pnas201218525 3229..3236 - PNAS-2013-McFall-Ngai-3229-36.pdf

PERSPECTIVE

Animals in a bacterial world, a new

imperative for the life sciences

Margaret McFall-Ngai

a,1

, Michael G. Had

fi

eld

b,1

, Thomas C. G. Bosch

, Hannah V. Carey

, Tomislav Domazet-Lo



Angela E. Douglas

, Nicole Dubilier

, Gerard Eberl

, Tadashi Fukami

, Scott F. Gilbert

, Ute Hentschel

, Nicole King

Staffan Kjelleberg

, Andrew H. Knoll

, Natacha Kremer

, Sarkis K. Mazmanian

, Jessica L. Metcalf

Kenneth Nealson

, Naomi E. Pierce

, John F. Rawls

, Ann Reid

, Edward G. Ruby

, Mary Rumpho

, Jon G. Sanders

Diethard Tautz

, and Jennifer J. Wernegreen

Department of Medical Microbiology and Immunology, University of Wisconsin, Madison, WI 53706;

Kewalo Marine Laboratory, University

of Hawaii, Honolulu, HI 96813;

Zoological Institute, Christian-Albrechts-University, D-24098 Kiel, Germany;

Department of Comparative

Biosciences, University of Wisconsin, Madison, WI 53706;

C

er Bo



skovi



c Institute, HR-10000 Zagreb, Croatia;

Department of Entomology

and Department of Molecular Biology and Genetics, Cornell University, Ithaca, NY 14853;

Max Planck Institute for Marine Microbiology,

Symbiosis Group, D-28359 Bremen, Germany;

Lymphoid Tissue Development Unit, Institut Pasteur, 75724 Paris, France;

Department of

Biology, Stanford University, Stanford, CA 94305;

Biotechnology Institute, University of Helsinki, Helsinki 00014, Finland;

Julius-von-Sachs

Institute, University of Wuerzburg, D-97082 Wuezburg, Germany;

Molecular and Cell Biology, University of California, Berkeley, CA 94720;

Singapore Centre on Environmental Life Sciences Engineering, Na

nyang Technological University, Singapore 637551, and Centre for

Marine Bio-Innovation and School o

f Biotechnology and Biomolecular Sciences, Univ

ersity of New South Wales, Sydney 2052, Australia;

Botanical Museum, Harvard University, Cambridge, MA 02138;

Division of Biology, California In

stitute of Technology, Pasadena

CA 91125;

Biofrontiers Institute, University of Colorado, Boulder CO 80309;

Department of Earth Sciences, University of Southern California,

Los Angeles, CA 90089;

Department of Organismic and Evolutionary Biology, Harvard University, Cambridge, MA 02138;

Cell Biology and

Physiology, University of North Carolina, Chapel Hill, NC 27599;

American Academy of Microbiology, Washington, DC 20036;

Department of

Molecular and Cell Biology, University of Connecticut, Storrs, CT 06269;

Department of Evolutionary Genetics, Max Planck Institute for

Evolutionary Biology, D-24306 Plön, Germany; and

Nicholas School and Institute for Genome Sciences and Policy, Duke University, Durham,

NC 27708

Edited by David M. Karl, University of Hawaii, Honolulu, HI, and approved January 17, 2013 (received for review December 2, 2012)

In the last two decades, the widespread application of genetic and genomic approaches has revealed a bacterial world astonishing in its

ubiquity and diversity. This review examines how a growing knowledge of the vast range of animal

–

bacterial interactions, whether in shared

ecosystems or intimate symbioses, is fundamentally altering our understanding of animal biology. Speci

fi

cally, we highlight recent techno-

logical and intellectual advances that have changed our thinking about

fi

ve questions: how have bacteria facilitated the origin and evolution of

animals; how do animals and bacteria affect each other

’

s genomes; how does normal animal development depend on bacterial partners; how

is homeostasis maintained between animals and their symbionts; and how can ecological approaches deepen our understanding of the

multiple levels of animal

–

bacterial interaction. As answers to these fundamental questions emerge, all biologists will be challenged to broaden

their appreciation of these interactions and to include investigations of the relationships between and among bacteria and their animal

partners as we seek a better understanding of the natural world.

bacterial roles animal origins

|

reciprocal effects animal

–

bacterial genomics

|

bacteria-driven development

|

microbiome and host physiology

|

nested ecosystems

Biologists have long appreciated the roles that

microbes play in the two distinct disciplines

of pathogenesis and ecosystem cycling. How-

ever, it was not until the late 1970s that Carl

Woese and George Fox opened a new re-

search frontier by producing sequence-based

measures of phylogenic relationships, reveal-

ing the deep evolutionary history shared by

all living organisms (1). This game-changing

advance catalyzed a rapid development and

application of molecular sequencing technol-

ogies, which allowed biologists for the

fi

rst

time to recognize the true diversity, ubiquity,

and functional capacity of microorganisms

(2). This recognition, in turn, has led to a

new understanding of the biology of plants

and animals, one that re

fl

ects strong interde-

pendencies that exist between these complex

multicellular organisms and their associated

microbes (3).

Although the biosphere comprises many

diverse taxonomic groups, our focus here is

principally on the interactions between one

group of microorganisms, the domain Bac-

teria, and one group of complex multicellular

organisms, the animals. Although we chose

to focus on animal

–

bacterial interactions, we

expect the application of new technology to

reveal similar trends among and between Ar-

chaea, fungi, plants, and animals. We begin

by describing what we know about the evo-

lution of animals and their interactions with

bacteria and about the in

fl

uence that these

relationships have had on the present-day

genomic makeup of the partners. We review

the wealth of new data on the roles of bacte-

ria in animal development and physiology

and conclude with a discussion of the nesting

of animal

–

bacterial relationships within their

larger ecological frameworks. We argue that

interactions between animals and microbes

Author contributions: M.M.-N., M.G.H., T.C.G.B., H.V.C., T.D.-L.,

A.E.D., N.D., G.E., T.F., S.F.G., U.H., N. King, S.K., A.H.K., N. Kremer,

S.K.M., J.L.M., K.N., N.E.P., J.F.R., A.R., E.G.R., M.R., J.G.S., D.T.,

and J.J.W. wrote the paper.

The authors declare no con

fl

ict of interest.

This article is a PNAS Direct Submission.

To whom correspondence may be addressed. E-mail: mjmcfallngai@

wisc.edu or had

fi

eld@hawaii.edu.

This article contains supporting information online at

www.pnas.

org/lookup/suppl/doi:10.1073/pnas.1218525110/-/DCSupplemental

www.pnas.org/cgi/doi/10.1073/pnas.1218525110

PNAS

February 26, 2013

vol. 110

no. 9

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–

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PERSPECTIVE

are not specialized occurrences but rather

are fundamentally important aspects of

animal biology from development to sys-

tems ecology.

In addition to the references of the main

text of this article, we include a list of useful

citations to provide the reader a broad open-

ing to the subtopics covered in this contribu-

tion (

SI Suggested Readings

Bacteria and the Origin of Animals

Understanding how associations among

bacteria and animals

fi

rst evolved may reveal

the foundations of ecological rules that

govern such interactions today. Animals

diverged from their protistan ancestors 700

–

800 Mya, some 3 billion years after bacterial

life originated and as much as 1 billion years

after the

fi

rst appearance of eukaryotic cells

(4) (Fig. 1). Thus, the current-day relation-

ships of protists with bacteria, from pre-

dation to obligate and bene

fi

cial symbiosis

(5, 6), were likely already operating when

animals

fi

rst appeared. Attention to this

ancient repertoire of eukaryote

–

bacterial

interactions can provide important insights

into larger questions in metazoan evolution,

from the origins of complex multicel-

lularity to the drivers of morphological

complexity itself.

Based on molecular and cellular data,

animals and choano

fl

agellate protists are

now considered sister groups, descended

from a common choano

fl

agellate-like an-

cestor (Fig. 1) (7). The major underpinnings

of animal

–

bacterial interactions

—

nutrition,

recognition, cell adhesion, and signaling

—

guide two types of choano

fl

agellate behavior

thatmayhavebeenkeytotheoriginof

animals: predation (8) and colony formation

(9). Extant choano

fl

agellates have homologs

of animal signaling and adhesion proteins

(e.g., cadherins and C-type lectins) that may

have arisen as critical facilitators of bactivory

(8). Diverse animals respond to bacterial

signals as triggers for morphogenesis or be-

havior (e.g., larval settlement). Thus, the

discovery that at least one choano

fl

agellate,

Salpingoeca rosetta,

responds to signals from

speci

fi

c bacteria to initiate colony formation

through cell division hints at an ancient

involvement of bacteria in the initiation of

multicellularity (9). It will be important to

learn whether intercellular cohesion in

sponges, which are known to harbor hun-

dreds of bacterial species (10

–

12), similarly

depends on the presence of bacteria. The

origin of multicellularity has been a topic of

intense debate in biology, and many hy-

potheses have been developed about how

this evolutionary milestone was achieved (13).

A microbial role in animal origins does not

obviate other perspectives on the evolution

of complex multicellularity but adds a nec-

essary functional and ecological dimension

to these considerations.

As early animals diversi

fi

ed, animal

–

bac-

terial interactions continued to shape evolu-

tion in new ways (Fig. 1

). Bacteria took on a

new role in animal nutrition, serving not only

as prey but also as producers of digestible

molecules in the animal gut. This role may

have become more diverse with the evolution

of a tubular gut, with one-way passage of

food from mouth to anus. Bacterial in

fl

uence

on gut evolution certainly intensi

fi

ed with the

subsequent origin of the coelom, a body

cavity in which the organs are suspended.

The advent of the coelom made gut elon-

gation and regional specialization possible,

facilitating both massive ingestion and stor-

age for later digestion. Although the degree

to which microbes have driven gut evolution

is unknown, the radiation of several animal

groups (e.g., ruminants) was undoubtedly

enabled by alliances with their gut-asso-

ciated microbiota. The evolution of form

andfunctioninotherorgansystems(e.g.,

respiratory, urogenital) may have also been

fl

uenced by interactions with bacterial

partners (14). Furthermore, it is likely that

the evolution of these organ system niches

drove radiation of particular clades of ani-

mal-associated bacteria (15), such as the

genus

Helicobacter

in vertebrate guts (16).

Evolution with animals, whether in sym-

biosis or via shared habitats, has also in

fl

enced the distribution and diversi

fi

cation of

bacteria. For example, 90% of the bacterial

species in termite guts are not found else-

where (17). Such specialization, while in-

creasing ef

fi

ciency, comes with a cost: for

every animal species that goes extinct, an

unknown number of unique bacterial lin-

eages that have evolved to depend on this

animal niche disappear as well (18). On a

broader scale, the evolution of animals pro-

vided novel physical environments for bac-

terial colonization, such as aerated deep

sediments resulting from animal burrow-

ing. Finally, human activities, which make a

range of molecules not previously found in

Archaea

Bacteria

Eukaryotes

billion years ago

100

log % modern

Atmospheric O2

Choanoflagellates

Sponges

Cnidarians

Other Bilaterians

Vertebrates

cellular immunity

multicellularity

epithelia

gut cavity

organs

adaptive

immunity

predation

metabolism

morphogenesis

environmental cues

innate immunity

adaptive immunity

Fig. 1.

Animals through time. (

) Upper atmospheric oxygen concentration, as a percent of current levels, plotted

against geological time. (

) Phylogenetic history of life on Earth, scaled to match the oxygen timeline. Note that the

origin of the eukaryotes and the subsequent diversi

fi

cation of animals both correspond to periods of increasing at-

mospheric oxygen. (

)(

Left

) A phylogeny of choano

fl

agellates and selected animals, annotated to indicate the

evolution of characters particularly relevant to interactions with bacteria. (

Right

) Interactions between bacteria and

eukaryotes, corresponding to the phylogeny. Bacteria are prey, sources of metabolites, inducers of development in

symbiosis (morphogenesis) and in larval settlement (environmental cues), and activators of immune systems.

3230

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McFall-Ngai et al.

nature, such as halogenated hydrocarbons,

have driven selection on bacterial catabolic

pathways (19), leaving a signature of our

presence in microbial metabolism.

Intertwining Genomes

The long history of shared ancestry and

alliances between animals and microbes

is re

fl

ected in their genomes. Analysis of

the large number of full genome sequences

presently available reveals that most life

forms share approximately one third of their

genes, including those encoding central

metabolic pathways (20). Not surprisingly,

many animal genes are homologs of bacte-

rial genes, mostly derived by descent, but

occasionally by gene transfer from bacteria

(21). For example, 37% of the

∼

23,000 hu-

man genes have homologs in the Bacteria

and Archaea, and another 28% originated in

unicellular eukaryotes (20) (Fig. 2). Among

these homologous genes are some whose

products provide the foundation for signaling

between extant animals and bacteria (22).

The intertwining of animal and bacterial

genomes is not just historical: by coopting the

vastly more diverse genetic repertoire present

in its bacterial partners (23), a host can rap-

idly expand its metabolic potential, thereby

extending both its ecological versatility and

responsiveness to environmental change. For

instance, many invertebrates have intracel-

lular bacterial symbionts whose genes encode

metabolic capabilities lacking in animals,

such as the synthesis of essential amino

acids (24), photosynthesis (25), or chemo-

synthesis (26). Certain marine invertebrates

that feed on algae maintain algal plastids

as photosynthetically active symbionts,

abehaviorthatallowsthehosttouse

photosynthate as a food source for ex-

tended periods (27). These metabolic add-

ons allow the animal to thrive by adapting

to otherwise noncompetitive lifestyles

(e.g., feeding on nutrient-poor diets such

as plant sap) (28) or environments (e.g.,

oligotrophic habitats) (26). Further, such

phenomena

fi

tthede

fi

nition of epigenetic

features. Recent studies have revealed that

bacterial pathogens (29) and other envi-

ronmental factors (30) can alter the ac-

tivities of epigenetic machinery. It is to be

anticipated that such in

fl

uences will extend

to all types of animal

–

bacterial interactions,

including those described above.

Microbial communities in the vertebrate

gut respond to the host diet over both daily

and evolutionary time scales, endowing ani-

mals with the

fl

exibility to digest a wide va-

riety of biomolecules and cope with and even

fl

ourish under conditions of diet change (15,

31). For example, the gut microbiome of

most people in the United States is adapted

to digest a high-fat, high-protein diet,

whereas populations in rural Malawi and

the Amazonas of Venezuela have distinct

microbial consortia and functional gene

repertoires optimized for breaking down

complex carbohydrates (32). The gut micro-

biome adapts to changing diets and con-

ditions not only by shifting community mem-

bership but also by changing gene content via

horizontal gene transfer. For instance, the gut

bacterium

Bacteroides plebeius

,foundinsome

Japanese people, bears a gene transferred

horizontally from the marine bacterium

Zobellia galactanivorans,

giving the gut

symbiont the capacity to degrade seaweed

polysaccharides (33). More generally, human-

associated bacteria have a 25-fold higher rate

of gene transfer than do bacteria in other en-

vironments, highlighting the important role

of gene transfer in host-associated bacterial

communities (34).

Bioinformatic analyses have revealed that

interactions with animals also in

fl

uence the

size and content of the genomes of their

bacterial partners. Although not all genome-

size reduction occurs in symbiosis, a long

history of intimate association with insects

has resulted in highly reduced genomes in

their intracellular symbionts; for example, the

endosymbiont

Candidatus

Hodgkinia cicadi-

cola of the Arizona cicada has a genome size

<

144 kb, smaller than that of some organelles

(35). Recent studies have shown that genome

reduction also occurs in segmented

fi

lamen-

tous bacteria (

Candidatus

Savagella), mem-

bers of the mammalian microbiota that are

critical for the maturation of the immune

system (36). Conversely, in

Bacteroides

thetaiotaomicron

, another member of the

mammalian intestinal microbiota, adapta-

tion to a gut habitat rich in complex car-

bohydrates has driven the expansion of at

least two gene families: glycan-utilization

genes, which constitute 18% of this species

’

genome (37), and diverse sulfatases that al-

low

B. thetaiotaomicron

to digest host mu-

cin (38). The genomic basis for other

microbial adaptations among gut microbes

is less clear. One possible selection pressure is

host temperature. In aquatic environments

such as the deep sea, host

fi

shes and

invertebrates conform to the temperature

of the environment, so temperature-driven

coevolution would be unlikely in these

habitats. In contrast, terrestrial environ-

ments often have broad, short-term (daily)

and long-term (seasonal)

fl

uctuations in

temperatures. It is in these habitats that

endothermy (maintaining a constant body

temperature by metabolic means) evolved

as a shared character in birds and mammals.

Most enteric bacteria of birds and mammals

have growth optima at

∼

40 °C, suggesting

the unexplored possibility that this trait

resulted from coevolution of these bacteria

with their endothermic hosts. The reciprocal

may also be true, i.e., an animal

’

s microbial

partners may have played a role in selecting

for the trait of endothermy. Constant high

temperature speeds up bacterial fermenta-

tion, providing rapid and sustained energy

input for the host. These bene

fi

ts are ap-

parent when comparing conventional to

germ-free mammals, which require one-

third more food to maintain the same body

mass (39). Keeping their microbes working

at optimum ef

fi

ciency likely offered a strongly

positive selection pressure for the evolution of

genes associated with the trait of endothermy

in birds and mammals.

Partners in Animal Development

Animal development has traditionally been

viewed as an autonomous process directed by

the genome. Because it both originated and

evolved in a microbe-rich environment, ani-

mal development deserves a reexamination,

at least in part, as an orchestration of animal-

encoded ontogeny and interdomain com-

munication (40, 41). Although relatively few

studies have been reported until recently,

these early data lead us to anticipate that

microbes play a role in providing signals for

multiple developmental steps.

From their earliest stages of development,

animals use sophisticated mechanisms to

manage their microbial environment. Physi-

cal barriers, such as capsules, chorions, and

mucus, protect eggs by excluding microbes,

and chemical barriers, including antimicro-

bial peptides (AMPs), shape the composition

Fig. 2.

The ancestry of humans re

fl

ected in the geno-

mic signature. A phylogenetic analysis of the human

genes reveals the relative percentage of the genome that

arose at a series of stages in biological evolution (20).

McFall-Ngai et al.

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February 26, 2013

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PERSPECTIVE

of the associated microbiota (42). Conversely,

several animals recruit speci

fi

cbacteriato

their embryonic surfaces to provide protec-

tion against potential pathogens (43). For ex-

ample, the shrimp

Palaemon macrodactylus

is protected from the fungus

Lagenidium

callinectes

by 2,3-indolinedione that is pro-

duced by an

Alteromonas

sp. on the embryo

’

surface (44). Although many animals, in-

cluding a wide variety of insects, have

transovarial (i.e., via the egg to the embryo)

transmission of bacterial partners (28, 45),

we have no persuasive evidence to date that

these microbes or their metabolites in

fl

uence

embryogenesis. Whereas developmentally

important symbioses have been documented

throughout the postembryonic (larval and

juvenile) stages of vertebrate and arthropod

life cycles, the roles of symbiotic microbes

during normal embryonic development are

just beginning to be studied. Unlike verte-

brates whose embryos develop inside enclo-

sures that physically block bacterial asso-

ciations, many invertebrates acquire their

symbionts through the female germ line.

Here, we may expect to

fi

nd regulatory sig-

nals being generated by microbes and inter-

actions between host and symbiont develop-

ment (46). It is apparent that evolution has

selected for anatomical, cellular, and molec-

ular determinants that act during this period

to prepare newborn animals for interactions

with the microbial world.

Ample evidence shows that microbes act

directly as agents of postembryonic develop-

ment. For example, fucosyltransferases deco-

rate the surface of the embryonic mammalian

intestine with fucose residues that provide a

nutrient source for gut microbes, including

B. thetaiotaomicron,

as they colonize the

newborn (47). In the squid-vibrio system, a

complex organ forms during embryogene-

sis that facilitates subsequent colonization

by the symbiotic bacterium

Vibrio

fi

scheri

(48). The products of horizontally acquired

microbes can be essential for a range of

developmental functions, including in

fl

ences on larval growth rate and body size in

invertebrates (49), postembryonic matura-

tion and renewal of epithelia in invertebrates

and vertebrates (50

–

53), development and

speci

fi

cation of the gut-associated lymphoid

tissues in vertebrates (54), activation of the

immune system in tsetse

fl

ies (55), and nor-

mal brain development in mammals (56, 57).

Intriguingly, the host regulatory pathways

that control immune responses to microbes

appear also to have central roles in animal

development, underscoring the intimate

relationships between development and

host

–

microbe interactions (58, 59).

Perhaps the most pervasive example of

microbial signaling in animal development

is the induction of settlement and metamor-

phosis of many marine invertebrate larvae

(60). This transition is an absolute require-

ment for completion of the animal

’

slife

cycle and is contingent on induction by ex-

ogenous morphogenetic cues, many of which

are produced by bacteria associated with a

particular environmental surface (60). Ma-

rine invertebrate metamorphoses offer valu-

able modelsforexploringthebasisofbacterial

signaling in animal development in a setting

where the very persistence of marine eco-

systems depends on it.

Coming full circle, the in

fl

uence of

microbes on animal reproduction can be

observed with particular clarity in inver-

tebrates (61). Most insect orders carry

vertically transmitted parasites that can af-

fect the processes of sexual determination,

maturation, and reproductive success. For

example, various

Wolbachia

strains femi-

nize crustacean genetic males, kill males, or

induce clonal production of females in some

insects (62). However, in one case, the as-

sociation with a

Wolbachia

strain has be-

come essential for reproduction; the wasp

Asobara tabida

requires this microbe for

egg maturation (63). Recent studies have

shown that, in both invertebrates and ver-

tebrates, the microbiota can even in

fl

uence

reproductive behavior (64). Changes in

cuticular-hydrocarbon pro

fi

les linked to

speci

fi

c bacterial symbionts in the gut of

Drosophila melanogaster

correlate with mate

choice (65), and several lines of evidence

suggest that olfactory cues associated with

mate choice in vertebrates are produced by

their resident microbiota (66).

Interdomain Communication

Although animals and bacteria have different

forms and lifestyles, they recognize one an-

other and communicate in part because, as

described above, their genomic

“

dictionar-

ies

”

share a common and deep evolutionary

ancestry. One modality of interdomain com-

munication, that occurring during bacterial

pathogenesis, has been extensively explored

for over a century. However, how might

bacterial signaling structure the biology

of the healthy host?

Biologists now know that bacteria have

social behaviors, communicating with each

other through chemical signaling, such as

quorum sensing (67, 68); more recently,

interdomain quorum signaling between bac-

teria and their eukaryotic partners has be-

come evident (22, 69

–

71). In addition to

quorum signals, bacteria use cell surface

–

derived molecules to communicate with

their hosts, affecting host processes both at

the cellular level [e.g., apoptosis, Toll-like

receptor (TLR) signaling (52, 72)], as well as

at the organ-system level (Fig. 3). Con-

versely, host-derived signal molecules like

nitric oxide (NO) can be sensed directly by

microbes (73). It is intriguing to consider

that these kinds of communication evolved

to maintain an association

’

s balance with its

hundreds of bene

fi

cial species and that

pathogens have

“

hijacked

”

these conversations

to enhance their

fi

tness through disease. For

example,

Salmonella typhimurium

has adap-

ted the quorum-sensing regulator QseC to act

as a receptor for the host hormone norepi-

nephrine and thereby tie the regulation of

virulence genes to the hormone

’

spresencein

thetissue(74).Somehosts,suchasthemarine

macroalga

Delisea pulchra,

respond to quo-

rum-signaling pathogens by producing halo-

genated furanones that act as signal mimics,

blocking the microbes

’

communication (75).

The gut is likely the site of the most dy-

namic and consequential bacteria signaling

that bene

fi

ts animal hosts, because of the

sheer numbers and diversity of its microbes

and the inherent permeability and sensitivity

of the gut epithelium. For example, acetate,

a short-chain fatty acid (SCFA) produced by

the gut bacterium

Acetobacter,

stimulates in-

sulin signaling in

D. melanogaster

,thereby

promoting host growth rates and reducing

sugar and lipid levels (49). In mammals,

SCFAs affect fat deposition, appetite-related

hormone titers, and food consumption,

which in turn can modulate the compo-

sition of the microbiota and have major

consequences for health and behavior (76,

77). Not surprisingly, the composition of

the gut microbiota and its SCFA produc-

tion are in

fl

uenced by diet. The resultant

interplay among diet, the microbiota, and

their metabolites is, in turn, implicated in

the development of major metabolic dis-

orders including obesity and diabetes (78).

As much as a third of an animal

’

s metab-

olome

—

i.e., the diversity of molecules car-

ried in its blood

—

has a microbial origin;

thus, the circulatory system extends the

chemical impact of the microbiota through-

out the human body (79), transporting meta-

bolites that in

fl

uence the physiology and

metabolism of distant organs and perhaps

other bacterial communities (80, 81). Some

dietary constituents can be modi

fi

ed by gut

microbiota into deleterious compounds; for

example, the conversion of dietary phos-

phatidylcholine into the proatherosclerotic

metabolite, trimethylamine, can jeopardize

cardiovascular health (82). Furthermore,

recent studies link the gut microbiota to

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McFall-Ngai et al.

brain physiology and animal behavior (83).

For instance, germ-free mice have defects in

brain regions that control anxiety (57), and

feeding probiotic bacteria to normal mice re-

duces depression-like behaviors (84, 85). The

fi

nding that TLRs, which transduce bacterial

signals to host cells, are present on enteric

neurons reveals one mechanism by which

microbiota can communicate with the cen-

tral nervous system through the brain

–

gut

axis (72). Thus, maintaining homeostasis

with the normal microbiota is essential to a

healthy nervous system.

As the guardian of an animal

’

s internal

environment, its immune system coordi-

nates cellular and biochemical responses to

alterations in the molecular landscape (86,

87), creating a robust equilibrium between

the healthy host and its normal microbiota.

The complexity of components that com-

prise this system re

fl

ects the great chemical

diversity present in the microbial world.

Pattern-recognition receptors (PRRs) of the

innate immune system can have enormous

repertoires, particularly in the invertebrates.

PRRs recognize microbe-associated molec-

ular patterns (MAMPs), such as bacteria-

speci

fi

c cell surface molecules (88). For ex-

ample, peptidoglycan (PGN), a cell wall

constituent of bacteria, interacts with PRRs

to induce developmental processes in ver-

tebrates and invertebrates (52, 54). The gut-

associated lymphoid tissues of mammals

mature with the presentation of peptidogly-

can monomer by the gut microbiota during

their early establishment, and the same

molecule induces the regression of a juvenile-

speci

fi

c epithelium that facilitates coloni-

zation by the symbiont in the squid

–

vibrio

system. Similarly, a polysaccharide produced

and exported by

Bacteroides fragilis,

acon-

stituent of the normal microbiota, signals

the PRRs of immune cells to suppress gut

fl

ammation (89). Disturbance of equilib-

ria maintained by MAMP

–

PRR interactions

can lead to a wide variety of pathologic

states, including in

fl

ammatory bowel disease

and diabetes (90, 91). Further, SCFAs pro-

duced by gut bacteria help the host defend

against enteric infections (92), revealing

molecular symbiosis between the micro-

biota and the immune system. Finally, im-

munologists are beginning to examine the

possibility that, in addition to a role in

pathogenesis, a principal selection pressure

acting on the form and function of the

adaptive immune system is the need to

maintain balance among the complex, co-

evolved consortia that form persistent

symbioses with the mucosal surfaces of

several organ systems in the vertebrate

host (86, 93

–

95).

Nested Ecosystems

Since the dawn of metazoan evolution, the

ecology of animals has depended on bac-

terial communities. The fossil record pro-

vides evidence that some animal forms in

the Ediacaran grazed on dense assemblages

of bacteria on hard substrates (96) and that

burrowing animals originated in association

with microbial mats (97). Biologists in-

creasingly recognize that, in extant animals,

developmental and physiological signaling

are processes whose understanding bene

fi

from an ecological perspective (98).

Viewing animals as host

–

microbe ecosys-

tems has given us new insights into the

maintenance of human health. The appli-

cation of ecological approaches, including

successional assembly and diversity analy-

sis, has proven valuable in understanding

how animal

–

microbial alliances function

(99

–

101). For example, human infants born

vaginally have a very different succession

during the early phases of gut colonization

and possibly long-term composition of their

microbiota than those delivered by Caesar-

ean section (102). The effects of this differ-

ence in infant delivery on adult health remain

to be discovered. We know that imbalances

in the mature human microbiome have

been correlated with a spectrum of diseases,

including obesity and diabetes (77). A re-

cent metacommunity analysis of the gut

microbiota of obese and lean twins revealed

that obesity is associated with a signi

fi

cantly less stable and more variable mi-

crobial community (103). Although most

research on consortia is currently focused

on humans and vertebrate model systems,

such as mice and zebra

fi

sh, similarly com-

plex interactions occur in all animal species.

Viewing bacterial colonization of animals as

an ecological phenomenon adds clarity to an

understanding of the mechanisms and

routes by which phylogenetically rich and

functionally diverse microbial communi-

ties become established and evolve on

and within animal hosts.

An ecological perspective in

fl

uences not

only our understanding of animal

–

micro-

biome interactions but also their greater role

in biology. The ecosystem that is an indi-

vidual animal and its many microbial com-

munities [i.e., the holobiont (104)] does not

Fig. 3.

Signaling within and between the animal and its microbiota. Members of the microbiota, such as those in

and on the gut, oral cavity, and skin, communicate among themselves and exchange signals with the animal

’

s organ

systems, participating in the body

’

s homeostasis. Some of the signals promoting this balance are mentioned in the

text (green), whereas other representatives are not (black;

Tables S1

and

). The microbiota also in

fl

uences animal

behavior, creating a direct interface with other organisms. AMP, antimicrobial peptides; LPS, lipopolysaccharide; PGN,

peptidoglycan; PSA, polysaccharide A; SCFA, short-chain fatty acids; TMA, trimethylamine oxide.

McFall-Ngai et al.

PNAS

February 26, 2013

vol. 110

no. 9

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PERSPECTIVE

occur in isolation but is nested within com-

munities of other organisms that, in turn,

coexist in and in

fl

uence successively larger

neighborhoods comprising ever more com-

plex assemblages of microbes, fungi, plants,

and animals (Fig. 4). Hydrothermal vent

communities illustrate the role of animal

–

microbe associations in such nested eco-

systems. At vents and other reducing hab-

itats, chemoautotrophic symbionts provide

organic nutrients for animal hosts in at least

seven different phyla. The activities of these

individual symbioses contribute to larger

communities that include nonsymbiotic

animal and microbial species that are able

to exist through the symbiotic primary pro-

duction that is not driven by solar energy but

rather by sul

fi

de, hydrogen, methane, and

other reduced energy sources (26, 105).

Similarly, nested within broader terrestrial

ecosystems, bacterial communities in

fl

oral

nectar can in

fl

uence the way animals such

as pollinators interact with plants. In

these instances, the bacteria change the

chemical properties of the nectar, making

it more or less attractive to the pollinator,

which changes the pollinator

–

plant dy-

namic (106).

Bacteria are critical determinants of animal

population and community structures, even

in ecosystems where intimate symbioses are

not the driving force. Recent studies demon-

strate that the larvae of many benthic marine

invertebrates require speci

fi

c microbial cues

for their recruitment from the plankton, and

these larval responses to bacteria in

fl

uence

the structuring of many marine benthic

communities (60, 107). For example, certain

strains of the bio

fi

lm-forming bacterium

Pseudoalteromonas luteoviolacea

produce

chemical cues that stimulate settlement and

metamorphosis by

Hydroides elegans

polychaete worm that fouls docks and the

hulls of ships worldwide (60, 108), as well

as a sea urchin (109) and a coral (107).

Surface bio

fi

lms on many marine animals

serve important functions in determining

the very nature of the animals

’

ecological

interactions with other organisms (110).

Similarly, the acquisition of an appropriate

microbiome at critical life history stages of

many animals affects their subsequent be-

havioral patterns and thus the stability of

their ecological roles in their communities

(64). Bacteria feeding on dead animals in

the sea, and likely on land, repel animal

scavengers by producing noxious metabo-

lites; these products allow the bacteria to

effectively outcompete organisms 10,000

times their size (111).

Conversely, invasive animals can alter the

activities of indigenous bacteria, with signi

fi

cant effects on their shared habitat. For ex-

ample, rats introduced onto small Paci

fi

islands decimated seabird populations, re-

sulting in decreased sea-to-land transport of

nutrients (guano) and altered decomposition

and nutrient cycling by soil microbes (112).

In another study, European earthworm spe-

cies introduced to North American hard-

wood forests led to signi

fi

cant changes in soil

microbial biomass and the metabolic quo-

tient of the soil ecosystem (113). In each of

these situations, an introduction led to a

substantial reduction in ecosystem producti-

vity. Applying metacommunity and network

analyses (114) to such animal

–

bacterial in-

teractions will be essential for the design of

effective strategies for managing ecosystems

in the face of the environmental perturba-

tions, such as pollution, invasive species, and

global climate change, that challenge the

biosphere.

Challenges

For much of her professional career, Lynn

Margulis (1938

–

2011), a controversial vi-

sionary in biology, predicted that we would

come to recognize the impact of the mi-

crobial world on the form and function of

the entire biosphere, from its molecular

structure to its ecosystems. The weight of

evidence supporting this view has

fi

nally

reached a tipping point. The examples come

from animal

–

bacterial interactions, as de-

scribed here, and also from relationships be-

tween and among viruses, Archaea, protists,

plants, and fungi. These new data are de-

manding a reexamination of the very concepts

of what constitutes a ge

nome, a population, an

environment, and an organism. Similarly,

features once considered exceptional, such as

symbiosis, are now reco

gnized as likely the

rule, and novel models for research are emerg-

ing across biology. As a consequence, the

New Synthesis of the 1930s and beyond must

be reconsidered in terms of three areas in

which it has proven weakest: symbiosis, de-

velopment, and microbiology (115). One of

these areas, microbiology, presents particular

challenges both to the species concept, as

formulated by Ernst Mayr in 1942, and to the

concept that vertical transmission of genetic

information is the only motor of selectable

evolutionary change.

It is imperative that human societies rec-

ognize the centrality of the relationships be-

tweenmicrobesandotherorganismsforthe

health of both individuals and the environ-

ments in which they live. The current focus

on studies of humans and their microbiota

has provided compelling evidence that the

composition and activity of resident microbes

play crucial roles in shaping the metabolic

and regulatory networks that de

fi

ne good

health, as well as a spectrum of disease states.

Nonetheless, the underlying ecological

mechanisms are still poorly de

fi

ned, and

the development of tools to translate this

understanding into novel therapies pres-

ents an ongoing challenge.

In broader-scale ecosystems, evidence is

mounting that seemingly minor environ-

mental perturbations have major long-

term impacts. A full understanding of the

consequences will require us to expand our

investigations of the associated changes in

microbial communities in soil, freshwater,

and marine habitats. How are such micro-

bial assemblages affected by the introduc-

tion of nonnative species of plants and

animals, the increases in temperature due

to global climate change, and the acidi

fi

ca-

tion of the oceans? Although a few studies

(116, 117) have revealed its importance, the

impact of acidi

fi

cation has thus far focused

largely on eukaryotic calci

fi

cation processes

(118). This emphasis leaves us still ignorant

of how marine ecosystems may be changed

if small shifts in seawater pH or tempera-

ture alter the compos

itions of bacterial

Fig. 4.

Nested ecological inter

actions of animals and

bacteria and their underlying metabolic bases. (

forest canopy insect illustr

ates the cascading effects

of animal-bacterial interact

ions across multiple spatial

scales. Bacterial symbionts (

Left

), residing in the gut

(

Center Left

), are essential to nutritional success of insect

species (

Center Right

) in tropical forest canopies (

Right

where they often make up a majority of animal biomass.

(

) Diversity of energy metabolism in bacteria and ani-

mals. Animals can ferment and aerobically respire but are

unable to perform the vast diversity of other, ecologically

vital, energy-harvesting processes. Beyond phototrophy,

which they share with plants, bacteria can also contribute

to primary production by using inorganic energy sources

(lithotrophy) to

fi

xCO

. Animals are directly or indirectly

dependent on bacteria for extracting energy and cycling

biomolecules, whereas animals actively contribute to

bacterial productivity through bioturbation, nutrient pro-

visioning, and as habitats for colonization and shelter.

3234

www.pnas.org/cgi/doi/10.1073/pnas.1218525110

McFall-Ngai et al.

communities that are crucial for recruit-

ment of the next generations of plants and

animals into their native habitats. The

maintenance and restoration of ecosystems

that support sustainable agriculture and

carbon-neutral energy production depend

on recognition of the interactions between

microorganisms and animals, plants, and

fungi, and the robustness of these rela-

tionships in response to anthropogenic and

other perturbations. Whether an ecosystem

is de

fi

ned as a single animal or the planet

’

biosphere, the goal must be to apply an under-

standing of the relationships between microbes

and other organisms to predict and manipulate

microbial community structure and activity

so as to promote ecosystem health.

These challenges present a vast and exciting

frontier for the

fi

eld of biology and call on life

scientists to alter signi

fi

cantly their view of the

fundamental nature of the biosphere. Ambi-

tious large-scale interdisciplinary research ef-

forts, such as the Human Microbiome Project

and the Earth Microbiome Project, aim to

provide a basic understanding of microbial

variation across a wide range of body and en-

vironmental habitats in both the normal and

perturbed states. Effective project design and

the resulting large data sets are driving ad-

vances in quantitative methods, such as the

creation and re

fi

nement of techniques to im-

prove approximation algorithms, dimension-

ality reduction, and visualization of the results

(119). These efforts have highlighted the need

for genomic standards, open-source integrated

analysis pipelines, and increased low-cost

computational power. A compelling goal for

the future is to apply these technologies, the

resultant data, and the emerging intellectual

framework to a wide array of biological

questions. Such a synthesis promises to gen-

erate a more accurate vision of life on earth.

Successful development of research on our

microbial world will result only with the

breakdown of existing intellectual barriers,

not only between the subdisciplines of bi-

ology, but also across the natural sciences,

mathematics, computer science, and engi-

neering. Such integration will be fostered

by the active promotion of cross-disciplinary

units at universities, collaboration among

professional societies, and novel approaches

by the funding agencies to support the de-

velopment of this new frontier (120). The

progress of change across the

fi

eld will also

require reformulation of educational goals,

including development of ways of teaching

biology that are as revolutionary as those

that occurred in the 1950s in the wake of

both the New Synthesis and the launch of

Sputnik. Because of advances described here,

we foresee a day when microbiology will be

a centerpiece not only of biological research,

but also of high school, undergraduate, and

graduate biology education.

ACKNOWLEDGMENTS.

We thank N. Glasser for assis-

tance with graphics and D. Haraway and E. A. C. Heath-

Heckman for helpful discussion and comments on the

manuscript.The work of this group was supported by

National Science Foundation Grant EF-0905606 to

the National Evolutionary Synthesis Center (NESCent).

This effort was also supported by fellowships to M.M.-N.

from the John Simon Guggenheim Foundation and the

Gordon and Betty Moore Foundation Visiting Scholar

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vol. 110

no. 9

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