In Defense of the Hologenome

In a perspective article published in Zoology, we recently wrote:

"The hologenome concept of evolution specifies that the animal's genome, mitochondria and microbiome are an aggregate of genes that together form a unit of natural selection (Zilber-Rosenberg and Rosenberg, 2008). The evidence motivating this concept spans the essential roles of the microbiome in eukaryotic fitness (McFall-Ngai et al., 2013), including digestion, immunity, olfaction, organ and neuronal development, etc. However, the hologenome concept is controversial because many biologists view the microbiome as extrinsic to the host animal and therefore unable to co-evolve sensu stricto with the host genome."

As an illustration of the controversy, I am posting an email dialogue that we recently had with a prominent expert in evolutionary biology and a postdoc on our paper on the hologenomic basis of speciation. The discourse below is meant to highlight the points and counter-points of the dialogue and perhaps convert a few skeptics.  I am blogging it because I believe it will be a far more effective way of advancing this discourse into the community, rather than just keeping it between a few scientists with limited benefit. 

And for more information, I posted the video from a google+ hangout on the hologenome below that was comprised of several evolutionary biologists and microbial ecologists (link to blog post). After we got the kinks worked out for the video chat, we had a productive one hour chat. 

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Dear XXX and YYY,

Thanks again for getting in touch with your commentary.  We  appreciate the opportunity to address your concerns, and we pledge to make this process productive so that we can all learn something in the discourse. You’ve listed a number of issues in this short commentary, but we think that we can demarcate them into four discrete areas to discuss. Do correct us if there’s anything critical that you feel that we’ve missed. 

Rob and I can start by saying that a short Science paper doesn’t do much justice for convincing the community in one shot. Thus, we expect  and welcome this discussion;  we have encouraged such in our preceding blog posts, google hangout discussions with other scientists (skeptics and supporters alike), and our Zoology article.  And there are more experiments to be published, of course. We also think that posting this discourse on our blogs (either anonymously or not) would be helpful so that the broader community can see the pros and cons of the arguments.

1. " The claim that coadapted gut bacteria cause hybrid lethality and speciation in Nasonia requires that bacterial gut assemblages of Nasonia recapitulate their hosts’ phylogeny, are species-specific, and are coadapted to their hosts in the sense that “foreign” microbiota lead to lower fitness."

There are three issues that you question in the above statements. First, the Nasonia gut microbiome recapitulates the hosts’ phylogeny. We demonstrated this pattern in our Evolution paper, this article, and a third set of experiments that we are currently working on.  So we think your commentary here stems from a misinterpretation of what phylosymbiosis is, rather than its lack of evidence. Here’s how we view it operationally. Phylosymbiosis is simply the reconstruction of the host’s phylogeny with a UniFrac inference method used to compare microbial community relationships. Like phylogenies, UniFrac trees are statistical inferences based on the weighted (Fig 2B) and unweighted abundances (Fig 2C) of OTUs. Remarkably, both the weighted and unweighted Unifrac trees are in complete agreement with a phylosymbiotic microbiome.  We suspect you are laser focused on the pie charts, which create the illusion that G and V have very similar communities based on the single, dominant Providencia genus. The individual OTUs are collapsed and simplified in the pie charts into a broader genus category. If we were to reconstruct the tree based on one OTU, like Providencia, you might be correct; however, these widely-accepted UniFrac trees developed by the microbiome field are reconstructed based on all OTUs in the microbial communities. Take for example the simple comparison that the younger species, G and L, share 41% of the OTUs in their communities while the more divergent species, G and V, share  24% of the OTUs in their communities. 

Your second claim is that the Nasonia gut microbiome is not species-specific.  In the same light as above, any one OTU can be present in all of the species but the metric that we use here is the total microbial community. It is species-specific because (i) there are a number of OTUs (besides Providencia) that are specific to each Nasonia species and (ii) the divergent Nasonia genomes select upon the microbiome diversity that can inhabit them. Because we have placed all of the Nasonia in our studies under identical environmental conditions, the variation we see in species’ microbiomes is explainable by gene-microbe interactions. 

Finally, the third claim that foreign bacteria should cause hybrid lethality is not a requirement at all. In fact “foreign” is an unusual term to describe this process. What matters is the ability of the host genome to recognize and thus epistatically interact with the microbiome. So, an OTU could be foreign in one generation, but still be part of the recognizable microbiota in subsequent generations. Perhaps a simple way of looking at this is from a genetic perspective. Like a beneficial gene within species that breaks down in hybrids, we expect that beneficial bacteria or those closely related to the native microbiome in Nasonia should cause lethality. In contrast, an unrecognizable “foreign” bacteria should not cause lethality because it is not part of the of  recognizable microbial community and thus epistasis within species that breaks down in hybrids. Ongoing experiments in our lab currently support these predictions. 

In conclusion, we urge caution because the claim that "none of these are established" or later that "these data...do not meet these criteria" (and throughout the top paragraph of page 3) dismisses widely accepted means of analysis and interpretation for microbial communities. 

2. "“Phylosymbiosis” connotes relative stability of the gut bacterial communities within species."....Brucker and Bordenstein (1) provide no evidence that the gut bacteria of Nasonia are sufficiently constant and species-specific to contribute to speciation."

Some clarification of the phylosymbiosis model is required in our response here because it seems to us that you have extended the term to something that we do not adhere to. As defined in the Science paper, it is “a term...to denote the microbial community relationships that recapitulate the phylogeny of their host”. Phylosymbiosis is a pattern that is not necessarily derived from a continuous process of stable OTUs each generation. Instead, phylosymbiosis derives from the host genome interacting with the environmental microbiome to collect an assemblage of (similar) microbes whose community relationships parallel the evolution of the host genome each time they are measured. We come to this conclusion because the null hypothesis for a totally random or diet-centric collection of microbes in a host is that phylosymbiosis would not be evident in the microbiome data.

Like phylogenomics, phylosymbiosis is the total microbiome metric of the host’s evolutionary        relationships. As we outlined in our 2012 TREE article, phylosymbiosis is a pattern that emerges under controlled conditions and will be subject to change with environmental dynamics.  Surely, bacterial OTUs can vary with environmental conditions and that variation is in fact one route to speciation by symbiosis that we raised in our TREE article in 2012. Different diets can unquestionably drive different microbiomes. The variation in microbiomes that were observed in our publications and ongoing research stems from variations in fly host microbes that the Nasonia are reared upon and the method of observing the microbiome (Evolution paper was cloning and sequencing, Science paper was Next-Gen). Furthermore, when we observe the bacterial sequences across our laboratory experiments and even in published genome sequences of Nasonia, we observe a conserved set of OTUs within the microbiome that spans nearly a decade of lab rearing; specifically Proteus, Providencia, and Acinetobacter.

In this current work and that of our 2012 Evolution paper, we independently demonstrate that when wasps are raised under the same environmental conditions, phylosymbiosis emerges.  But the phylosymbiotic microbiome itself does not have to be identical between environments, just as gene expression or epigenetics does not have to be identical between generations or environments. The remarkable thing in Nasonia is precisely that the UniFrac tree analysis finds phylosymbiosis independently in two published studies at different times and one more unpublished study in our lab. Thus, the phylosymbiosis pattern itself is stable, but the specific OTUs present in Nasonia do not have to be. We see this clarification not as a problem  but as a reflection of the natural way by which the host genome interacts within the microbiome.

We also are the first to admit that like any laboratory study of speciation, the processes observed in the laboratory are isolated observations whose importance is not whether they occur in nature, but that nature has the potential to operate and drive speciation events with them.

3. "Under their hypothesis, restoring the hybrid community to that of the parents (both of which have similar gut bacteria) should reduce lethality.  However, when the authors perform this experiment, they do not find significantly increased hybrid viability (Figure S1B; crosses g/v and v/g)"

We understand your position here. It would seemingly make sense that the resident microbial community from one of the parents could rescue hybrid inviability because they now have a normal microbiome.  But let's look at the data and the reasoning behind the experiments. First, Providencia is only one of the many species that naturally occur in Nasonia parentals as well as conventionally-reared hybrids. Thus, the mono-inoculation experiment here is not testing the specific prediction that a full, multi-faceted microbiome can rescue the hybrids. We previously tried this experiment by enriching for bacterial communities from parentals, but ran into several confounding problems of host material being present with the enriched microbial communities.  

Second, we're now skeptical that this prediction will actually hold water. Consider the gene-gene analogy from a speciation genetic perspective. You can only rescue hybrid mortality when you knock-out the epistatic genes, which is what we analagously did with the microbiome. But placing the "correct" gene in a hybrid background would only reinstate the epistasis that underscores the hybrid incompatibility. Thus placing the correct microbe, either specifically or a close relative, should recapitulate the epistatic interaction and mortality. Under this reasoning, your claim that a normal microbiome should rescue lethality is not supported either in our hologenomic view or a nuclear-centric view of hybrid incompatibilities 

4. " When the authors perform this experiment, they do not find a significant difference between Nasonia inoculated with their normal bacterial community (Providencia) or with a completely novel bacteria (Enterococcus)"

Some background on these two bacteria might be helpful here. Enterococcus is actually present in both hybrids and non-hybrids (we state that in the third page of the main text, also see our supplemental tables) and thus we expected it to kill because the host genome would recognize it. Ongoing experiments with very distantly related bacterial species that Nasonia may not be able to recognize do not seem to kill the hybrids, which is what we would predict. Thus, we assert that your reasoning here is backwards.

In summary, a major part of our effort is to convince evolutionary biologists that the microbiome arises from an element of host control and under host control they are part of an extended phenotype with genomes themselves. Microbial ecologists have weighed this evidence to be quite self-evident in the past few years, though not every study bears out an element of host control. Oddly enough, both fields accept the intimate interactions between the host genome and ancient endosymbionts / organelles; but clearly they emerged from predecessors that were more free-living and “farmed” from the environment until they became obligate and germline transmitted. What we are seeing here is a continuum of symbiotic interactions.

Guest Blog Post On The Story Behind A New Phylosymbiosis Paper

Below is a guest blog post by Soeren Franzenburg (Twitter: @naturfokussiert) who recently migrated from Kiel/Germany to do a postdoc at Cornell University on animal-microbe symbioses in Angela Douglas' laboratory. When Soeren's paper came out, my jaw dropped to the table. Without hesitation, it is one of the best papers that I've read all year. It is a major contribution to an emerging theme in biology that the host genome, and specifically the immune system, "farm" the microbiome from the environment in specific ways. Hat tip to Greg Hurst @TheLadybirdman for the analogy of hosts farming the microbes from the heterogenous environment into their bodies.

If you're interested in microbial ecology, microbiomes, evolution, immunity, the hologenome concept, speciation, phylosymbiosis and more, this story is one you want to keep up on.  I have also gotten to know one of the senior authors on the paper, Thomas Bosch, over the last few months. It seems that conferences in evolutionary biology, the microbiome, or symbiosis might want to capture this emerging theme with a symposia on the genome-microbiome interface.

Finally, if anyone wants to write a guest blog post on the story behind their recent publication, do not hesitate to get in touch and we'll set it up.

The story behind: “Distinct antimicrobial peptide expression determines host species-specific bacterial associations”

Seth asked me, if I would like to write a guest blog post about my paper “Distinct antimicrobial peptide expression determines host species-specific bacterial associations” which was recently published in PNAS. Since the job of a scientist should not only be to perform research, but also to communicate science to a broader audience, I gladly accepted this challenge.

Picture of Hydra (Source: Wikipedia)

In this publication, we investigated the mechanisms underlying the assembly of bacterial communities associated with seven closely related species of the freshwater cnidarian Hydra.

It became increasingly evident in the past years that bacterial associates are essential for the host’s health, as supported by severe fitness disadvantages in axenic or certain gnotobiotic animals. As a consequence it would be advantageous for a host to actively select for suitable bacterial associates and maintain a defined host-bacterial homeostasis. This ability should be genetically fixed in the host’s genome. If the host’s genotype matters for microbiota composition, closer related host species should be colonized by more similar bacterial communities compared to distantly related species. This recapitulation of host phylogeny by microbiota compositions was recently termed a phylosymbiotic relationship.

However, besides host phylogeny, diet was shown to be one major determinants of microbiota composition in the wild and distantly related species with similar lifestyles can show convergence in their microbial communities. That is why observing phylosymbiotic host-microbe relationships and investigating the underlying host-mechanisms relies on studies with closely-related host-organisms and very well controlled environmental conditions, including diet. These issues were also discussed recently in a scientific Google⁺ Hangout, organized by Seth.

In our recent paper, we were able to show phylosymbiotic host-microbe relationships in seven species of the freshwater cnidarian Hydra. The studied species were separately reared in simple, water containing plastic-dished for up to 30 years under identical environmental conditions, including standardized diet. Nevertheless, their microbial composition differed substantially and revealed a highly phylosymbiotic pattern. Impressively, after three decades of identical cultivation, each species still maintained its specific, bacterial fingerprint.

Subsequently, we were able to pinpoint a group of species-specific antimicrobial peptides, called arminins, as critical determinants of the microbiota assembly. When axenic Hydra polyps were inoculated with bacterial communities characteristic for different Hydra species, the recipient host selected for bacterial taxa resembling its native microbiota, just like in an elegant reciprocal microbiota transfer experiments between zebrafish and mice conducted by Rawls et al. (2006). However, arminin loss-of-function polyps significantly lost this selective potential and ended up with untypical bacterial communities. When inoculated with their native bacterial colonizers, control- and arminin deficient Hydra were colonized equally, indicating that the species-specific microbiota is partially resistant to the antimicrobial peptides expressed by its host. Thus, the host’s immune system indeed plays a major role in selecting the bacterial associates. Since Hydra is a phylogenetically old organism, these observations are likely to be valid for more complex animals as well.

The paper ends with a short perspective on the role of symbiosis in speciation. Several studies have shown that the microbiota can act as a “metabolic organ”, allowing the host to feed on otherwise insufficient diet. For example, the symbiotic bacterium Buchnera provides its aphid host with essential amino acids, allowing the utilization of nutrient poor phloem sap as food source. Generally, changing the microbial partners can confer new traits to the host much faster than evolution of the host genome alone. These traits might open a new ecological niche and thus accelerate sympatric speciation.

The crucial question is: How do animals change their microbial partners? Our publication indicates that changes in fast evolving antimicrobial peptides are sufficient to drastically alter host-associated bacterial communities. Coupling fast evolving genes with the adaptive potential of changed bacterial partners could be a potential promoter for speciation.

Congrats to Soren et al for such a wonderful piece of symbiosis work.

Related blog posts:

1. The science of the superorganism: Host-microbiota specificity (November 28, 2012)

2. The large immune effect - vlog 2 (December 18, 2012)

3. Is the microbiota species specific? Cell paper weighs in (July 23, 2012)

Got mom's bacteria? Take 2. Easy-reading summary in DoubleXScience

The universality of maternal microbial transmission (i.e., babies getting not just their genes and mitochondria from their mom, but their helfpul bacteria too) is steadily gaining attention. I previously blogged about this topic and our article (link). Here's an excellent, lay summary (source) of the routes that bacteria take to get transmitted to human babies, with a few quotes from me.

The secret ingredient in breast milk

Posted on September 26, 2013 by DXS Contributor

The surprising hitchhikers that we pass to our babies

By Beth Skwarecki

“Maternity.” A study by Henrique Bernardelli (1880-1890).

There’s no question that human milk is a biological powerhouse. In addition to providing excellent nutrition for a baby, this maternally produced sustenance includes antibodies, immune cells, natural antibiotics, and antivirals.

It also contains “prebiotic” food for the bacteria and other tiny organisms — collectively known as microbes — that live in a baby’s gut, nourishing them and keeping the colonies healthy. Maybe you’ve already heard about that. But did you know that breast milk also contains the microbes themselves? Wait until you learn where they come from.

Scientists are finding microbes everywhere — and that’s because they are everywhere. TheHuman Microbiome Project, a federally funded project aimed at identifying all of the bugs that call the human body home, sampled 18 body sites and found truly humbling numbers: We harbor 10,000 microbial species that collectively express 8 million genes. Just to give some perspective, humans express just over 20,000 genes.

And while gut microbes get the lion’s share of the headlines (after all, most of our microbial buddies live in the intestines) the microbes in the other parts of the body do some amazing things too. These tiny friends swarm in the human mouth, nose, vagina, and armpit — and the exact makeup of our microbes differs from person to person.

Actually, “everywhere” doesn’t mean only the surfaces of our bodies. We used to think that healthy urine was bacteria free, but that’s not necessarily true. Microbes have also been found in brain tissue. We used to think healthy lungs were sterile, too, but nope. Until about 2008, the accepted dogma was that a fetus, floating tranquilly inside its mother’s uterus, was in a microbe-free, pure state. And we thought mother’s milk, the first thing many babies consume, was also sterile, that it picked up any bacteria from surfaces during transfer from mother to child.

We thought wrong. Instead of sterility being the rule, passing down germs from mother to child may be an essential part of reproduction.

“Babies are born without a fully developed intestinal mucosa, and need interaction with bacteria to basically jump start their immune system,” says Lisa Funkhouser, who co-authored a paper with Seth Bordenstein last month on the many ways mothers across the animal kingdom transmit microbes to offspring.

What’s most fascinating about the microbes in breast milk and those that a fetus harbors before birth is where they originate. The idea is still in its, um, infancy, but evidence is accumulating that cells in the bloodstream pick up microbes from the intestine and transport them to destinations in milk-producing breast tissue … and across the placenta to the developing fetus.

The evidence for hitchhiking bacteria

This idea first appeared in the work of a group of Swiss and French scientists who put a few facts together: First, babies’ guts contain the “good” species of bacteria even though the infant immune system isn’t really up to the task of sorting out good from bad. Second, bacteria occur even in human milk that’s been collected with a sterilized pump and an antiseptic on the mother’s skin. And third, white blood cells readily travel from the gut and lungs into breast milk, which suggested that microbes could be delivered by hitching a ride on these cells.

So these researchers put this idea to the test. They found matching strains of bacteria in maternal feces, blood, and aseptically collected milk. Switching to mice for more invasive tests, they found that 70% of pregnant animals had bacteria in their lymph nodes, versus just 10% of non-pregnant mice. After birth, bacteria could be found in their milk ducts, and the patches of immune-system tissue in their intestines (one hypothesized boarding station for gut microbes) were enlarged.

This idea of an “enteromammary pathway” connecting the gut and breast is not without precedent. In 1979, researchers identified a similar pathway that brought antibodies from the gut to breast tissue. But antibodies are small particles compared to bacteria, and the concept that microbes might also use such a pathway came much later.

The cells making up our intestines are sewn tightly to each other in what are called tight junctions. In 2001, researchers found that white blood cells can reach an “arm” through the junction and take samples of the bacteria in our partially digested food slurry. A perspective article cheekily referred to this as “Periscope up!”, and this behavior gained a reputation as the immune system’s regular patrol for pathogens, including taking a prisoner to pick apart.

But when the white blood cells capture bacteria, bacterial death isn’t always the goal. The bacteria in breast milk are alive. A study published earlier this month filled in some of the puzzle of how they evade death. The researchers, led by microbiologist Christophe Lacroix, used a constellation of techniques to show that identical strains of bacteria are found in the maternal gut, maternal milk, and the infant gut and that these bacteria are viable in all three places.

A weakness in most new studies of our microbiome is that they rely on DNA sequencing technology that can’t tell the difference between living and dead microbes, Lacroix says. So the best evidence for live bacteria being carried through the blood comes from a combination of sequencing and culture-based methods.

Think of culture as smearing some bacteria on a Petri dish, as microbiologists have been doing since Fanny Hesse taught her husband to make agar gel in 1882. Now consider the many differences between that dish and the inside of the human intestine. A few microbes, like E. coli, grow well in both environments, but plenty of bacteria (not to mention fungi, archaea, and viruses) have been identified only from scraps of their DNA. Some have called these bacteria unculturable, but Lacroix just sees them as more of a challenge.

Lacroix’s study demonstrates how tough it is to figure out just which microbes are riding around in the body. It was hard to find matching bacteria in all three samples, possibly because of technical difficulties. Because trillions of bacteria live in the gut, any sample is likely to underemphasize a lot of lower-abundance species. In fact, the investigators got a triple match in only one of their seven mother–baby pairs, although several pairs showed matches in two out of three. Still, the results are unlikely to be a fluke. Since contamination from skin or surfaces could have thrown them off, Lacroix’s team took the clever step of focusing on bacteria that can’t grow on surfaces exposed to air.

Lacroix concludes that evidence for bacteria hitching rides from the gut to milk tissue is “quite strong,” but what would really seal the deal would be to find living bacteria in blood samples, basically catching the bacterial passengers on their commute.

Microbes in the womb?

Microbes in breast milk are only half the story. Evidence also suggests that mothers pass microbes to fetuses in the womb, as well. For one thing, live bacteria have been found in the umbilical cord blood of healthy babies. Bacteria can also be found in meconium, the clinical name for baby’s first poop. Meconium consists of the leftovers of what the fetus accumulated while in the womb: dead cells, mucus, bile, and other appetizing ingredients which would include bacterial leftovers if bacteria are present in the womb. Indeed, in a particularly exciting mouse study, researchers fed tagged bacteria to pregnant females. After delivering the pups by sterile c-section, the investigators found the tagged bacteria in the pups’ meconium.

Obviously, that kind of research can’t be done quite that way in humans, “but it really raises the bar about how much further we have to delve into these questions,” says Bordenstein. “We’re at a tipping point of our knowledge, and the current paradigms will probably change in the next few years. There’s a surface of bubbling interest in the idea the womb is not sterile.”

That’s great, but … why?

“In the breast milk you have quite a broad ecosystem. So why would there be such an ecosystem if it has no real features for delivering some sort of property to the baby?” Lacroix asks. Based on ongoing experiments, he thinks that a special group of microbes is important for setting up a healthy ecosystem in the infant gut. Some of their products fuel our own cells while others support yet more microbes. After all, an ecosystem has many layers, and Lacroix believes these tiers establish themselves during the first six months of life.

But are human cells deliberately capturing these microbes (and perhaps selecting specific species) in the “taking prisoner” approach of white blood cells reaching through intestinal gaps, or are the microbes hijackers? Clues suggest that animals do the selecting. Bordenstein has tested species of insects that, despite the same diet, develop predictably different microbial ecosystems. “There’s got to be a host control of the microbiome at some level,” he says, probably directed by the immune system.

Of course, babies fill out their microbiomes through other means, too. We know that infants born vaginally have a different microbe profile from those born by caesarian section, and that formula-fed babies have a different profile from those who are breastfed. If these differences are relevant to infant and child health, it might be possible to use microbes as a preventative therapy for kids at risk. Many studies on breast milk bacteria (including Lacroix’s) are funded by Nestle, which is particularly interested in developing a better probiotic-enhanced formula. There are probiotic formulas on the market already, but they have shown limited and mixed results on infant health. Enhancing the number of species or their mode of delivery could help, for example, premature infants who can’t breastfeed.

Time will tell. “We are in this stage of perpetual discovery and exploration,” Bordenstein says. “The microbiome is at a stage where we were a hundred years ago with genetics.”

Beth Skwarecki is a science writer based in Pittsburgh, PA. She previously wrote for Double X Science about the seasonality of human birth. Find her on twitter @BethSkw.

This entry was posted in Health and tagged Beth Skwarecki, breast milk, breastfeeding, gut bacteria,maternal transfer, microbiome, microbiota by DXS Contributor. Bookmark the permalink.