding to 8.2% of bacterial standing stock in the epilimnion of the reservoir. HNF were the major bacterivores responsible for 73% of total protistan bacterivory, while ciliates grazing was more important at the beginning and end of the campaign (Additional file 7 ), coinciding with relatively low numbers of Cladocerans (Fig.  2 b).

Community composition of microbes in the water column

From 51 DNA samples, 47 and 43 were successfully amplified and sequenced for prokaryotic and eukaryotic analyses, respectively. Datasets were rarefied to 31,011 (prokaryotes) and 13,202 (protists) reads per sample (Additional file 8 ). Both taxonomic entities displayed similar temporal developments based on their ASV dynamics. After mixing, we observed a fast separation of epilimnion samples from the deeper lake strata and undirected fluctuations and delayed differentiation of meta- and hypolimnetic communities (Fig.  3 ). Reduction of diversity over the time was noticed in both prokaryotes and protists especially in the epilimnion (Fig.  3 ). Despite similar dynamic patterns observed at highly resolved taxonomic level, prokaryotes showed more unified composition at family to phylum levels than protists (Fig.  4 a). Specifically, Actinobacteriota, Verrucomicrobiota, Chloroflexota and several families of Gammaproteobacteria (Comamonadaceae, Burkholderiaceae and Methylophilaceae) were distributed at comparable relative abundances in all samples from all layers. In contrast, among protists only Katablepharida and Ciliophora showed comparable distributions (Fig.  4 b, Additional file 9 ).

Nonparametric multidimensional scaling plots reflecting dynamics of prokaryotic ( a ) and protistan ( b ) communities at three depths of Římov reservoir. Plots are based on Bray–Curtis’s distance matrices calculated on rarefied ASV datasets. Kruskal’s stress values are 0.096 and 0.079 for prokaryotic and protistan plots respectively. The sampling start is indicated with the letter ‘s’, and the end is indicated with the letter ‘e’. The lines are connecting communities according to the temporal course of the study. Diameters of the circles correspond to the Shannon–Wiener diversity index (SI)

Prokaryotic and protistan community composition at three depths of Římov reservoir during the study. The gaps indicate missing samples. a Prokaryotic community. The majority of groups are resolved at phylum to class levels, with the exception of Gammaproteobacteria where several families were shown due to high heterogeneity in their distribution. b Protistian community. The groups are resolved at phylum to class level, with the exception of Supergroup Excavata, which was dominated by kinetoplastea. Ciliophora were excluded from the analysis of protists due to homogeneous distribution with high proportions in all the three layers. The figure including ciliophora is available in Additional file 9

The epilimnion was characterised by a high contribution of Bacteroidota (max. 35%) and increasing read counts of Alphaproteobacteria (max. 26%) and Armatimonadota (max. 3%) towards the study end (Fig.  4 a). The eukaryotic community was dominated by cryptophytes with the highest ASV contribution of 70% on 18th April (Fig.  4 b). Synurophytes also contributed a significant number of reads, especially towards the study end (up to 30%). Apicomplexa, almost absent at the beginning, increased in relative abundances as the study progressed and reached 10% on 25th April.

Among prokaryotes in the metalimnion, a high proportion of Planctomycetota was present throughout the study period, in contrast to negligible counts in the upper water layer. Moreover, populations of Acidobacteriota, Gallionellaceae, Nitrosomonadaceae, Legionellaceae and TRA3-20 established in the metalimnion albeit at low percentages (Fig.  4 a, Additional file 8 ). Similar to the epilimnion, cryptophytes were the most dominant eukaryotic group and represented up to 40% on 25th April and 5th May (Fig.  4 b). In contrast, Dinophyta, Chrysophyta, Cercozoa, Telonema and Excavata had significantly higher contributions to the metalimnetic community (Additional file 8 ).

In the hypolimnion, we observed a collapse of Bacteroidota population after 25th April, the date corresponding to the breakpoint clearly separating the meta- from hypolimnion according to nMDS analysis (Fig.  3 ). While the majority of bacterial groups were common to both metalimnetic and hypolimnetic samples (Fig.  4 a), Methylomonadaceae and Solimonadaceae were present exclusively in the hypolimnion. The eukaryotic community in the hypolimnion was dominated by Perkinsozoan sequences (max. 30%), especially towards the study end (Fig.  4 b). Similar to Bacteroidota, cryptophyte sequences drastically dropped after 25th April which reflected the separation of hypolimnion from the metalimnion (Figs. 3 , 4 b). Chrysophytes had a relatively high contribution in the hypolimnion during the early phase of the campaign but dropped considerably towards the study end. Cercozoa, Telonemia and Dinophyta had relatively high and stable proportions throughout the campaign.

Dynamics of important groups of microbial eukaryotes in the water column

Cell abundances of eight eukaryotic groups were quantified by CARD-FISH using specific probes (Table  1 , Fig.  5 , Additional files 9 , 10 ). Cryptophytes dominated in the epilimnion for about three weeks from late April to early May (max. 4.3 × 10 3  cells ml −1 ) and decreased towards the study end. Their abundances in the meta- and hypolimnion remained relatively low (< 1.4 × 10 3  cells ml −1 ). Epilimnetic abundances of the aplastidic CRY1 lineage of cryptophytes initially increased to 1.6 × 10 3  cells ml −1 (34% of the total eukaryotes) on April 13th (Fig.  5 , Additional file 10 ) and decreased to 0.4 × 10 3 cells ml −1 towards the study end. In the metalimnion, abundances of this lineage showed similar but less pronounced dynamics and reached maxima on 20th April with 1.1 × 10 3 cells ml −1 (27% of total eukaryotes). Their abundance in the hypolimnion was relatively low (< 0.6 × 10 3 cells ml −1 , < 22% of total eukaryotes).

Absolute abundances of particular flagellate groups at three depths of Římov reservoir obtained by CARD-FISH. Left to right from top-total eukaryotes by DAPI staining, cryptophytes targeted by probe Crypto B, CRY1 lineage of cryptophytes targeted by probe Cry1-652, katablepharids targeted by probe Kat-1452, katablepharid clade 2 targeted by probe Kat2-651, kinetoplastids targeted by probe Kin516, Perkinsozoa clade 1 targeted by probe Perkin01, Telonemids targeted by probe Telo-1250, and Cercozoa Novel Clade 10 targeted by probe NC10-1290. A figure representing relative abundances of flagellates can be found in the supplemental material (Additional file 10 )

Katablepharids targeted by probe Kat-1492 were not abundant in the epilimnion with exception of two peaks of ca. 0.2 × 10 3 cells ml −1 (Fig.  5 ). In the metalimnion, their abundance gradually increased and represented up to 7% of total eukaryotes (Additional file 10 ). In the hypolimnion, this lineage showed low numbers throughout the study period, in contrast to the katablepharids detected with probe Kat2-651 which were found exclusively in the hypolimnion with up to 0.3 × 10 3 cells ml −1 (Fig.  5 ).

Kinetoplastida, Perkinsozoa, Telonema and Cercozoa Novel Clade 10 were detected only in the meta- and hypolimnion (Fig.  5 ). In the metalimnion, Kinetoplastida increased with some oscillations till the study end with maxima of 0.18 × 10 3 cells ml −1 . Similarly, their abundances fluctuated considerably with maxima of 0.21 × 10 3 cells ml −1 in the hypolimnion. Perkinsozoans targeted by probe Perkin01 had relatively low abundances in the metalimnion (max. 0.1 × 10 3 cells ml −1 ), while they reached up to 0.5 × 10 3 cells ml −1 in the hypolimnion (Fig.  5 ). Telonema were abundant in meta- and hypolimnion samples with the highest peaks recorded on 25th April with 0.17 × 10 3 cells ml −1 and 0.26 × 10 3 cells ml −1 , respectively. The Novel Clade 10 of Cercozoa showed similar abundance patterns to those observed for Telonema (Fig.  5 ). However, maximal abundances were two–three times lower, i.e., 0.09 × 10 3  cells ml −1 and 0.10 × 10 3  cells ml −1 in meta- and hypolimnion, respectively.

Microbial interactions in different water layers

We performed a network analysis to examine the connections between and within the eukaryotic and bacterial communities (Fig.  6 ). The majority of potential interactions were located in two large clusters consisting of three modules each. The modules of the smaller cluster reflected the temporal development within the epilimnion. Phototrophic eukaryotes, such as Cryptomonas (Cryptophyta), Chlamydomonas (Chlorophyta) and Chrysophyta had central positions and were excessively linked to Flavobacteriales, Chitinophagales and Sphingobacteriales, which contributed the highest proportion of nodes in this cluster (Fig.  6 , Additional file 11 ). Ciliates within the epilimnetic network cluster, such as Vorticella sp., were associated with colonial chrysophytes, while Rimostrombidium and Strombidium spp., exhibited extensive connections to bacterial nodes. The modules in the large meta- and hypolimnion associated cluster showed a limited temporal resolution. The module four represented the earliest part of the sampling period and consisted mainly of protists affiliated to Bicosoecida, Cercozoa and Ciliophora organized around centric diatoms (Fig.  6 , Additional file 11 ). Heterotrophic flagellated protists Bicosoecea and Cercozoa along with Ciliophora ( Vorticella, Tokophrya, Tintinnidium etc.) comprised the lion share of this module. Chrysophytes and dinophytes were additional microbial eukaryotic groups detected in module four. A highly interconnected bacterial node in this part of the network was affiliated with Pirellulaceae (Planctomycetota) and was linked to diatoms, bacterivorous cercozoans and ciliates. Interestingly, four Legionella spp. ASVs were part of the hypolimnion network and were linked to eukaryotes. Moreover, in two cases, this was a selective connection to single eukaryotic ASVs (a ciliate and a cercozoan) and a simultaneous association with Verrucomicrobiota (Fig.  6 , Additional file 11 ). Rather than showing a temporal distinction, modules five and six exhibited a depth-related separation (Fig.  6 , Additional file 11 ). Module five was more aligned with the hypolimnion, while module six represented the community interactions in both meta- and hypolimnion. Similar to module four, the microbial eukaryotes present in module five were mainly excavates and cercozoans. Excavates such as kinetoplastids represented two nodes in the network modules five and also module six (Fig.  6 , Additional file 11 ). One additional node in module five was represented by the predatory protist group Telonema. Planctomycetota prevalent in module six were mainly represented by members of Nemodikiaceae (CL500-3 group). Ammonia oxidizers such as Nitrosomonadaceae were also present in this module and were connected to nitrite oxidizers, e.g. Nitrotoga sp. (Gallionellaceae) and ASVs affiliated with Methylobacter.

Network analysis based on the most abundant ASVs from protistan and prokaryotic communities. a Network: upper cluster represents the community dominating in the epilimnion, lower cluster represents the community dominating in the hypolimnion. Prokaryotic nodes are displayed as circles, protistan nodes as diamonds. b The main modules detected in the network. The arrows indicate directions of temporal shifts between modules. Differentiation between modules H 5 and H + M 6 is based on spatial parameters as module 6 was also well represented by members of the metalimnion community

High-frequency sampling, reflecting the typical doubling time of microbes, allowed us to follow tightly the community assembly at three depths during the transition from mixis to stratification in the water column. Interestingly, both protistan and prokaryotic community development showed strikingly similar dynamics in the different water strata of the reservoir (Fig.  3 ). The eukaryotic and bacterial communities in the epilimnion gradually diverged from those in the meta- and hypolimnion soon after the mixing, responding to the increase in temperature and light intensity. Moreover, towards the end of the campaign when the water column became stratified, we observed a clear separation between meta- and hypolimnetic communities.

Formation of water strata-associated communities and detected microbial interactions

The network analysis allowed us to follow the interaction between prokaryotes and protists and to shed light on their potential ecological and trophic roles in the epilimnion and hypolimnion of Římov reservoir (Fig.  6 ). Phototrophic eukaryotes found in the epilimnion cluster were the typical recurrently appearing members of spring blooms [ 34 ]. They were associated with Flavobacteriales, Chitinophagales and Sphingobacteriales known to be efficient decomposers of phytoplankton derived exudates and polymers during spring blooms [ 31 , 36 , 37 ] (Fig.  6 , Additional file 11 ). In the later phase of the study the protists exhibited increased connections with bacteria known as potential consumers of small molecular substances such as Polynucleobacter sp., Actinobacteriota and copiotrophic Comamonadaceae. These bacterial groups are well recognized members of spring bloom and disturbance successions [ 30 , 31 , 32 , 38 ]. Ciliates in this cluster were mainly represented by omnivorous and moderately efficient bacterial grazers such as Rimostrombidium and Strombidimum spp. [ 65 ], linked to bacterial nodes.Their presence was also confirmed microscopically, where they contributed substantially to high ciliate numbers and grazing rates at the beginning and the end of the study (Fig.  1 d, Additional file 7 ). Additionally, substrate attached ciliates, e.g. Vorticella sp. [ 68 , 69 ] were found in this part of the network associated to colonial chrysophytes.

The sessile or particle-attached lifestyle was even more pronounced in the large hypolimnion cluster. Modules five and six (Fig.  6 , Additional file 11 ) appear to be associated with lake snow (macroscopic organic aggregates or detritus particles) [ 70 ], while the module four was characterised by association between centric diatoms, which were probably sedimenting through the water column, with Vorticella sp., Tokophrya sp. and Bicosoecida (Fig.  2 a). The latter are characterized as mainly attached flagellates feeding on bacteria or more rarely free-living and feeding on particle-associated bacteria [ 8 , 71 ]. Planctomycetota were a dominant bacterial phylum in the hypolimnion cluster. Specifically, in module four they were represented by Pirellulaceae, particle attached bacteria previously detected in the hypolimnion of Římov reservoir during algal blooms [ 72 ]. On the other hand, the group CL500-3, prevalent in modules five and six is also recognised for its psychrophilic nature and particle-attached lifestyle [ 72 , 73 ]. These bacteria are capable of peptide degradation through the so-called ‘planctosome’ complex bound to the outer membrane [ 72 , 74 ]. Their activity might create micro-environments rich in labile amino acids [ 75 ] and ammonia beneficial for free-living Nanopelagicales (Actinobacteriota), specialists in the uptake of amino acids [ 38 , 76 ], dominant in module five. The extensive connection between Nitrosomonadaceae (ammonia oxidizers) and Gallionellaceae (nitrite oxidizers) in the same module indicates their combined potential of nitrate production in the deep waters. Nitrate produced by these bacteria was shown to positively influence the methane oxidation efficiency of Methylobacter spp. (Methylomonadaceae) [ 77 ]. Cercozoans detected in different modules within the hypolimnion cluster [ 16 , 23 ] were reported as feeders of bacteria and small eukaryotes [ 7 , 78 ] or phytoplankton parasites [ 23 , 79 ]. Both strategies corroborate the associations detected in the co-occurrence network (Fig.  6 ). Another putative parasitic relationship previously described in the literature [ 80 , 81 ] was detected between Legionella sp. and eukaryotic ASVs (Fig.  6 , Additional file 11 ).

Different feeding strategies could be responsible for lineage-specific distribution of protists in the water column, as one lineage of katablepharids (Kat2-651) was detected with CARD-FISH exclusively in the hypolimnion, while another lineage was abundant in the epi- and metalimnion (Fig.  5 ). Omnivorous and predatory strains of katablepharids are described in the literature [ 7 , 41 , 82 ]. Excavates (Kinetoplastea and Diplonemea) are typically found in the hypolimnion of freshwater lakes during summer [ 22 , 24 , 83 ], or in hypertrophic, shallow lakes rich in suspended organic particles [ 41 ]. In our study, the presence of kinetoplastids in the metalimnion and hypolimnion was confirmed by both sequencing and CARD-FISH data (Figs. 4 , 5 ) in line with reports of kinetoplastids feeding on bacteria associated with detritus particles [ 8 , 84 ] sinking from the epilimnion to deeper strata at the end of the spring bloom [ 83 ]. The presence of Telonema in this module also corroborated with reports from a wide range of freshwater habitats [ 18 , 20 , 24 , 85 ]. However, our study is the first to track their population dynamics using a specific oligonucleotide probe (Table  1 , Additional file 6 ), revealing that telonemids are almost absent in surface waters and mainly inhabit the cold deep-water layers (Figs. 4 , 5 ).

Dynamics of dominant protistan groups

Cryptophyta was the most abundant eukaryotic group dominating in all epi- and metalimnion samples, commonly detected by both sequencing and CARD-FISH methods (Figs. 4 , 5 , Additional files 9 , 10 ). Microscopic observations showed the high abundances of big (10–30 µm long) chloroplast-bearing but also small aplastidic cryptophytes. Highly abundant heterotrophic CRY1 lineage [ 7 , 41 ] accounted on average for 30% of the total cryptophytes targeted by the general Crypto B probe (Fig.  5 ). This lineage did not show clear associations within the network but contributed substantially to the first maximum of HNF observed in epi- and metalimnion and probably played an important role as bacterivores in line with recent findings of high bacterial uptake rates of CRY1 [ 41 , 42 ]. The balance between auto- and heterotrophic cryptophytes during the springtime is related to the availability of ample sunlight, nutrients and prokaryotic prey [ 30 , 42 , 86 ].

Perkinsozoa dominated the deep-water communities (Figs. 4 , 5 ) and were not present in the co-occurrence network. Perkinsozoa comprise putative parasitic protists widely distributed in marine and freshwaters [ 21 , 23 , 87 , 88 , 89 ]. In our study, the contribution of Perkinsozoan ASVs and cell abundance increased with the onset of stratification (Figs. 4 , 5 ), reaching up to 25% of total ASVs and 26% of total eukaryotes in the hypolimnion, similar to previous studies [ 21 , 23 , 31 ]. With CARD-FISH analysis, perkinsozoans were observed both free-living and inside of free-living protists (Additional file 6 ). An additional taxon well represented in the sequencing data and absent in the network analysis was Apicomplexa, a poorly understood group especially in freshwater environments. Apicomplexa were reported as obligate intracellular parasites mainly affecting fish and phytoplankton [ 90 ]. In our data set, they were more abundant in the epilimnion and significantly correlated with cryptophytes (r 2  = 0.600, p  = 2.719 × 10 −10 ). Apicomplexa and perkinsozoa were not part of the network probably due to their putative parasitic association with higher eukaryotes that were not included in analyses.

Microbial food web organization in spring

Algal blooms are the major factors shaping the spring microbial community not only in the epilimnion [ 91 , 92 ] but also in the deeper strata, due to the enhanced particle flux from the surface waters [ 83 , 93 ]. For an overview of the microbial food web in the Římov reservoir during spring, please see a schematic diagram (Additional file 12 ). The bloom of chrysophytes, diatoms and large cryptophytes in the reservoir at the beginning of the sampling campaign was likely decimated by zooplankton or viral lysis [ 31 ]. However, ciliates dominated by raptorial prostomes such as Urotricha spp. and Balanion planctonicum might also considerably contribute to the phytoplankton reduction [ 67 , 94 ] (Fig.  1 d). Cladocerans, especially large sized Daphnia spp. were highly abundant during the first 2 weeks of sampling and seemed to represent the main driver responsible for the decline of phytoplankton bloom in mid-April. The highest abundances of smaller grazers, i.e. Rotifera were observed close to the maxima of their corresponding favourite prey, i.e. chrysophytes and cryptophytes (Fig.  2 ). Polyarthra spp., which were shown to selectively feed on chrysophytes [ 95 ], were first in the succession and were followed by omnivorous Keratella sp. preferably feeding on chrysophytes and cryptophytes [ 96 ]. However, Cyclops vicinus seemed to drastically reduce the rotifer population, similar to previous observations of spring plankton succession in Římov reservoir [ 97 ]. The simultaneous establishment of a stable population of Eudiaptomus gracilis probably did not contribute much to the rotifers’ top-down control, but this copepod successfully replaced rotifers and daphnids as a powerful algal grazer in the later phase [ 98 ]. E. gracilis was also shown to exhibit strong influence on the lower food web organization due to a high clearance rate of ciliates [ 99 ]. The drop in ciliate densities, including high proportions of bacterivorous or omnivorous species, most likely resulted in a short-term increase in bacterial numbers (Fig.  1 d, f). Notably, ciliates were almost equally important bacterivores as HNF during the times of high ciliate abundances (Additional file 7 ). Ciliates, especially raptorial prostomes also contributed to the reduction of HNF in the epilimnion (Fig.  1 d, e). In addition, approximately half of the ciliate community during its peak abundance in early April was composed of typical flagellate hunters such as Balanion planctonicum and Urotricha spp. (data not shown) [ 7 , 94 , 100 ]. After the drop of ciliate abundance, the protistan bulk bacterivory rate was largely attributed to HNF (Additional file 7 ) dominated by aplastidic cryptophytes such as CRY1 lineage [ 41 , 42 ] and omnivorous katablepharids (Fig.  5 ) [ 7 , 41 ].

Communities in the deeper strata showed a clear dominance of heterotrophic groups although we did not follow the dynamics of higher trophic levels due to their low abundances. However, the highly complex network between prokaryotes and protists in the hypolimnion (Fig.  6 ) indicates a considerable increase of bacterivorous, parasitic, and detritivorous strategies in these strata.

In this study, we followed the dynamics of organisms < 200 µm in three different water column layers of a freshwater reservoir at high-temporal resolution during spring. The results unveiled parallel community assembly patterns for protists and prokaryotes, revealing an early separation of epilimnetic communities and subsequent differentiation between the meta- and hypolimnetic layers. Besides confirming a prevalence of phototrophic and predatory strategies among epilimnetic protists, we observed the emergence of organisms affiliated with Perkinsozoa, Telonemia, Kinetoplastida, and Cercozoa in the meta- and hypolimnion, indicating a dominance of particle-associated lifestyles and parasitic and detritivorous strategies in deeper strata during the spring period. Furthermore, we showcased diverse associations between bacterial and protistan taxa, ranging from substrate degradation-related to parasitic. These associations followed temporal successions and displayed depth-specific dynamics. Sequence-based and microscopic techniques allowed for the integration of protists into a holistic picture of the complex community dynamics during springtime. Such a hybrid approach appears to be a powerful tool for integrating various groups of organisms in temporal and spatial dynamics, enhancing our comprehension of microbial interactions and the functioning of freshwater ecosystems.

Availability of data and materials

The sequence data generated from the 16S and 18S rRNA gene amplicon sequencing was submitted to the European Nucleotide Archive (ENA) and are available under the BioProject: PRJEB66298, [ https://www.ebi.ac.uk/ena/browser/view/PRJEB66298 ].

Worden AZ, Follows MJ, Giovannoni SJ, Wilken S, Zimmerman AE, Keeling PJ. Environmental science. Rethinking the marine carbon cycle: factoring in the multifarious lifestyles of microbes. Science. 2015;347:1257594.

Article   PubMed   Google Scholar  

Caron DA, Countway PD, Jones AC, Kim DY, Schnetzer A. Marine protistan diversity. Ann Rev Mar Sci. 2012;4:467–93.

Arndt H, Dietrich D, Auer B, Cleven E-J, Grafenhan T, Weitere M, Mylnikov AP. Functional diversity of heterotrophic flagellates in aquatic ecosystems. Syst Assoc Spec. 2000;59:240–68.

Google Scholar  

Zubkov MV, Tarran GA. High bacterivory by the smallest phytoplankton in the North Atlantic Ocean. Nature. 2008;455:224–6.

Article   CAS   PubMed   Google Scholar  

Caron DA, Hu SK. Are we overestimating protistan diversity in nature? Trends Microbiol. 2019;27:197–205.

Piwosz K. Weekly dynamics of abundance and size structure of specific nanophytoplankton lineages in coastal waters (Baltic Sea). Limnol Oceanogr. 2019;64:2172–86.

Article   CAS   Google Scholar  

Simek K, Grujcic V, Mukherjee I, Kasalicky V, Nedoma J, Posch T, Mehrshad M, Salcher MM. Cascading effects in freshwater microbial food webs by predatory Cercozoa, Katablepharidacea and ciliates feeding on aplastidic bacterivorous cryptophytes. FEMS Microbiol Ecol. 2020;96:fiaa121.

Article   PubMed   PubMed Central   Google Scholar  

Boenigk J, Arndt H. Bacterivory by heterotrophic flagellates: community structure and feeding strategies. Antonie Van Leeuwenhoek. 2002;81:465–80.

Massana R, Terrado R, Forn I, Lovejoy C, Pedros-Alio C. Distribution and abundance of uncultured heterotrophic flagellates in the world oceans. Environ Microbiol. 2006;8:1515–22.

Obiol A, Muhovic I, Massana R. Oceanic heterotrophic flagellates are dominated by a few widespread taxa. Limnol Oceanogr. 2021;66:4240–53.

Article   Google Scholar  

Lopez-Garcia P, Rodriguez-Valera F, Pedros-Alio C, Moreira D. Unexpected diversity of small eukaryotes in deep-sea Antarctic plankton. Nature. 2001;409:603–7.

Moon-van der Staay SY, De Wachter R, Vaulot D. Oceanic 18S rDNA sequences from picoplankton reveal unsuspected eukaryotic diversity. Nature. 2001;409:607–10.

de Vargas C, Audic S, Henry N, Decelle J, Mahe F, Logares R, Lara E, Berney C, Le Bescot N, Probert I, et al. Ocean plankton. Eukaryotic plankton diversity in the sunlit ocean. Science. 2015;348:1261605.

Pasulka A, Hu SK, Countway PD, Coyne KJ, Cary SC, Heidelberg KB, Caron DA. SSU-rRNA gene sequencing survey of benthic microbial eukaryotes from Guaymas basin hydrothermal vent. J Eukaryot Microbiol. 2019;66:637–53.

Giner CR, Pernice MC, Balague V, Duarte CM, Gasol JM, Logares R, Massana R. Marked changes in diversity and relative activity of picoeukaryotes with depth in the world ocean. ISME J. 2020;14:437–49.

Lepere C, Boucher D, Jardillier L, Domaizon I, Debroas D. Succession and regulation factors of small eukaryote community composition in a lacustrine ecosystem (Lake Pavin). Appl Environ Microbiol. 2006;72:2971–81.

Article   CAS   PubMed   PubMed Central   Google Scholar  

Grossmann L, Jensen M, Heider D, Jost S, Glucksman E, Hartikainen H, Mahamdallie SS, Gardner M, Hoffmann D, Bass D, Boenigk J. Protistan community analysis: key findings of a large-scale molecular sampling. ISME J. 2016;10:2269–79.

Simon M, Lopez-Garcia P, Deschamps P, Moreira D, Restoux G, Bertolino P, Jardillier L. Marked seasonality and high spatial variability of protist communities in shallow freshwater systems. ISME J. 2015;9:1941–53.

Simon M, Lopez-Garcia P, Deschamps P, Restoux G, Bertolino P, Moreira D, Jardillier L. Resilience of freshwater communities of small microbial eukaryotes undergoing severe drought events. Front Microbiol. 2016;7:812.

Khomich M, Kauserud H, Logares R, Rasconi S, Andersen T. Planktonic protistan communities in lakes along a large-scale environmental gradient. FEMS Microbiol Ecol. 2017;93:fiw231.

Mukherjee I, Hodoki Y, Nakano S. Seasonal dynamics of heterotrophic and plastidic protists in the water column of Lake Biwa, Japan. Aquat Microb Ecol. 2017;80:123–37.

Mukherjee I, Salcher MM, Andrei AS, Kavagutti VS, Shabarova T, Grujcic V, Haber M, Layoun P, Hodoki Y, Nakano SI, et al. A freshwater radiation of diplonemids. Environ Microbiol. 2020;22:4658–68.

Lepere C, Masquelier S, Mangot JF, Debroas D, Domaizon I. Vertical structure of small eukaryotes in three lakes that differ by their trophic status: a quantitative approach. ISME J. 2010;4:1509–19.

Mukherjee I, Hodoki Y, Okazaki Y, Fujinaga S, Ohbayashi K, Nakano SI. Widespread dominance of kinetoplastids and unexpected presence of diplonemids in deep freshwater lakes. Front Microbiol. 2019;10:2375.

Lepère C, Domaizon I, Hugoni M, Vellet A, Debroas D. Diversity and dynamics of active small microbial eukaryotes in the anoxic zone of a freshwater meromictic lake (Pavin, France). Front Microbiol. 2016;7:171591.

Monjot A, Bronner G, Courtine D, Cruaud C, Da Silva C, Aury J, Gavory F, Moné A, Vellet A, Wawrzyniak I. Functional diversity of microbial eukaryotes in a meromictic lake: coupling between metatranscriptomic and a trait-based approach. Environ Microbiol. 2023;25:3406–22.

Oikonomou A, Pachiadaki M, Stoeck T. Protistan grazing in a meromictic freshwater lake with anoxic bottom water. FEMS Microbiol Ecol. 2014;87:691–703.

Šimek K, Grujčić V, Hahn MW, Horňák K, Jezberová J, Kasalický V, Nedoma J, Salcher MM, Shabarova T. Bacterial prey food characteristics modulate community growth response of freshwater bacterivorous flagellates. Limnol Oceanogr. 2018;63:484–502.

Moreira D, Lopez-Garcia P. Time series are critical to understand microbial plankton diversity and ecology. Mol Ecol. 2019;28:920–2.

Simek K, Nedoma J, Znachor P, Kasalický V, Jezbera J, Hornňák K, Sed’a J. A finely tuned symphony of factors modulates the microbial food web of a freshwater reservoir in spring. Limnol Oceanogr. 2014;59:1477–92.

Kavagutti VS, Bulzu PA, Chiriac CM, Salcher MM, Mukherjee I, Shabarova T, Grujcic V, Mehrshad M, Kasalicky V, Andrei AS, et al. High-resolution metagenomic reconstruction of the freshwater spring bloom. Microbiome. 2023;11:15.

Shabarova T, Salcher MM, Porcal P, Znachor P, Nedoma J, Grossart HP, Seda J, Hejzlar J, Simek K. Recovery of freshwater microbial communities after extreme rain events is mediated by cyclic succession. Nat Microbiol. 2021;6:479–88.

Sommer U, Gliwicz ZM, Lampert W, Duncan A. The PEG-model of seasonal succession of planktonic events in fresh waters. Arch Hydrobiol. 1986;106:433–71.

Sommer U, Adrian R, Domis LDS, Elser JJ, Gaedke U, Ibelings B, Jeppesen E, Lürling M, Molinero JC, Mooij WM, et al. Beyond the Plankton Ecology Group (PEG) model: mechanisms driving plankton succession. Annu Rev Ecol Evol Syst. 2012;43:429–48.

Park H, Shabarova T, Salcher MM, Kosova L, Rychtecky P, Mukherjee I, Simek K, Porcal P, Seda J, Znachor P, Kasalicky V. In the right place, at the right time: the integration of bacteria into the Plankton Ecology Group model. Microbiome. 2023;11:112.

Zeder M, Peter S, Shabarova T, Pernthaler J. A small population of planktonic Flavobacteria with disproportionally high growth during the spring phytoplankton bloom in a prealpine lake. Environ Microbiol. 2009;11:2676–86.

Eckert EM, Salcher MM, Posch T, Eugster B, Pernthaler J. Rapid successions affect microbial N-acetyl-glucosamine uptake patterns during a lacustrine spring phytoplankton bloom. Environ Microbiol. 2012;14:794–806.

Neuenschwander SM, Ghai R, Pernthaler J, Salcher MM. Microdiversification in genome-streamlined ubiquitous freshwater Actinobacteria. ISME J. 2018;12:185–98.

Piwosz K, Mukherjee I, Salcher MM, Grujcic V, Simek K. CARD-FISH in the sequencing era: opening a new universe of protistan ecology. Front Microbiol. 2021;12:640066.

Grujcic V, Nuy JK, Salcher MM, Shabarova T, Kasalicky V, Boenigk J, Jensen M, Simek K. Cryptophyta as major bacterivores in freshwater summer plankton. ISME J. 2018;12:1668–81.

Simek K, Mukherjee I, Nedoma J, de Paula CCP, Jezberova J, Sirova D, Vrba J. CARD-FISH and prey tracer techniques reveal the role of overlooked flagellate groups as major bacterivores in freshwater hypertrophic shallow lakes. Environ Microbiol. 2022;24:4256–73.

Simek K, Mukherjee I, Szoke-Nagy T, Haber M, Salcher MM, Ghai R. Cryptic and ubiquitous aplastidic cryptophytes are key freshwater flagellated bacterivores. ISME J. 2023;17:84–94.

Znachor P, Nedoma J, Hejzlar J, Seda J, Kopacek J, Boukal D, Mrkvicka T. Multiple long-term trends and trend reversals dominate environmental conditions in a man-made freshwater reservoir. Sci Total Environ. 2018;624:24–33.

Sherr EB, Sherr BF. Protistan grazing rates via uptake of fluorescently labeied prey. In: Handbook of methods in aquatic microbial ecology. CRC Press; 2018, pp. 695–701.

Brussaard CP. Optimization of procedures for counting viruses by flow cytometry. Appl Environ Microbiol. 2004;70:1506–13.

Lund JWG, Kipling C, Le Cren E. The inverted microscope method of estimating algal numbers and the statistical basis of estimations by counting. Hydrobiologia. 1958;11:143–70.

Hillebrand H, Dürselen C-D, Kirschtel D, Pollingher U, Zohary T. Biovolume calculation for pelagic and benthic microalgae. J Phycol. 1999;35:403–24.

McCauley E. The estimation of the abundance and biomass of zooplankton in samples. In A manual on methods for the assessment of secondary productivity in fresh waters 1984.

Koste W. Rotatoria. Die Rädertiere Mitteleuropas, begründet von Max Voigt. Überordnung Monogononta. Gebrüder Borntraeger, Berlin, Stuttgart. I. Text (673 pp) u II Tafelbd; 1978.

Parada AE, Needham DM, Fuhrman JA. Every base matters: assessing small subunit rRNA primers for marine microbiomes with mock communities, time series and global field samples. Environ Microbiol. 2016;18:1403–14.

Martin M. Cutadapt removes adapter sequences from high-throughput sequencing reads. EMBnet J. 2011;17:10–2.

Callahan BJ, McMurdie PJ, Rosen MJ, Han AW, Johnson AJ, Holmes SP. DADA2: high-resolution sample inference from Illumina amplicon data. Nat Methods. 2016;13:581–3.

Quast C, Pruesse E, Yilmaz P, Gerken J, Schweer T, Yarza P, Peplies J, Glockner FO. The SILVA ribosomal RNA gene database project: improved data processing and web-based tools. Nucleic Acids Res. 2013;41:D590-596.

Yilmaz P, Parfrey LW, Yarza P, Gerken J, Pruesse E, Quast C, Schweer T, Peplies J, Ludwig W, Glockner FO. The SILVA and “All-species Living Tree Project (LTP)” taxonomic frameworks. Nucleic Acids Res. 2014;42:D643-648.

Guillou L, Bachar D, Audic S, Bass D, Berney C, Bittner L, Boutte C, Burgaud G, de Vargas C, Decelle J, et al. The Protist Ribosomal Reference database (PR2): a catalog of unicellular eukaryote small sub-unit rRNA sequences with curated taxonomy. Nucleic Acids Res. 2013;41:D597-604.

Dixon P. VEGAN, a package of R functions for community ecology. J Veg Sci. 2003;14:927–30.

Hu A, Ju F, Hou L, Li J, Yang X, Wang H, Mulla SI, Sun Q, Burgmann H, Yu CP. Strong impact of anthropogenic contamination on the co-occurrence patterns of a riverine microbial community. Environ Microbiol. 2017;19:4993–5009.

Bastian M, Heymann S, Jacomy M. Gephi: an open source software for exploring and manipulating networks. In: Proceedings of the international AAAI conference on web and social media. 2009, pp. 361–362.

Pruesse E, Peplies J, Glockner FO. SINA: accurate high-throughput multiple sequence alignment of ribosomal RNA genes. Bioinformatics. 2012;28:1823–9.

Ludwig W, Strunk O, Westram R, Richter L, Meier H, Yadhukumar A, Buchner A, Lai T, Steppi S, Jobb G, et al. ARB: a software environment for sequence data. Nucleic Acids Res. 2004;32:1363–71.

Pruesse E, Quast C, Knittel K, Fuchs BM, Ludwig W, Peplies J, Glockner FO. SILVA: a comprehensive online resource for quality checked and aligned ribosomal RNA sequence data compatible with ARB. Nucleic Acids Res. 2007;35:7188–96.

Stamatakis A, Ludwig T, Meier H. RAxML-III: a fast program for maximum likelihood-based inference of large phylogenetic trees. Bioinformatics. 2005;21:456–63.

Yilmaz LS, Parnerkar S, Noguera DR. mathFISH, a web tool that uses thermodynamics-based mathematical models for in silico evaluation of oligonucleotide probes for fluorescence in situ hybridization. Appl Environ Microbiol. 2011;77:1118–22.

Sherr BF, Sherr EB, Fallon RD. Use of monodispersed, fluorescently labeled bacteria to estimate in situ protozoan bacterivory. Appl Environ Microbiol. 1987;53:958–65.

Šimek K, Grujčić V, Nedoma J, Jezberová J, Šorf M, Matoušů A, Pechar L, Posch T, Bruni EP, Vrba J. Microbial food webs in hypertrophic fishponds: omnivorous ciliate taxa are major protistan bacterivores. Limnol Oceanogr. 2019;64:2295–309.

Jezbera J, Horňák K, Šimek K. Food selection by bacterivorous protists: insight from the analysis of the food vacuole content by means of fluorescence in situ hybridization. FEMS Microbiol Ecol. 2005;52:351–63.

Posch T, Eugster B, Pomati F, Pernthaler J, Pitsch G, Eckert EM. Network of interactions between ciliates and phytoplankton during spring. Front Microbiol. 2015;6:1289.

Risse-Buhl U, Scherwass A, Schlüssel A, Arndt H, Kröwer S, Küsel K. Detachment and motility of surface-associated ciliates at increased flow velocities. Aquat Microb Ecol. 2009;55:209–18.

Gómez F, Wang L, Lin S. Morphology and molecular phylogeny of peritrich ciliate epibionts on pelagic diatoms: Vorticella oceanica and Pseudovorticella coscinodisci sp. Nov. (Ciliophora, Peritrichia). Protist. 2018;169:268–79.

Grossart HP, Simon M. Limnetic macroscopic organic aggregates (lake snow): occurrence, characteristics, and microbial dynamics in Lake Constance. Limnol Oceanogr. 1993;38:532–46.

Schoenle A, Hohlfeld M, Rosse M, Filz P, Wylezich C, Nitsche F, Arndt H. Global comparison of bicosoecid Cafeteria -like flagellates from the deep ocean and surface waters, with reorganization of the family Cafeteriaceae. Eur J Protistol. 2020;73:125665.

Andrei AS, Salcher MM, Mehrshad M, Rychtecky P, Znachor P, Ghai R. Niche-directed evolution modulates genome architecture in freshwater Planctomycetes. ISME J. 2019;13:1056–71.

Okazaki Y, Fujinaga S, Tanaka A, Kohzu A, Oyagi H, Nakano SI. Ubiquity and quantitative significance of bacterioplankton lineages inhabiting the oxygenated hypolimnion of deep freshwater lakes. ISME J. 2017;11:2279–93.

Wiegand S, Jogler M, Jogler C. On the maverick planctomycetes. FEMS Microbiol Rev. 2018;42:739–60.

Grossart H-P, Simon M. Significance of limnetic organic aggregates (lake snow) for the sinking flux of particulate organic matter in a large lake. Aquat Microb Ecol. 1998;15:115–25.

Salcher MM, Posch T, Pernthaler J. In situ substrate preferences of abundant bacterioplankton populations in a prealpine freshwater lake. ISME J. 2013;7:896–907.

van Grinsven S, Sinninghe Damste JS, Harrison J, Villanueva L. Impact of electron acceptor availability on methane-influenced microorganisms in an enrichment culture obtained from a stratified lake. Front Microbiol. 2020;11:715.

Piwosz K, Pernthaler J. Enrichment of omnivorous cercozoan nanoflagellates from coastal Baltic Sea waters. PLoS ONE. 2011;6:e24415.

Kuhn S, Medlin L, Eller G. Phylogenetic position of the parasitoid nanoflagellate Pirsonia inferred from nuclear-encoded small subunit ribosomal DNA and a description of Pseudopirsonia n. gen. and Pseudopirsonia mucosa (Drebes) comb. nov. Protist. 2004;155:143–56.

Molmeret M, Horn M, Wagner M, Santic M, Abu Kwaik Y. Amoebae as training grounds for intracellular bacterial pathogens. Appl Environ Microbiol. 2005;71:20–8.

Watanabe K, Nakao R, Fujishima M, Tachibana M, Shimizu T, Watarai M. Ciliate Paramecium is a natural reservoir of Legionella pneumophila . Sci Rep. 2016;6:24322.

Ok JH, Jeong HJ, Lim AS, Lee SY, Kim SJ. Feeding by the heterotrophic nanoflagellate Katablepharis remigera on algal prey and its nationwide distribution in Korea. Harmful Algae. 2018;74:30–45.

Mukherjee I, Hodoki Y, Nakano S. Kinetoplastid flagellates overlooked by universal primers dominate in the oxygenated hypolimnion of Lake Biwa, Japan. FEMS Microbiol Ecol. 2015;91:fiv083.

Caron DA. Grazing of attached bacteria by heterotrophic microflagellates. Microb Ecol. 1987;13:203–18.

Brate J, Klaveness D, Rygh T, Jakobsen KS, Shalchian-Tabrizi K. Telonemia-specific environmental 18S rDNA PCR reveals unknown diversity and multiple marine-freshwater colonizations. BMC Microbiol. 2010;10:168.

Dokulil M, Skolaut C. Succession of phytoplankton in a deep stratifying lake: Mondsee, Austria. Hydrobiologia. 1986;138:9–24.

Mangot J-F, Debroas D, Domaizon I. Perkinsozoa, a well-known marine protozoan flagellate parasite group, newly identified in lacustrine systems: a review. Hydrobiologia. 2011;659:37–48.

Jobard M, Wawrzyniak I, Bronner G, Marie D, Vellet A, Sime-Ngando T, Debroas D, Lepère C. Freshwater Perkinsea: diversity, ecology and genomic information. J Plankton Res. 2020;42:3–17.

Park MG, Yih W, Coats DW. Parasites and phytoplankton, with special emphasis on dinoflagellate infections. J Eukaryot Microbiol. 2004;51:145–55.

Morrison DA. Evolution of the Apicomplexa: Where are we now? Trends Parasitol. 2009;25:375–82.

Rösel S, Grossart H-P. Contrasting dynamics in activity and community composition of free-living and particle-associated bacteria in spring. Aquat Microb Ecol. 2012;66:169–81.

Paver SF, Hayek KR, Gano KA, Fagen JR, Brown CT, Davis-Richardson AG, Crabb DB, Rosario-Passapera R, Giongo A, Triplett EW, Kent AD. Interactions between specific phytoplankton and bacteria affect lake bacterial community succession. Environ Microbiol. 2013;15:2489–504.

Kagami M, Gurung TB, Yoshida T, Urabe J. To sink or to be lysed? Contrasting fate of two large phytoplankton species in Lake Biwa. Limnol Oceanogr. 2006;51:2775–86.

Müller H, Schlegel A. Responses of three freshwater planktonic ciliates with different feeding modes to cryptophyte and diatom prey. Aquat Microb Ecol. 1999;17:49–60.

Liang D, Luo H, Huang C, Ye Z, Sun S, Dong J, Liang M, Lin S, Yang Y. High-throughput sequencing reveals omnivorous and preferential diets of the rotifer Polyarthra in situ. Front Microbiol. 2022;13:1048619.

Gilbert JJ. Food niches of planktonic rotifers: diversification and implications. Limnol Oceanogr. 2022;67:2218–51.

Devetter M, Seďa J. Regulation of rotifer community by predation of Cyclops vicinus (Copepoda) in the Římov Reservoir in spring. Int Rev Hydrobiol. 2006;91:101–12.

Šorf M, Brandl Z. The rotifer contribution to the diet of Eudiaptomus gracilis (GO Sars, 1863) (Copepoda, Calanoida). Crustaceana. 2012;85:1421–9.

Kunzmann AJ, Ehret H, Yohannes E, Straile D, Rothhaupt K-O. Calanoid copepod grazing affects plankton size structure and composition in a deep, large lake. J Plankton Res. 2019;41:955–66.

Šimek K, Bobková J, Macek M, Nedoma J, Psenner R. Ciliategrazing on picoplankton in a eutrophic reservoir during the summer phytoplankton maximum: a study at the species and community level. Limnol Oceanogr. 1995;40:1077–90.

Metfies K, Berzano M, Mayer C, Roosken P, Gualerzi C, Medlin L, Muyzer G. An optimized protocol for the identification of diatoms, flagellated algae and pathogenic protozoa with phylochips. Mol Ecol Notes. 2007;7:925–36.

Bochdansky AB, Huang L. Re-evaluation of the EUK516 probe for the domain eukarya results in a suitable probe for the detection of kinetoplastids, an important group of parasitic and free-living flagellates. J Eukaryot Microbiol. 2010;57:229–35.

Mangot JF, Lepere C, Bouvier C, Debroas D, Domaizon I. Community structure and dynamics of small eukaryotes targeted by new oligonucleotide probes: new insight into the lacustrine microbial food web. Appl Environ Microbiol. 2009;75:6373–81.

Download references

Acknowledgements

We are thankful to Radka Malá for excellent laboratory assistance.

This study was supported by the research Grant 22-35826 K (Grant Agency of the Czech Republic) awarded to IM, which covered all the materials and the salaries of IM and KŠ. MMS was supported by the research Grant 20-12496X (Grant Agency of the Czech Republic). TS and PZ were supported by the research Grants 23-05081S and 22-33245S, respectively. The funding bodies had no role in study design, data collection and analysis, interpretation of data or preparation of the manuscript.

Author information

Authors and affiliations.

Biology Centre of the Czech Academy of Sciences, Institute of Hydrobiology, Na Sádkách 7, 37005, Ceske Budejovice, Czech Republic

Indranil Mukherjee, Michaela M. Salcher, Petr Znachor, Jaromír Seďa, Miloslav Devetter, Pavel Rychtecký, Karel Šimek & Tanja Shabarova

Department of Ecology, Environment and Plant Sciences, Stockholm University, Stockholm, Sweden

Vesna Grujčić

Faculty of Science, University of South Bohemia, 37005, Ceske Budejovice, Czech Republic

Petr Znachor & Karel Šimek

Biology Centre of the Czech Academy of Sciences, Institute of Soil Biology and Biogeochemistry, Na Sádkách 7, 37005, Ceske Budejovice, Czech Republic

Miloslav Devetter

You can also search for this author in PubMed   Google Scholar

Contributions

IM, VG and KŠ conceived the study. IM and TS wrote the manuscript with the input from all authors. IM performed CARD-FISH, data analysis and interpretation. VG performed sampling, CARD-FISH and data analysis. TS performed sampling, sequence data analysis and interpretation, and design of figures. MS performed sampling, phylogenetic analyses and construction of CARD-FISH probes. KŠ performed microscopic counts, bacterivory rates estimation, data analysis and interpretation. PZ, PR, JS and MD performed sampling and sample analysis. All the authors contributed to critical revisions and approved the final version of the manuscript.

Corresponding authors

Correspondence to Indranil Mukherjee or Tanja Shabarova .

Ethics declarations

Ethics approval and consent to participate.

Not applicable.

Consent for publication

Competing interests.

The authors declare that they have no competing interests.

Additional information

Publisher's note.

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary Information

Additional file 1:.

Hydrological data and sampling dates. Precipitation data and flow rates of reservoir were provided by the Water Authority of Vltava River (Povodí Vltavy).

Additional file 2:

Chemistry data. DOC Dissolved organic carbon, DN Dissolved nitrogen, DSi Dissolved silica, TP Total phosphorus, DP Dissolved phosphorus, DRP Dissolved reactive phosphorus, A254-400 absorbance measured at corresponding wavelength (nm).

Additional file 3:

Randomized axelerated maximum likelihood (RAxML) tree (100 bootstraps) of katablepharids. Branches with bootstrap support < 20% were multifurcated, probe targets are marked by different colors. Asterisks indicate sequences not targeted by probes.

Additional file 4:

Randomized axelerated maximum likelihood (RAxML) tree (100 bootstraps) of Cercozoa including Novel Clade 10 (NC10). Branches with bootstrap support < 20% were multifurcated, probe targets are marked by different colors. Asterisks indicate sequences not targeted by probe.

Additional file 5:

Randomized axelerated maximum likelihood (RAxML) tree (100 bootstraps) of Telonema. Branches with bootstrap support < 20% were multifurcated, probe targets are marked by different colors. Asterisks indicate sequences not targeted by the probes, # indicates sequence that is too short to be checked for the target region.

Additional file 6:

Microphotographs displaying: a different lineages of protists hybridized with CARD-FISH probes designed for this study (Kat2-651, Telo-1250 and NC10-1290), cell hybridized with Kat-1452 shown for comparison; b different lifestyles observed for Perkinsozoa hybridized with Perkin01 (upper row free-living, lower row protist-associated). The scale bar applies for all images. Microphotographs were produced using Zeiss Imager Z2, Carl Zeiss, Oberkochen, DE equipped with a Colibri LED system and the following filter sets: DAPI 49 (Excitation 365; Beamsplitter TFT 395; Emission BP 445/50), fluorescein 38 HE (Excitation BP 470/40; Beamsplitter TFT 495; Emission BP 525/50).

Additional file 7:

Total grazing by protists in the epilimnion.

Additional file 8:

Table of all eukaryotic and prokaryotic ASVs. Read numbers of ASVs in all samples of rarefied datasets.

Additional file 9:

Protistan community composition at three depths of Římov reservoir during the study. The gaps indicate missing samples. The groups are resolved at phylum to class level, with the exception of Supergroup Excavata, which was dominated by kinetoplastea.

Additional file 10:

Relative abundances of particular flagellate groups in three depths of Římov reservoir obtained with CARD-FISH analysis. Left to right from top- cryptophytes targeted with Crypto B probe, CRY1 lineage of cryptophytes targeted with Cry1-652 probe, katablepharids targeted with Kat-1452 probe, katablepharid clade 2 targeted with Kat2-651 probe, kinetoplastids targeted with Kin516 probe, Perkinsozoa clade 1 targeted with Perkin01 probe, Telonemids targeted with Telo-1250 probe, and Cercozoa novel clade 10 targeted with NC10-1290 probe.

Additional file 11:

Network analysis based on the most abundant ASVs from protistan and prokaryotic communities. a Network: upper cluster represents the community dominating in the epilimnion, lower cluster represents the community dominating in the hypolimnion. Prokaryotic nodes are displayed as circles, protistan nodes as diamonds. b The main modules detected in the network. The arrows indicate directions of temporal shifts between modules. Differentiation between modules H 5 and H+M 6 is based on spatial parameters as members of module 6 were better resented in the metalimnion communities. Prokaryotic and protistan nodes are organized into modules and listed below, accompanied by heatmaps based on Z scores calculated for each module. Samples are grouped according to water column layers, with the time course depicted from left to right.

Additional file 12:

Schematic figure of food web in Římov reservoir during the studied spring period. Arrows indicate the direction of carbon flow. DOM Dissolved organic matter, POM Particulate organic matter, HNF Heterotrophic nanoflagellates.

Rights and permissions

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/ . The Creative Commons Public Domain Dedication waiver ( http://creativecommons.org/publicdomain/zero/1.0/ ) applies to the data made available in this article, unless otherwise stated in a credit line to the data.

Reprints and permissions

About this article

Cite this article.

Mukherjee, I., Grujčić, V., Salcher, M.M. et al. Integrating depth-dependent protist dynamics and microbial interactions in spring succession of a freshwater reservoir. Environmental Microbiome 19 , 31 (2024). https://doi.org/10.1186/s40793-024-00574-5

Download citation

Received : 22 December 2023

Accepted : 30 April 2024

Published : 08 May 2024

DOI : https://doi.org/10.1186/s40793-024-00574-5

Share this article

Anyone you share the following link with will be able to read this content:

Sorry, a shareable link is not currently available for this article.

Provided by the Springer Nature SharedIt content-sharing initiative

• 18S and 16S amplicon sequencing
• Microbial food webs
• Metalimnion
• Hypolimnion
• Spring succession

Environmental Microbiome

ISSN: 2524-6372

• Submission enquiries: [email protected]

Collage of wetland-dependent species

• Thumbnail (130 x 130) 42.0K
• Medium (650 x 650) 812.6K
• Large (1300 x 1300) 2.8M
• Extra Large (2400 x 2400) 8.4M
• Original (2400 x 2400) 8.4M

You are exiting the U.S. Fish and Wildlife Service website

You are being directed to

We do not guarantee that the websites we link to comply with Section 508 (Accessibility Requirements) of the Rehabilitation Act. Links also do not constitute endorsement, recommendation, or favoring by the U.S. Fish and Wildlife Service.