By Tobin Hammer, Jon Sanders, & Noah Fierer
May 18th, 2018
NOTE: A more complete discussion of these ideas is now available in the corresponding publication (available here).
Have you-like us-ever written a paper or grant proposal with a statement along the lines of “All animals host microbial symbionts that play critical roles in many aspects of host ecology, behavior, and development”? If so, this blog post is for you. We argue that this statement is not a fact, but a hypothesis, and one that is not actually based on firm evidence. We suggest that this “universal microbiome paradigm” should be tested through careful analyses, using methods that go beyond conventional 16S rRNA gene amplicon surveys, as well as sampling of a more diverse array of potential host taxa.
It is widely assumed that all animals are hosts to resident microbiomes, and it is why many animals are considered dirty or disease-ridden, especially rodents insects, and larger pests too. You will hear of many sites like https://www.pestcontrolexperts.com/local/michigan/royal-oak/ which people will seek out to remove these creatures from close proximity. However it is not all of what it seems on the surface, and not everything you read is true. You see there are good and bad things about the resident’s microbes within many animals, and actually, it is quite an interesting idea. As just one example, consider the first principle of hologenome theory, as formulated in a recent review: “All animals and plants harbor abundant and diverse microbiota and are thus considered holobionts” (1). However, this statement is simply not well-supported by data. We don’t yet have robust measurements of microbial abundances for a diverse enough suite of macrobes to even get close to approximating the diversity of all animals and plants. For example, there are more species of jumping spiders than mammals, yet when was the last time you read a paper about jumping spider microbiomes? Even a more conservative statement like “most animals harbor abundant microbiota…” must reckon with the enormous abundance and diversity of groups like caterpillars and ants, many (if not most) of which have few resident microbes (2, 3).
We already have examples of animal taxa that lack a resident microbiome in certain tissues (e.g., guts of aphids (4)). Some animals lack a resident microbiome in certain life stages (e.g., larvae of honeybees (5)). Likewise, and in contrast to the implications of some claims (see this quote [i] from ref. 6), not all microbial taxa are abundant and active in a given animal. For example, fungi are only transiently present in the human gut (7). We already know that central biological functions, like digestion, can be mediated by microbes in some animals, but not in others. Is it then that much of a stretch for there to be animal taxa that simply do not harbor a resident, functional microbiome in any tissues or life stages? There is already evidence that such animals are common if nature, if not yet widely reported or recognized as such in the microbiome research community (2, 3, 8, 9).
Because microbial contaminants, transients, and parasites/pathogens are ubiquitous, any animal could be said to “have a microbiome”. Therefore, it may be more productive to instead ask, for said animal: how strongly does it depend on microbes, and which aspects of its biology (if any) are influenced by microbial activities? Although here we are emphasizing the extreme hypothetical case of an animal fully independent of microbes, the degree of microbial dependence-as might, in an ideal world, be measured by fitness reduction upon microbiome removal under a range of conditions-will vary as a continuum across animal taxa (e.g. (10)).
Below we highlight a few reasons why we think the paradigm that all animals have abundant and necessary microbiomes remains so strongly entrenched in the microbiome research community:
Contaminants. Prior to sequencing, we typically dunk our samples in a series of reagents that contain microbial DNA. Thus, you’ll always be able to sequence something in any tissue of any animal you choose. The lower the microbial biomass in your sample, the more easily it will be contaminated by Propionibacterium on your skin or Acinetobacter in your DNA extraction kit (11). This wouldn’t be a problem if all animals hosted abundant microbes, but they don’t. Often, these contaminant taxa are common in water, soil, and plants, and so they can masquerade as real symbionts, and it’s easy to come up with stories about what they might do for the animal of interest.
Transient microbes. Let’s say that the DNA you sequenced was derived from live microbes that were actually present in the sample, and not from your skin bacteria, or from lab reagents. The question then becomes: were these microbes transiently present (e.g., ingested with food and then excreted), or resident? By “resident” microbes, we mean those populations that are reasonably stable [ii] and capable of growing on or in the host (i.e., symbionts). In some cases, like the bean bug Riptortus pedestris, ingested microbes may colonize at high densities and provide beneficial functions (12), but in other cases they may simply pass through inactive, or be digested themselves.
Parasites and pathogens. Even a thriving assemblage of symbionts, consisting of taxa common among host individuals, isn’t necessarily beneficial. To use an example from another host-symbiont system, take plants and their folivorous caterpillars. In Guanacaste, Costa Rica, over 70% of caterpillars recorded from the tree Enterolobium cyclocarpum belong to a single species, Coenipeta bibitrix (data from http://janzen.sas.upenn.edu/). But regardless of its core status, C. bibitrix is a bona fide parasite, not a mutualist. Likewise, in animal microbiome surveys, core microbes could simply be common parasites, or microbes commonly ingested with food or water (3, 7). This is why observational data can only take you so far-it is often necessary to conduct experiments that explicitly test the effect of a given microbe, or whole microbiome, on host fitness (keeping in mind that these effects can be context-dependent).
Literature bias. Researchers naturally gravitate to model systems, and in general, to hosts that have interesting symbioses with microbes. So right off the bat, animals lacking resident microbiomes will be neglected. And when they are studied, those data are likely to get shelved – maybe the PCRs “didn’t work” or the data looked contaminated – and never published. For example, several years ago one of us (Noah) worked on a project with other collaborators investigating the microbiome of Timema stick insects. Even after weeks of lab work and protocol tweaking, the samples failed to yield any usable microbial data-an outcome that now makes sense in light of stick insects’ lack of resident gut microbiome (8). The project was shelved and ultimately forgotten because no one wants to write or read a paper with unusable data. We were working under the assumption that all animals have abundant microbiomes, so clearly we must just have been doing something wrong. There are likely many other studies that fell into this dustbin of history. The outcome of all this is that the published literature gives an unrepresentative view of the extent of abundant and functional microbiomes across animals.
How, then, to recognize whether an animal has, and needs, resident microbes? Here we list some suggestions, with an eye to researchers studying non-model systems or animal taxa that have not yet had their microbiomes characterized:
- View microbiome data skeptically: your animal may not work the same way as aphids, humans, cows, or bobtail squid. Be open to the diversity of ways in which animals interact with microbes. And please publish “negative” results!
- Take precautions to reduce potential sources of contamination, like sterile sampling techniques and sequencing of your negative controls. Most importantly, keep a copy of Salter and colleagues’ paper (11) on your desk at all times; this alone would prevent a lot of mistakes from entering the literature (such as the human placental microbiome (13)).
- Measure the absolute abundance of microbes, e.g., with qPCR, microscopy, and/or culturing (if most of the microbes are culturable under your conditions); see (2, 3) for two recent examples. This does not have to add that much extra time and expense, yet it is strangely uncommon among host-microbiome surveys. Comparisons of microbial composition and total counts between the environment (e.g., food) and the animal (e.g., guts) are especially useful in distinguishing between transient and resident microbes (3, 7, 14).
- Do experiments to test for microbial effects on host fitness – an abundant core symbiont may actually be a garden-variety pathogen. Keep in mind that microbial function in vitro or in silico may not apply in vivo: for example, a cellulose-degrading or nitrogen-fixing microbe could be isolated from an animal (or its genes detected in a metagenome), but it may not be performing those functions-or even doing anything at all-in the animal itself.
The concept of microbial symbionts as a universal force mediating the ecology and evolution of all larger organisms-and backlash against it-is not new. Over 60 years ago, Paul Buchner, a principal founder of symbiosis research, complained that “again and again there have been authors who insist that endosymbiosis is an elementary principle of all organisms…” (p. 69, (15)). Following in the tradition of Buchner, we’d like to encourage the microbiome research community to question this paradigm and to start a productive discussion about how we define and study microbiomes across the diversity of macrobes. Yes, there have been loads of fascinating, surprising, ground-breaking discoveries made on the roles of microbiomes in animal biology. And these findings have led to an awareness that microbiomes *could* be involved in virtually any biological process of virtually any animal. But let’s hit the brakes a bit so we don’t cross over into the tacit assumption that microbial symbionts actually *are* necessary to all of animal life.
[i] “Today we realize that any multicellular organism must be considered a metaorganism comprising the macroscopic host and its synergistic interdependence with bacteria, archaea, fungi, and numerous other microbial and eukaryotic species including algal symbionts.” (6)
[ii] In more precise terms, a stably associated microbial population’s replication rate inside the host equals or exceeds the rate of loss due to cell death or excretion. We also note that macrobial community ecologists have long placed an important distinction on transient versus resident (or “persistent”, “breeding”, “self-maintaining”, etc.) populations (16).
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