Microbiome Science and Medicine
Research Article • DOI: 10.2478/micsm-2013-0001 • MICSM • 2013 • 1–9
Invertebrate systems for hypothesis-driven microbiome
research
Abstract
A number of novel, invertebrate systems have emerged as excellent
models for the study of microbiomes. Due to their small size, evolutionary
diversity, ease of culture, and – in many cases – relatively simple gut
communities, invertebrates of many different orders can be tools to drive
hypothesis-driven microbiome research. In this review we highlight several
host systems amenable to microbiota analyses and specific questions
that can be easily addressed in those systems. These questions address
functional equivalence across similar habitats, host-specificity and
coevolution of host-microbe interactions, and acquisition and transmission
dynamics of host-associated communities. We propose that host systems
be chosen based on the question of interest, and that insect systems are
excellent tools for the vast behavioral, ecological, and genetic diversity that
allows them to address a variety of these questions.
Irene L.G. Newton*,
Kathy B. Sheehan,
Fredrick J. Lee,
Melissa A. Horton,
Randy D. Hicks
Department of Biology,
Indiana University,
1001 E 3rd Street, Bloomington,
IN 47405, USA
Keywords
Nasonia • Apis • Bombus • Porifera • Atta • Drosophila • Symbiosis • Mutualism
• Metabolism
Received 23 December 2012
Accepted 11 March 2013
© Versita Sp. z o.o.
Introduction:
Since the discovery that rRNA genes could be isolated from
the environment, sequenced, and compared phylogenetically in
order to identify previously uncultured organisms [1], microbial
ecologists have been probing the composition and function
of environmental communities. Two basic questions drive
research in microbial ecology: Who is there? and What are they
doing? In the years since Pace’s seminal work, many different
methods have been developed to address these questions.
Because the vast majority of microbes are not yet culturable,
researchers have utilized sequencing, in situ hybridizations and
functional genomics to identify the composition and characterize
the function of dynamic and complex microbial communities
[2-5]. Over the years, several culture-independent experimental
approaches have been employed with great success in a
number of different habitats where microbes are recalcitrant to
conventional methods including: acid mine drainage [6], the deep
subsurface [7] and the open ocean [8]. Extended research efforts
have seemingly culminated in the massive attempt by the Earth
Microbiome Project to characterize the diversity and function of
microbial communities in different biomes on the Earth [9,10].
No organism lives in isolation – indeed, we are all holobionts
[11,12] – and many researchers had been investigating hostassociated microbial communities before the launch of the
National Institute of Health’s Human Microbiome Project [13]
and the application of next-generation sequencing to 16S
rRNA gene amplicons [14,15]. However, the funding available
has arguably altered the field and direction of investigation
promoted by the Human Microbiome Project. The techniques
and tools implemented in the study of free-living, environmental
assemblages were employed to study microbes that reside within
and upon eukaryotic hosts, with an emphasis on humans and
model systems. Initial analyses were largely descriptive, utilizing
the 16S rRNA gene as a taxonomic tag to identify bacteria
present in humans [16-18]. These primary studies developed
new methods, explored limitations of existing techniques
(from sample collection and processing to bioinformatics), and
established a discovery-based foundation for hypothesis-driven
research. The idea that microbial signatures could be used as
indicators of disease or could be incorporated into personalized
medicine drove many of the studies on human microbiota
[13,19-21]. However, certain fundamental questions about hostassociated microbial communities cannot be easily answered
in humans. This difficulty arises either because of ethical or
monetary constraints, or simply due to the nature of the host and
the complexity of the system, which hamper a scientist’s ability
to perform completely controlled experiments.
In this review, we highlight systems in which researchers can
more easily address these important questions because of the
life-history of the hosts, the availability of genetic tools, and/or
the simplicity of microbial communities. We focus on alternative
invertebrate model systems in which to investigate microbiome
function and composition. Invertebrate systems have several
* E-mail: irene.newton@gmail.com
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advantages when compared to mammalian models: insects are
small and easy to rear, they are easy to manipulate and massively
sample, their genetics and environment can often be closely
controlled and monitored, and their associated communities
are generally simpler than those found in mammals. Because
a great number of microbial communities have been sampled
and characterized from a diverse group of invertebrate hosts, it
is not possible to include them all in this review. We apologize
for the unknowing exclusion of any research programs. This
review highlights only the most thoroughly investigated systems
that employ deep or next-generation sequencing (Figure 1). For
completeness, we include a current list of microbiome projects
in alternative, non-human models, organized by host phylogeny
(Table 1).
In this review, sets of different host-microbiome systems
have been organized based on research addressing four
fundamental questions: Is there functional equivalence in
microbial communities across similar habitats? Are the groups
present specific to their hosts? Do these microbes co-evolve or
differentiate with their hosts? What is the transmission strategy
for host-associated communities?
But, do we find similar microbes performing similar metabolic
tasks across different lignocellulosic communities? Research
on the microbial metabolisms involved in the digestion of plant
material has revealed both congruence and dissimilarities across
different animal systems [32-38]. The cow rumen and associated
Table 1. H
ost taxonomy, common name, and associated citations for
microbiome studies on novel systems. Only studies involving large
scale and high-throughput projects are included in the table.
Taxonomy
Animalia
Porifera
Animalia
Bilateria
Deuterostomia
Chordata
Actinopterygii
Amniota
Diapsida
Synapsida
Functional equivalence between lignocellulosic
habitats
Animalia
Bilateria
Lophotrochozoa
There are many entirely herbivorous insects -- those that rely on
a limited, plant-based diet for nutrition [22-25]. Several of these
organisms subsist on lignocellulose-heavy diets [26-28] and
could provide powerful model systems in which to understand
the complexity of functions found within lignocellulose-degrading
communities [29]. Lignocellulosic plant biomass is composed
of lignin (a phenylpropanoid polymer), cellulose (crosslinked
and hydrogen bonded glucose polymers) and hemicellulose
(various beta-(1->4)-linked polysaccharides) and is relatively
recalcitrant to enzymatic degradation [30,31]. Animals that
subsist exclusively on plant biomass must necessarily employ
a digestive strategy that effectively attacks the chemical and
physical structure of these complex polysaccharides [32].
Animalia
Bilateria
Ecdysozoa
Nematoda
Arthropoda
Arachnida
Insecta
Hymenoptera
Diptera
Siphonaptera
Blattodea
Hemiptera
Common Name
Citations
Sponge
[63,64,96-98]
Zebrafish
Guppy
[99-101]
[102]
Hoatzin
Chicken
Iguana
Python
Bat
[36,38,103-104]
[105-109]
[110]
[111]
[112]
Earthworm
[113]
Round worm
Tick
[114-116]
Honey bee
Bumble bee
Parasitic wasp
Leaf-cutter ant
Turtle ant
Fruit fly
Mosquito
Flea
Termite
Firebugs
[73,75,76]
[74]
[70]
[28,44,50]
[60,61]
[87-89]
[117-120]
[116]
[26,27,121]
[122]
Figure 1. S
chematic illustration of some of the insect systems used as models in microbiome research. Complexity of the systems varies from
a handful of bacterial species (in Drosophila melanogaster) to hundreds of bacteria (in termites). Images taken with permission from:
http://en.wikipedia.org/wiki/File:Drosophila_melanogaster_-_side_(aka).jpg;
http://en.wikipedia.org/wiki/File:Cephalotes_atratus.jpg;
http://commons.wikimedia.org/wiki/File:Bee-apis.jpg;
http://commons.wikimedia.org/wiki/File:The_stronger_of_the_two.jpg;
http://en.wikipedia.org/wiki/File:Workertermite1.jpg.
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Invertebrate systems for hypothesis-driven
microbiome research
microbiota have been extensively researched, and this habitat
is home to an extreme diversity of microbes, including a large
eukaryotic component (anaerobic protozoa and fungi [33,39-41])
performing a diverse set of functions.
Datasets produced from amplicon-based, metagenomic
and metatranscriptomic studies can be analyzed by homology
searches using several well-established, curated databases.
Among these are the Kyoto Encyclopedia of Genes and
Genomes (KEGG), which links enzymes to pathway maps and
Enzyme Commission numbers describing specific biochemical
transformations; Clusters of Orthologous Groups (COG), which
attempts to phylogenetically classify orthologous gene sets
across representative genomes; and Carbohydrate Active
Enzymes (CAZy), which is a database of homologous enzymes,
modules, and enzyme classes that all act on glycosidic bonds.
From studies utilizing these bioinformatics resources, we know
that the rumen is an extremely diverse habitat – including members
from all three domains of life – that has not been exhaustively
cataloged. However, the question remains whether this large,
diverse consortium is a signature of lignocellulosic habitats.
Perhaps the degradation of complex plant polysaccharides
requires microbes performing similar functions. In addition to
comparative work in other non-agricultural ruminant systems
[34,35,42], these questions have also been explored deeply in
several insect model systems, discussed below.
Leaf-cutter ants
Leaf-cutter ants (Hymenoptera:Formicidae) maintain an external,
fungal symbiont long presumed to assist in the digestion of plant
material foraged by worker ants (but see [43]). The ants cultivate
a fungal garden, macerating and cutting plant material to add
to the garden, meticulously removing intruding microbes, and
feeding on the fungus [44]. The garden is a complex symbiosis
between the ants, their fungal symbiont, and several protective
bacterial strains [44-49]. Metagenomic analysis of the fungal
symbiont garden revealed a diversity of microbes performing
functions similar to those of microbes found in other plantdegrading habitats, especially the rumen [28,50]. Functional
analysis of sequences from the garden using the KEGG, COG
and CAZy databases identified a high representation of enzymes
involved in sugar metabolism, B-vitamin and amino-acid
biosynthesis, and oligosaccharide degradation. Interestingly,
bacteria were found to encode >50% of the enzymes in this latter
category, with over half of the cellulases and hemicellulases,
and other glycoside hydrolases being of bacterial origin [50].
Functional similarity to rumen microbiota, however, did not
translate into an equally diverse microbial community. Ampliconbased census of the bacterial community present in the fungal
garden using 16S rRNA gene amplification identified a simpler
bacterial community than that found in the rumen. Whereas
upwards of 700 operational taxonomic units (or OTUs, estimated
based on metagenomic data [51]) were identified in the bovine
rumen, fewer than 300 OTUs (based on 97% identity) were
found in the fungal garden, with the dominant members being
γ-Proteobacteria and Firmicutes [28]. Additionally, the ability
to degrade different forms of cellulose was found in bacterial
isolates from the fungal garden [28], suggesting a more complex
syntrophic interaction than previously imagined – these bacteria
may assist the fungal symbiont in degrading the leaves collected
by their ant hosts. Indeed, because the ability of the fungal
symbiont to degrade cellulose directly has been questioned [43],
it may be that the complex community, including the bacterial
partners, is required for full enzymatic digestion of the leaf litter.
Termites
Both the so-called “higher” and “lower” termites
(Isoptera:Termitoidae) are excellent model systems in which
to analyze microbial function in plant biomass degrading
communities. Some higher termites form a symbiosis with a
basidiomycete fungus and cultivate the microbe to presumably
assist in degradation of plant forage [26]. The lower termites,
in contrast, maintain protists in their guts, where the eukaryotic
microbes are thought to assist in the mobilization of complex
plant polysaccharides [29]. Metagenomic and proteomic analysis
of the higher termite gut revealed that the bacterial members are
largely dominated by the Fibrobacteres and Spirochaetes phyla
[27]. A functional analysis using the CAZy database determined
the presence of more than 700 genes or gene modules with
homology to glycoside hydrolases, with more than 100 related
to cellulose or hemicellulose hydrolysis and some of these
enzymes taxonomically affiliated with bacterial groups [27,52].
In the lower termites, a metatranscriptomic library of the gut
microbial community was utilized to characterize the metabolism
of the parabasalian protists inhabiting the gut [53]. Interestingly, a
fraction of the reads (<15%) were affiliated with bacterial groups.
As expected, functional analysis of the lower termite gut utilizing
the KEGG database identified pathways related to carbohydrate
metabolism, including starch and sucrose metabolism, pyruvate
metabolism, glycolysis, and the citrate cycle, and ranked these
processes as abundant [53]. Searches using CAZy identified
extensive expression of glycoside hydrolases predicted to
be involved in lignocellulose degradation and taxonomically
affiliated with either protists or bacterial groups [53]. The large
diversity of cellulases, hemicellulases and pectinases are
predicted to be involved in the degradation of lignocellulose.
From the above analyses of three distinct insect systems
(ants, “higher” and “lower” termites) and results from the study
of ruminants, we can make generalizations and formulate
hypotheses for future research in lignocellulosic habitats. For
example, although the contribution of the eukaryotic members
in these communities is not fully understood, each insect
forms a specific symbiosis with distinct fungal or protistan
groups, suggesting the importance of these microbes, which
are overlooked by purely 16S rRNA gene-based surveys. The
complexity of glycoside hydrolases and the diversity of the
community members seem to be necessary components of
lignocellulosic communities. In the future, alterations in diet
composition (shifting ratios of different plant polysaccharide
components), perturbations in the community (using antibiotics
or antifungal agents), or metabolic analyses of cultured isolates,
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acquired a novel bacterial group hypothesized to have expanded
the ability of the hosts to survive on a plant based diet [60,61].
However, the presence of Rhizobiales correlated with host
diet does not alone prove function in the context of herbivory;
indeed, Rhizobiales are often plant associated and ants may be
inoculated with the bacteria simply due to herbivory. Specific
metabolic tests or genomic sequencing of Rhizobiales isolates
from the turtle ants will allow for a better understanding of
function of this particular bacterial group. Additionally, tests of
host fitness in ants modified to lack Rhizobiales symbionts may
allow assessment of the contribution of the bacteria to host
health.
may identify specific contributions from each domain of life in
the processing of lignocellulose.
Specificity of
communities
host-associated
microbial
Microbiome community membership may be species specific
– that is, microbial composition can be used to resolve
taxonomically distinct hosts [54-56]. This suggests that
membership in microbial communities is not random but instead
relevant to host biology and evolution. Early work correlated
both diet and host phylogeny with bacterial composition
in mammalian guts [54]. Most interestingly, the taxonomic
composition of the panda microbiome clustered this herbivorous
mammal with its omnivorous relatives [54]. However, more recent
metagenomic analyses of the panda gut and other herbivorous
mammals suggest that the microbial communities, as predicted,
have the ability to effectively degrade plant material [37,57].
There may be species specificity in which organisms are present
in a host’s microbiome although the function provided by that
community may change based on environment and/or diet.
This result presents an intriguing possibility – that existing,
host-specific, community members can acquire new functions
through horizontal gene transfer. Indeed, this kind of functional
expansion in specific host-associated groups has been noted in
bacteria from the guts of human adults. The acquisition of novel
carbohydrate-active enzymes by human-specific Bacteroides
is an excellent example [58,59]. The fact that a Bacteroides
strain was not simply replaced by another bacterium capable
of the same metabolic process suggests constraints on
community membership either through host immune responses
or interactions between microbial members. In order to explore
function and composition with regards to host specificity and
diet, one requires a system with a broad diversity of hosts, where
diets and habitats vary across the phylogeny. Two novel systems
are emerging as excellent models in this respect: turtle ants and
marine sponges.
Marine Sponges
Because of their phylogenetic and morphological diversity,
marine sponges have been used to identify microbial signatures
unique to the genetic background of hosts and to probe the
functionality of the community members. Morphologically
similar, but genetically divergent sponges were sampled and
bacterial communities within each species were analyzed using
metagenomics [63]. Most interestingly, taxonomic profiles of
bacterial groups present differed although metabolic functions
of the groups were similar across sponge species. The microbial
signatures found in six sponge species and three water samples
collected near the sponges were compared, and these planktonic
microbial communities differed significantly from the spongeassociated bacterial fractions [63]. Although host-phylogeny in
the sponges does not correlate with microbial composition, a
large, species-specific community was found to be present in
each sponge host [64]. These species-specific communities
seemed to be performing equivalent functions in their hosts
including denitrification and ammonium oxidation [63]. In the
sponges, therefore, it seems microbiome taxonomy is specific
to each host species, while function may be dictated by habitat.
Transmission and exchange of microbial
communities between populations and
individuals
Turtle ants
The question of host diet, microbiome membership, and
evolution has been explored in an herbivorous group of ants,
the turtle ants (Hymenoptera:Formicidae) [60,61]. In one study,
authors were able to screen over 283 ant species from 141
genera [60]. Representatives from the genera were chosen and
the 16S rRNA gene was sampled revealing a relatively broad
community, dominated by the Proteobacteria and including
ant-specific bacterial groups [60]. This ant system, however, is
much less complex than the mammalian guts (tens to hundreds
of OTUs found in the turtle ants compared to hundreds to
thousands found in mammals [57,62]) allowing the authors to
better correlate patterns across the phylogeny [61]. For example,
herbivory in these ants (in the Cephalotes and Procryptocerus
genera) correlated with presence of a specific α-Proteobacteria
clade: Rhizobiales. Instead of altering the function of existing
microbial community members, the herbivorous ants had
One fundamental question that remains unanswered in
microbiome studies is: From where does the microbiome arise?
Precluding vertically transmitted mutualists and parasites, most
animals develop in a sterile environment. However, either during
the birth process or after hatching, animals are quickly colonized
by microbes from their surroundings. Studies of microbial
succession in human infants suggest that this dynamic process
takes months to complete and that bacterial species present in
the mother may or may not be dominant in the infant [65-67].
Environmental contributions, not transmission from conspecifics,
may have a dramatic impact on community composition. Studies
involving humans [68] and non-human primates [56] indicate that
diet and host geography can significantly impact microbiome
composition. Transmission and exchange of microbiota have
been studied in the parasitic wasp Nasonia and several bee
species. In these insect models, questions of colonization and
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Invertebrate systems for hypothesis-driven
microbiome research
The parasitic wasp, Nasonia (Hymenoptera:Pteromalidae),
develops within another insect – the Sarcophaga blowfly.
Nasonia mothers lay eggs within the pupae of the blowfly, where
the Nasonia larvae grow and pupate, digesting the immobilized
Sarcophaga as they develop [69]. Because the parasitic wasp
spends part of its life within another insect, we can ask whether
or not this primary environment plays a role in shaping the
adult Nasonia microbiome. Researchers sampled three Nasonia
species, throughout wasp development, each infecting the same
Sarcophoga host [70]. As the Nasonia develop, the diversity of the
microbial community also increases – pupal microbiome diversity
was between two and six times greater than larval microbiome
diversity [70]. Also, the authors found microbiota similarity varied
along a developmental gradient – larval microbiota more closely
resembled that of the Sarcophaga host, while the microbiota of
both the pupae, which reside within the Sarcophaga, and the
adults, which emerge from the host, diverged in community
composition. Due to the behavior of the parasitic wasp, the
Nasonia-Sarcophaga system will prove useful to the study of
transmission dynamics between insects. For example, both
Nasonia and Sarcophaga share genetically similar strains of the
intracellular parasite Wolbachia [71], suggesting that in the recent
past, and due to their close physical interaction, Wolbachia was
transmitted from one insect to another.
better able to survive pathogen challenge than those with altered
flora [74]. Although the mechanism behind this antagonism has
yet to be determined, a normal and robust microbiome in the
bumble bee is clearly healthful. Because bumble bees are social
insects, social transmission of microbiota between the bees may
inoculate future generations, providing a protective benefit to the
community [74].
The mating system of the eusocial honey bee (Apis mellifera)
provides an interesting opportunity to explore microbiota
transmission and exchange within the society. The honey bee
gut microbiome is comprised of a well-defined set of bacterial
groups [76,77], some of which are also present in the related
bumble bee [78], and some of which may be transmitted
between caste members during development [79]. The honey
bee differs from many other eusocial insects in that queens
mate promiscuously, mating with many males and producing
genetically diverse colonies [80]. Interestingly, genetically diverse
colonies are better able to protect themselves against pathogens
compared to genetically uniform colonies (where the queen
mates with a single male) [80-83]. Honey bee colonies made up
of a diverse group of worker bees are also better able to defend
themselves against artificial infection by important bacterial
pathogen, Paenibacillus larvae, and have also been found to
succumb less frequently to infection in the field as compared to
colonies produced from queens mated singly [75,84-86]. Due to
the interesting mating system of the honey bee and the ability
to experimentally control and manipulate the social environment
questions about the origins and development of the microbiome
in a society can be addressed in Apis.
Bees
The advantage of a genetically tractable host
Several different species of bees, hymenopteran insects within
the Apoidea family, have been utilized as models in which to
probe microbiota transmission and exchange. The life-history
diversity within the Apoidea, which includes social and solitary
insects, allows one to ask questions about the social context of
microbiome transmission. One comprehensive study identified
environmental sources (such as plants) for lactobacilli found
in sweat bees, suggesting that the flowers from which the
bees forage may be the source for some microbial community
members [72]. Additionally, intergenerational transmission might
proceed via nest inoculation because the pollen and other plant
material within sweat bee nests were dominated by these same
lactobacilli [72].
Several studies have focused on other important pollinators,
investigating possible benefits to the microbiota found in bees
[73-76]. Microbiota found within bumble bee colonies, for
example, may protect against specific eukaryotic parasites [74].
The authors attempted to create germ-free bumble bees and
although unsuccessful, they managed to severely alter the normal
microbiota present in the insects [74]. Bumble bees harboring
the altered microbial community were then challenged with
Crithidia bombi parasites. Their survival was then compared to
bees that had been allowed access to fecal material from normal
bumble bees. Bumble bees inoculated with fecal material were
Among the invertebrate systems currently being used to
investigate microbiome function, the Drosophila model
(Diptera:Drosophilidae) stands out as potentially transformative.
The gut associated microbes found in Drosophila have been
reviewed elsewhere [87], and comprise a very small community
of bacteria and yeasts [88,89]. However, this simple community
has been shown to dramatically impact host metabolism,
immune function and behavior. For example, Drosophila
developmental and metabolic homeostasis is significantly
altered by the introduction of a mutant microbiome member
in the gut and can be reversed by enhancing host insulin/IGF
signaling pathways [90]. Also, Drosophila mating preference
is affected by the presence of a single microbial member [91],
likely via the alteration of cuticular hydrocarbons on the fly host
[92]. Drosophila can be made axenic, and microbiome function
analyzed in gnotobiotic flies [93]. Finally, the power of Drosophila
genetics can be leveraged to understand effects of specific
host proteins and pathways on gut microbe interactions. For
example, the molecular sensor PGRP-LE seems specific to
immune tolerance of microbiota and that sensor modulates
the ability of different pathogens and mutualists to colonize the
gut [94]. Given the enormous array of Drosophila mutants that
exist, and the vast number of genetic tools available, the use of
transmission can be cleanly addressed due to the more simple
gut community and the ability to massively sample these
tractable, small hosts.
Nasonia
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Drosophila as a model system for dissection of host-microbiota
cross talk is seemingly unlimited.
associated gut communities are the most heavily studied across
all systems, regardless of complexity. Although this allows more
direct comparative work, restricting analyses to gut communities
can artificially increase the relative importance of diet in shaping
community membership and ignore other, potentially distinct,
microbial environments within hosts. Indeed, averaging the
microbial composition across many different host-associated
habitats by extracting nucleic acids from entire animals
would blur the importance of specific communities upon any
experimental variable investigated.
Regardless, invertebrate systems are emerging as excellent
models in which to explore hypothesis-driven research with
respect to microbiomes. Any system has limitations and
caveats – for example, the ability to genetically manipulate
Drosophila allows for the investigation of specific questions not
possible in the other insect systems mentioned in this review.
Additionally, it may be exceedingly difficult to create truly
gnotobiotic eusocial insects for the investigation of simplified
gut communities, as the complex development of these hosts
involves trophylaxis between colony members. However, the
systems described herein hold promise – due to the hosts’
ecology, evolution, or behavior – for answering specific, welldefined questions.
Future Work and Perspectives
Unfortunately, “microbiome” has largely become synonymous
with the bacterial community – most projects ignore or only
touch on the archael, viral, and eukaryotic community members.
We expect that as comparative databases are assembled and
curated, and as the tools for amplicon study in non-bacterial
microbial domains are improved, researchers will be able to
generate a more complete view of the microbiome. Although
originally coined to specifically refer to host-associated microbial
communities [95], the word “microbiome” is now utilized broadly
to refer to any habitat. That being said, it is unclear whether the
deviation in the usage of the word “microbiome” would change
the field of investigation or alter the framing of research questions.
While superficially it may seem that the host-associated habitat
is but one of several kinds of microbial environments there is a
vital difference: the host can evolve in response to the microbes
present. It is likely that this important property serves to limit
the kind and diversity of microbes present, as well as to select
for particular groups coevolved with their hosts. Finally, host-
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