Symbiosis as a General Principle in Eukaryotic Evolution

Symbiosis as a General Principle in EukaryoticEvolution

Angela E. Douglas

Department of Entomology, Cornell University, Ithaca, New York 14853

Correspondence: [email protected]

Eukaryotes have evolved and diversified in the context of persistent colonization by non-pathogenic microorganisms. Various resident microorganisms provide a metabolic capabil-ity absent from the host, resulting in increased ecological amplitude and often evolutionarydiversification of the host. Some microorganisms confer primary metabolic pathways, suchas photosynthesis and cellulose degradation, and others expand the repertoire of secondarymetabolism, including the synthesis of toxins that confer protection against natural enemies.A further route by which microorganisms affect host fitness arises from their modulation ofthe eukaryotic-signaling networks that regulate growth, development, behavior, and otherfunctions. These effects are not necessarily based on interactions beneficial to the host, butcan be a consequence of either eukaryotic utilization of microbial products as cues or host–microbial conflict. By these routes, eukaryote–microbial interactions play an integral role inthe function and evolutionary diversification of eukaryotes.

Eukaryotes do not live alone. They bear livingcells of bacteria (Eubacteria and Archaea),

and often eukaryotic microorganisms, on theirsurfaces and internally without any apparentill effect. Furthermore, there is now persuasiveevidence that all extant eukaryotes are derivedfrom an association with intracellular bacteriawithin the Rickettsiales that evolved into mi-tochondria (Williams et al. 2007), with theimplication that this propensity to form persis-tent associations has very ancient evolutionaryroots. In this respect, the eukaryotes are differ-ent from the bacteria, among which only a sub-set associate with eukaryotes, specifically mem-bers of about 11 of an estimated 52 phyla ofEubacteria (Sachs et al. 2011) and a tiny minor-ity of Archaea (Gill and Brinkman 2011).

The current interest in the microbiota as-sociated with eukaryotes stems from key tech-nological advances for culture-independentanalysis of microbial communities, especiallyhigh-throughput sequencing methods to iden-tify and quantify microorganisms (Caporasoet al. 2011; Zaneveld et al. 2011). The HumanMicrobiome Project (commonfund.nih.gov),MetaHIT (metahit.eu), and other initiativesare yielding unprecedented information on thetaxonomic diversity and functional capabilitiesof microorganisms associated with humans,other animals, and also plants, fungi, and uni-cellular eukaryotes (the protists), as well as abi-otic habitats (Qin et al. 2010; Muegge et al. 2011;Human Microbiome Project 2012a; Lundberget al. 2012; Bourne et al. 2013). Much of this

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research has focused on the Eubacteria, but eu-karyotic members of the microbiota, especiallythe fungi, are increasingly being investigated(Iliev et al. 2012; Findley et al. 2013).

Although driven by technological change,these culture-independent studies of the micro-biota of humans and other eukaryotes are hav-ing profound consequences for our conceptualunderstanding. In particular, there is a growingappreciation that the germ theory of disease,which has played a crucial role in improvingpublic health and food production throughthe 20th century, has also led to the widespreadbut erroneous belief that all microorganismsassociated with animals and plants are patho-gens. This outmoded perception is increasinglybeing replaced by the recognition that eukary-otes are chronically infected with benign andbeneficial microorganisms, and that diseasecan result from disturbance to the compositionor activities of the microbiota (McFall-Ngaiet al. 2013; Stecher et al. 2013).

This article reviews the pervasive impact ofsymbiosis with microorganisms on the traitsof their eukaryotic hosts and the resultant con-sequences for the evolutionary history of eu-karyotes. For the great majority of associations,the effects of symbiosis can be attributed totwo types of interaction. The first interac-tion—“symbiosis as a source of novel capabili-ties”—is founded on metabolic or other traitspossessed by the microbial partner but not theeukaryotic host. By gaining access to these ca-pabilities, eukaryotes have repeatedly derivedenhanced nutrition, defense against natural en-emies, or other selectively important character-istics. The second interaction—“the symbioticbasis of health”—comprises the improved vigorand fitness that eukaryote hosts gain throughmicrobial modulation of multiple traits, includ-ing growth rates, immune function, nutrientallocation, and behavior, even though the effectscannot be ascribed to specific microbial capa-bilities absent from the host. There is increasingevidence that the health benefits of symbiosisare commonly a consequence of microbial mod-ulation of the signaling networks by which thegrowth and physiological function of eukaryotehosts are coordinated.

This article comprises three sections: thetwo types of interaction are considered in turn,with the key patterns and processes illustratedby specific examples from a range of symbiosesin animals, plants, and other eukaryotes; andthe concluding comments address some keyunanswered questions about symbiosis in eu-karyotes. This article does not review the fulldiversity of associations made in this article onthe general principles of symbiosis in eukary-otic evolution; interested readers are referredto Douglas (2010).

SYMBIOSIS AS A SOURCE OF NOVELCAPABILITIES

The Ancient Evolutionary Roots of SymbiosesFounded on Metabolism

Eukaryotes have repeatedly gained access tocomplex metabolic capabilities by forming sym-bioses with other organisms that possess thesefunctions. By acquiring the organism (not justthe genes), eukaryotes have gained not only thegenes coding specific enzymes, but also the ge-netic and cellular machinery for their regulatedexpression.

The capacity to acquire metabolic capabili-ties by symbiosis is evident in the common an-cestor of all extant eukaryotes and can be linkedto the metabolic limitations in the lineage giv-ing rise to the eukaryotes. In particular, thislineage lacked the capacity for autotrophiccarbon fixation and aerobic respiration; thesetraits had evolved in the Eubacteria, presumablymore recently than the common ancestor ofthe two groups. The association with the a-pro-teobacterial ancestor of mitochondria probablyevolved 1–2 billion years ago. All extant eukary-otes have mitochondria or are descended frommitochondriate ancestors, raising the possibili-ty that the acquisition of mitochondria definedthe evolutionary origin of eukaryotes (Embleyand Martin 2006). (We cannot, however, ex-clude the alternative scenario that the ancestorof mitochondria was acquired by bona fide eu-karyotic cells, with the subsequent extinction ofall amitochondriate eukaryotic lineages.) Fur-thermore, the mitochondria have undergone

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substantial evolutionary diversification, includ-ing the transformation into hydrogenosomes,functionally distinct organelles that generateATP with the production of hydrogen and lackoxidative phosphorylation (Hjort et al. 2010).Hydrogenosomes have evolved multiple timesin secondarily anaerobic eukaryotes, includingvarious ciliate protists and the lorificeran ani-mals in anoxic marine sediments (van der Gie-zen et al. 2005; Danovaro et al. 2010). The evo-lutionary history of eukaryotic acquisition ofoxygenic photosynthesis by symbiosis with thecyanobacterial ancestor of chloroplasts is alsocomplex. Unlike mitochondria, which evolvedjust once, chloroplasts have evolved from twodifferent cyanobacterial groups (Keeling 2013).One cyanobacterial-derived chloroplast is alliedto Synechoccus and has been reported in the rhi-zarian amoeba Paulinella chromatophora (Na-kayama and Ishida 2009; Nowack and Grossman2012). The other lineage is in the very successfulArchaeplastids, including both algae (e.g., Rho-dophytes, Chlorophytes) and terrestrial plants,with the subsequent evolution of some algaeinto secondary, and even tertiary, chloroplastsin further groups of eukaryotes (Keeling 2013).

Without doubt, the evolutionary and eco-logical success of the eukaryotes is founded ontheir acquisition of aerobic respiration and pho-tosynthesis by symbiosis, with the transition ofbacterial symbionts to organelles. Without thesecapabilities, the eukaryotes as a group wouldhave been excluded from oxic habitats (occu-pied by the photosynthetic cyanobacteria andaerobic bacteria) and restricted to the low-ener-gy lifestyle dictated by hypoxia and anoxia. Thelocalization of electron transport chain in aero-bic respiration and photosynthesis to the intra-cellular compartments of mitochondria andplastids, respectively, provided additional evo-lutionary opportunities for eukaryotes. The re-striction of energy production to the organellarmembranes, separate from cell membrane func-tion, permitted large cell size and associated re-duction in the ratio of (cell membrane surfacearea)/(cell volume) without prejudicing energyproduction (Lane and Martin 2010). (Bacterialcells with the respiratory chain localized to thecell membrane are generally restricted to small

size with a high surface area:volume ratio, al-though some bacteria can attain relatively largecell sizes through various adaptations (Schulzand Jorgensen 2001). Furthermore, the bac-terial-derived organelles represent additionalsubcellular compartments with key roles in me-tabolism (e.g.,b-oxidation of fatty acids, steroidhormone synthesis) and signaling (e.g., apopto-sis, calcium signaling), thereby enhancing cellu-lar efficiency and capacity to mediate complexinteractions.

Symbioses Founded on Primary Metabolismof Microbial Symbionts

Symbiosis with microorganisms is not restrictedto associations involving eubacterial endosym-bionts that evolved in the distant ancestors ofmodern eukaryotes. Rather, the capacity to ac-quire bacterial or eukaryotic microorganisms isa persistent theme in the biology of the eukary-otes and a major factor shaping adaptation ofeukaryotes to different habitats and lifestyles, aswell as the evolutionary diversification of manygroups. Particularly striking examples are pro-vided by the acquisition of photosynthetic algae(eukaryotic microorganisms that are, them-selves, the product of symbiosis) by fungi, gen-erating the lichens that account for nearly half ofall Ascomycete fungal species, and by variousanimals, including the corals, whose capacityto form reefs in the photic zone is symbiosisdependent.

Furthermore, aerobic respiration and pho-tosynthesis are not the only complex metabolictraits acquired by eukaryotes through symbio-sis, although none of the microbial partnerscontributing these other traits are known tohave evolved into organelles. Nitrogen fixationand chemosynthesis are two metabolic capa-bilities that are apparently absent from the an-cestral eukaryote and have been acquired bymultiple eukaryotic groups by symbiosis. An-giosperm plants of the eurosid clade are partic-ularly predisposed to associate with nitrogen-fixing bacteria, notably the rhizobia (includingRhizobium and Bradyrhizobium in the a-pro-teobacteria) and the actinobacteria Frankia.The access to atmospheric nitrogen in these

Symbiosis in Eukaryotic Evolution

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plants facilitates their utilization of nitrogen-poor soils, for example, in early succession com-munities, and is recognized as a contributoryfactor to the evolutionary diversification of le-gumes, with their large, nitrogen-rich seedsprotected by nitrogen-containing toxins, in-cluding alkaloids (Houlton et al. 2008; Corbyet al. 2011). Nitrogen-fixing symbioses have alsobeen reported in protists, for example, in ma-rine diatoms (Foster et al. 2011), and some an-imals, notably the wood-feeding shipwormsand some termites (Lechene et al. 2007; Desaiand Brune 2012).

Chemosynthesis (the capacity to fix carbondioxide using the energy derived from the oxi-dation of reduced substrates, such as hydrogen,hydrogen sulfide, and methane) is widely ex-ploited by animals. The habitats where theseassociations flourish are zones of oxic/anoxicmixing, including aquatic sediments, hydro-thermal vents, and hydrocarbon seeps. Manyanimals in these habitats bear chemosyntheticsymbionts on their body surface (e.g., annelidworms), in gills (e.g., bivalve mollusks), or in acentral tissue called the trophosome (in pogo-nophoran worms), and many display reducedcapacity for feeding and a digestive tract that ismuch reduced or absent (Petersen et al. 2011;Roeselers and Newton 2012).

Various eukaryotes have compounded themetabolic limitations of their eukaryotic inher-itance by the loss of further metabolic capabil-ities. Reduction of metabolic scope is particu-larly evident in the animals, which, as a group,lack the capacity to synthesize at least nine of the20 protein amino acids (the essential amino ac-ids) and various cofactors required for the func-tion of enzymes central to metabolism (e.g.,various vitamins). The arthropods have, addi-tionally, lost the capacity for sterol synthesis.Generally, these lost metabolic capabilities com-prise many reactions and tend to be energetical-ly demanding (Wagner 2005). Organisms withan ample dietary supply of amino acids or co-factors would gain no advantage from retainingthese capabilities (relaxed selection) and argu-ably the selective advantage of energetic effi-ciency from losing them. As predicted fromthese considerations, the sponges and choano-

flagellates (the most basal animals, and closestrelative of animals, respectively) feed on livingorganisms, especially bacteria, which provide anutritionally balanced diet.

Repeatedly in the evolution of animals, sym-biosis with microorganisms has enabled ani-mals to escape from the nutritional constraintof feeding on a nutritionally balanced diet.Many microbial symbionts of animals haveprobably evolved by the progressive delay in an-imal digestion of food-associated microorgan-isms, raising the evolutionary scenario of a shiftin the animal to a poor-nutrient diet coupled tothe transition of nutrient acquisition by diges-tion to biotrophic release from intact microbialcells. Two diets for which animals require nu-tritional support from microbial symbiontsare vertebrate blood and plant sap, as indicatedby the possession of microbial symbionts byall animals feeding on these diets through thelife cycle (Buchner 1965). For example, symbi-otic microorganisms are borne in the gut orspecialized cells of the obligately blood-feedingleeches, ticks, lice, and bedbugs, and they arebelieved to provide their animal hosts with Bvitamins. Plant sap feeding has also evolvedmultiple times, but only among insects of theorder Hemiptera (including whiteflies, aphids,cicadas, and planthoppers). These insects deriveessential amino acids, nutrients in short supplyin the plant phloem and xylem sap, and pos-sibly vitamins from their microbial partners(Douglas 2009).

Microbial symbioses have also played an im-portant role in animal utilization of structuralplant material, including lignocellulose. Her-bivory has evolved multiple times in the verte-brates and, where investigated, the extraction ofenergy from the ingested cellulose is dependenton cellulolysis by symbiotic bacteria in an an-aerobic portion of the gut, known as the fer-mentation chamber. The waste products of thebacterial fermentation are short-chain fatty ac-ids, such as acetic acid and butyric acid. Theseproducts are transported into the epithelial cellsof the animal and via the blood to other organs,where they are used as a substrate for aerobicrespiration (Karasov and Douglas 2013). Micro-bial degradation of ingested lignocellulose and

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fermentation of cellulose are generally less im-portant for invertebrate, especially insect, her-bivores, but can make an important contribu-tion to the carbon economy of insects, such astermites, wood roaches, wood wasps, and somebeetles, that feed on wood products of very lownutritional content (Geib et al. 2008; Suen et al.2010; Adams et al. 2011). Multiple factors maycontribute to the greater contribution of mi-crobial symbionts to vertebrates than insectsfeeding on structural plant material. They in-clude the greater anatomical barriers to a fer-mentation chamber in a small, highly aerobicinsect than in a larger vertebrate with a predom-inantly anoxic gut lumen, and the widespreadoccurrence of intrinsic cellulases (i.e., of ani-mal origin) in invertebrates, including many in-sects, but apparently in no vertebrates (Davisonand Blaxter 2005; Calderon-Cortes et al. 2012).Consequently, microbial symbiosis is a generalprinciple for the evolution of herbivory in ver-tebrates, but its significance for invertebrate an-imals has to be assessed on a case-by-case basis.

Symbioses and Secondary Metabolism

Symbiotic microorganisms can also contributeto the secondary metabolism of their eukary-otic hosts. “Secondary metabolism” refers tometabolic reactions and pathways that do notcontribute directly to organismal growth andreproduction but that can be crucial to the fit-ness of the organism in the natural environ-ment. Secondary metabolism includes the syn-thesis of toxins, antibiotics, pheromones andallomones, and pigments, as well as the degra-dation of toxins and other secondary metabo-lites. The capacity of eukaryotes for secondarymetabolism varies widely, even among closelyrelated species, and the technical difficulties inworking with secondary metabolism has led tovarious failures to recognize the role of micro-organisms in the secondary chemistry ofsome eukaryotes, as well as some unsubstanti-ated claims of symbiotic secondary metabolism.

Two complementary approaches provide anexcellent test for the role of microbial symbiontsin secondary metabolism: identification of therelevant genes in the sequenced genome of the

microbial partner, and perfect correlation be-tween expression of the secondary metaboliccapability in the host and presence of the mi-crobial partner with these genes. This can beillustrated by the contribution of bacterial sym-bionts to the production of a biologically activeclass of secondary compounds, the polyketides.For example, the beetle Paederus bears bacte-ria of the genus Pseudomonas, which have thegenes for polyketide synthase and linked re-actions required for the production of the poly-ketide profile of these animals (Piel et al. 2004a),and elimination of the Pseudomonas in Paederusresults in loss of polyketide production (Kell-ner 2001). Similarly, many grasses are protectedfrom herbivory by alkaloids that are synthe-sized by clavicipitaceous fungal endophytesthat ramify through the plant tissues (Fletcherand Harvey 1981), and the genetic basis of thefungal synthesis of one key alkaloid loline by thefungal endophyte in the grass Lolium has beenestablished (Spiering et al. 2005). Secondarymetabolites synthesized by microbial symbiontshave also been shown to contribute to the pro-tection against predators and pathogens in mul-tiple benthic marine animals, including spong-es (Piel et al. 2004b), bryozoans (Sharp et al.2007), and tunicates (Kwan et al. 2012).

Just as secondary chemistry is very diverse,so are the routes by which eukaryotes havegained access to these often-complex biosyn-thetic pathways for secondary metabolite syn-thesis. This is illustrated by recent research onthe source of carotenoid pigments in animals.Carotenoids have antioxidant properties andare also used as pigments. Animals, generally,are unable to synthesize carotenoids, and manyderive carotenoids from their diet. However, thecarotenoid profile of some insects is indepen-dent of a dietary supply. In one group of in-sects, the whiteflies, the carotenoids are of sym-biotic origin, with the carotenoid biosynthesisgenes coded by its bacterial symbiont Portiera(Sloan and Moran 2013). In two other groups,the aphids and spider mites, the carotenoidbiosynthesis genes are encoded in the animalgenome, following independent lateral transfersfrom fungi (Moran and Jarvik 2010; Altinciceket al. 2012; Novakova and Moran 2012).





There is one further aspect to secondarymetabolism that deserves careful consideration:the contribution of resident microorganismsto the detoxification of secondary metabolites.This is currently a “hot topic” in biomedicalresearch, in the light of evidence that the activ-ities of the gut microbiota can determine thehalf-life and metabolic fate of orally adminis-tered drugs, with clinically important impli-cations for drug efficacy and toxicity (Haiserand Turnbaugh 2012; Holmes et al. 2012; Haiseret al. 2013). An analogous issue has been ofpersistent concern in the discipline of herbi-vory for decades. Although herbivorous ani-mals generally possess a diverse array of detox-ifying enzymes, including cytochrome P450monooxygenases, glutathione S-transferases,and esterases (Despres et al. 2007), symbioticmicroorganisms have long been invoked to me-diate the detoxification of plant allelochemicals(Jones 1984; Berenbaum 1988; Dillon and Dil-lon 2004). Supportive evidence comes from afew systems. The microbiota in the foregut fer-mentation chamber (the rumen) of ruminantmammals has the potential to detoxify ingestedallelochemicals in the food, as illustrated by thereported capacity of the rumen microbiota ofindigenous Indonesian cattle to degrade thetoxic amino acid mimosine in the tropical le-gume Leucaena leucocephala (Cheeke 1994).Similarly, the capacity of reindeer to use rein-deer moss (a lichen, Cladonia rangiferina) hasbeen attributed to a bacterium Eubacteriumrangiferina in the reindeer rumen, which canmetabolize usnic acid, an abundant metabolitein the lichen that is toxic to most animals(Sundset et al. 2008, 2010). Plant allelochemicaldetoxification has also been implicated in cer-tain insect symbioses, notably bark-feeding bee-tles and leaf-cutting ants. The gut microbiota ofthe mountain pine beetle Dendroctonus ponder-osae include bacteria of the genera Pseudomo-nas, Serratia, Rahnella, and Burkholderia thatbear genes involved in the degradation of ter-penes, the principal defensive compounds ofthe trees infested by these beetles (Adams et al.2013). The leaf-cutting ant Acromyrmex echina-tior maintains, and feeds on, the symbiotic fun-gus Leucocoprinus gongylophorus in its nest. The

fungus genome has multiple genes for laccases(a type of phenoloxidase). Enzymatically ac-tive laccase enzyme is passed through the gutof ants feeding on the fungus and releasedonto plant material, where it can degrade plantcompounds, such as tannins and flavonoids (DeFine Licht et al. 2013).

THE SYMBIOTIC BASIS OF HEALTH

The function of all living organisms is orches-trated by signaling networks that coordinateprocesses within individual cells and, for mul-ticellular forms, also among cells, tissues, or-gans, and so on. It has long been known thatthese regulatory networks can be manipulatedby pathogens and parasites, with various con-sequences ranging from the reorganization ofthe cytoskeleton and intracellular traffickingto the restructuring of host growth patterns, re-productive schedules, and behavior (Gandonet al. 2002; Bhavsar et al. 2007; Hughes et al.2012). It is now becoming increasingly clearthat the nonpathogenic resident microorgan-isms can also modulate the signaling networksof their eukaryotic hosts and that these manip-ulations are generally advantageous for the host.Although the data are still fragmentary, variouslines of evidence are consistent with the hy-pothesis that eukaryotes derive health benefitsfrom symbiosis because their regulatory net-works are structured to function in the contextof interactions with the resident microbiota.

The health benefits of symbiosis can beillustrated by plant-growth-promoting rhizo-bacteria (PGPRs), including strains of Azo-spirillum, Bacillus subtilis, Pseudomonas putida,and Enterobacter cloacae. As the term suggests,PGPRs are associated with plant roots and pro-mote plant growth. The underlying processesare complex and variable, but very commonlyinvolve the capacity to synthesize signaling mol-ecules, including plant hormones, for example,the auxin indole-3-acetic acid (IAA), and vola-tiles, for example, 2,3-butanediol (Spaepenet al. 2007; Lugtenberg and Kamilova 2009;Roca et al. 2013). The best-studied interactionrelates to PGPR-derived IAA, which triggersincreased root branching and a higher densi-

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ty of root hairs. This altered root morphologyenhances nutrient uptake from the soil, there-by promoting plant growth (Dobbelaere et al.1999; Steenhoudt and Vanderleyden 2000). Theimportant implication of these results is thatthe plant regulatory networks controlling thepattern of plant growth are not structured togenerate optimal growth of microbe-free plants,which is achieved only in the context of signalexchange with associated rhizosphere bacteria.

The IAA-producing PGPRs are an exampleof resident microorganisms that influence hostgrowth by the release of molecules that are alsoan integral part of the host-signaling network(in this case, a plant hormone). Other com-pounds produced by microbial symbionts arenot known elements of the host-signaling net-work but, nevertheless, act to modulate (ampli-fy or dampen) host signaling. One examplecomes from research on the association betweenthe Drosophila fruit fly and its gut microbiota.When the microorganisms are eliminated, theDrosophila display depressed development ratesand elevated levels of lipid and circulating glu-cose, similar to the phenotypic traits of flieswith impaired insulin signaling (Shin et al.2011). Flies infected with a mutant of a domi-nant bacterial symbiont, Acetobacter pomorum,that cannot produce the enzyme pyrroloquino-line quinone-dependent alcohol dehydrogenase(PQQ-ADH) also display these traits, togetherwith reduced expression of the genes for insu-lin-like peptides (dilp-3 and dilp-5). PQQ-ADHmediates the oxidation of ethanol to acetic acid.When acetic acid was supplied in the food, de-velopment rates, lipid and glucose contents, anddilp gene expression in flies bearing the mutantbacteria shifted to values found in conventionalflies. These data suggest that acetic acid pro-duced by wild-type A. pomorum stimulates in-sulin signaling in the fly. Although the processesby which acetic acid interacts with insulin sig-naling are unknown, the resultant speeding oflarval development is advantageous for the in-sect, which develops in rotting fruit and mustcomplete larval development before the fruitresources are exhausted. As with the IAA-pro-ducing PGPRs associated with plant roots, theacetic-acid-producing bacteria in fruit fly guts

promote host health and vigor by increasing theamplitude of host signaling.

The hyperlipidemia and hyperglycemiadisplayed by Drosophila containing mutant A.pomorum are reminiscent of the elevated lip-id and glucose levels in the laboratory mousetreated with antibiotics that alter the composi-tion of the gut microbiota (Cho et al. 2012). Thegut microbiota of the mouse can also be alteredby diet or mutation, especially of the mouseimmune system and nutrient signaling, withcorrelated phenotypic lesions, especially in nu-trient allocation and immune function (Mas-lowski and Mackay 2011; Claesson et al. 2012;Maynard et al. 2012). Transplant studies sug-gest that the altered microbial community like-ly contributes to the altered host phenotype.When introduced to germ-free control mice(reared on the standard diet/of wild-type geno-type), the recipients display similar deleteriousphenotypic traits (Vijay-Kumar et al. 2010;Smith et al. 2013). These various data sets re-inforce the growing appreciation that the com-position and activities of the resident micro-biota are important for host health, such thatperturbation of the microbiota can contributeto chronic ill health, a condition known as “dys-biosis.” The concept of dysbiosis, first coined acentury ago (Metchnikoff 1910), has gained rel-evance in the context of hypotheses put forwardto account for the increase in immunologicaland metabolic disease in humans, with reducedexposure of children to environmental micro-organisms (hygiene hypothesis) or to specificmembers of the human resident microbiota(disappearing microbiota hypothesis) as pos-sible drivers (Strachan 1989; Blaser and Falkow2009).

Given the fitness costs of dysbiosis, the sus-ceptibility of the regulatory circuits of animalsand plants to modulation by their resident mi-crobiota appears as a very real vulnerability.How is it that eukaryote-signaling networksare not insulated from the influence of residentmicroorganisms, but appear to be under jointhost–microbial control?

Two sets of processes may contribute to thesymbiotic basis of health. The first is that micro-organisms provide a reliable cue for current or





future environmental conditions. Eukaryotesthat respond appropriately to the microbialproducts, that is, display regulated changes ingrowth or developmental patterns, behavior,and the like, would be at a selective advantage.For example, the profile of metabolic end-prod-ucts from bacteria associated with rotting fruitmay provide a reliable cue for the longevity ofthe fruit resource, enabling Drosophila larvae totitrate their developmental time to environmen-tal circumstance. Specifically, the amplified in-sulin production in response to Acetobacter-de-rived acetic acid increases developmental rates,and this may be highly adaptive in the naturalenvironment. Microbial products are used ascues by various eukaryotes. For example, a sul-fonolipid released from specific bacteria, Algo-riphagus machipongonensis, induces colony for-mation in choanoflagellates (Alegado et al.2012); N-acyl homoserine-lactones (quorum-sensing molecules) released from Vibrio bacteriapromote settling of motile zoospores of thegreen alga Enteromorpha (Joint et al. 2002);and compounds released from the bacteriumPseudoalteromonas associated with the substratein marine environments promote the settlingand metamorphosis of barnacle larvae (Had-field 2011).

I describe these various compounds as cuesbecause their production by microorganisms isindependent of the response of the eukaryotichost, that is, whether and how a eukaryote de-rives information from microbial compoundshas no effect on their production by the micro-organisms. If a microbe-derived compound is areliable cue, the signaling network of the eu-karyote may evolve to increasing dependenceon that cue through reduced responsiveness toother environmental factors, and this may resultin dependence on specific microbial productsfor sustained function of the signaling networkand, ultimately, host health.

The alternative basis for microbial impactson host-signaling networks derives from con-flict between the microorganisms and host.Conflict is inevitable because of incomplete se-lective overlap between a eukaryotic host and itsresident microbiota. A eukaryote is a nutrient-rich patch in which microorganisms tolerant of

the immune system and other defenses canproliferate, and it is also frequently a route fordispersal. Consequently, many microorganismshave no selective interest in the reproductiveoutput of their host. (Exceptionally, the variousmaternally inherited forms are in conflict withthe host over host sex ratio.) Thus, microorgan-isms in the animal gut may favor hyperphagiadespite its negative consequences for host fit-ness, modulate gut peristalsis rates to optimizetheir residence time at the expense of optimalnutrient absorption by the host, and dampenimmune responses to favor their persistence(de La Serre et al. 2010; Round et al. 2011; Mat-sumoto et al. 2012; Maynard et al. 2012).

Counterintuitively, host–microbial conflictover the amplitude of host-signaling pathwayscan lead to host dependence, but only where theprevalence of the interacting microorganismsin the host population is very high. This impor-tant effect was first shown by research on therelationship between a parasitic wasp, Asobaratabida, and the vertically transmitted bacteriumWolbachia. Several lines of evidence suggestthat Wolbachia induces oxidative stress and alinked dampening of apoptotic signaling, prob-ably as a result of disruption of iron metabo-lism (Kremer et al. 2009). As a likely conse-quence, elimination of Wolbachia by antibiotictreatment results in massive apoptosis (pro-grammed cell death) of the ovaries, leaving thewasp reproductively sterile. It has been arguedthat, over evolutionary time, the wasp apoptoticpathways have gained heightened responsive-ness, in compensation for the inhibitory manip-ulation by Wolbachia (Pannebakker et al. 2007).Importantly, this effect is constitutive (presum-ably because Wolbachia is always present undernatural conditions), with the consequence thatappropriate apoptotic signaling requires themanipulative intervention of the Wolbachia bac-teria. Analogous host compensation for micro-bial manipulation may contribute to the mul-tiple demonstrations of interactions betweenthe resident microbiota and the developmental,nutritional, neurological, and immunologicalhealth of animals (Stappenbeck et al. 2002;Smith et al. 2007; Bravo et al. 2011; Olszaket al. 2012). Additionally, host dependence on

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microbial products for sustained health may beretained in eukaryotic lineages, where the con-flict is resolved because of the sheer complexityof signaling networks. Elimination of microbialmodulation of one pathway may be selectedagainst because the multiple, knock-on effectson linked signaling pathways are highly delete-rious. Ultimately, the symbiotic basis of hosthealth may only be explicable in the context ofthe deep evolutionary history of interactionsbetween eukaryotes and their resident micro-biota.

CONCLUDING COMMENTS

It has taken a long time for biologists gener-ally to appreciate the significance of associa-tions with symbiotic microorganisms for eu-karyotes. Despite the discovery of the dualnature of lichens (fungi and algae/cyanobacte-ria) and near-ubiquitous associations betweenplant roots and mycorrhizal fungi in the 1800s,symbioses were treated as mere curiosities ofnature through much of the last century. Theoverwhelming molecular evidence for bacterialorigins of mitochondria and plastids, togetherwith the realization that the resident microbio-ta is vital for the human health and productivi-ty of crops and livestock has, at last, placed sym-biosis in the mainstream of biology. And yet,multiple questions central to our understand-ing of the function and evolution of symbiosesremain.

Tremendous opportunities are being pro-vided by the latest sequencing technologies. Al-though currently used mostly to catalog thecomposition and activities of bacterial com-munities (Human Microbiome Project 2012b),these methods are starting to be applied to in-vestigate both the diversity of eukaryotic micro-bial symbionts and how microbial communitiesare structured (Costello et al. 2012). A key un-resolved question is whether the functionaltraits of resident microorganisms can be pre-dicted from their taxonomic composition.Perhaps such a relationship is frequently con-founded by microbe–microbe and microbe–host interactions, resulting in spatiotemporalvariation in the traits of microorganisms. Re-

cent advances in single-cell imaging and sin-gle-cell sequencing (Pamp et al. 2012; Perniceet al. 2012; Lasken 2013) offer unprecedentedopportunities to investigate such heterogeneity.

These ecological issues segue into long-standing evolutionary questions, particularlywhether the microbiota can influence the spe-ciation patterns and evolutionary diversifica-tion of their hosts (Brucker and Bordenstein2013). In certain instances, evolutionary changemay be facilitated by among-microbe transfer ofsymbiosis-related gene clusters via “symbiosisislands” (Finan 2002), equivalent to pathoge-nicity islands in pathogenic bacteria, or by thedisplacement of one microbial partner by an-other taxon with different traits (Koga et al.2013; Toju et al. 2013). With the dramaticallyimproving genomic technologies, it is becom-ing increasingly feasible to answer these funda-mental questions.

We should also recognize that most currentresearch is focused on a tiny subset of the di-versity of symbioses, notably a few phyla of Eu-bacteria associated with animals, especially hu-mans and laboratory mice. A major outstandingissue is the taxonomic and functional diversityof symbioses across the evolutionary radiationof eukaryotes. In particular, relatively little isknown about symbioses involving protists aseither host or symbiont, even though the pro-tists account for most of the evolutionary diver-sity of eukaryotes. Some of these associationsare apparently without parallel in animals orplants, for example, motility conferred on largeprotists by spirochete ectosymbionts (Clevelandand Grimstone 1964), and protection frompredators by extrusive structures associatedwith bacterial symbionts of the ciliate Euplodi-nium (Petroni et al. 2000). Reinvigorated re-search on symbioses involving protists has thepotential to expand and modify our concept ofthe general principles of symbiosis.

ACKNOWLEDGMENTS

This work is supported by NIH grant1R01GM095372-01, NSF grant BIO 1241099,and the Sarkaria Institute for Insect Physiologyand Toxicology.





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2014; doi: 10.1101/cshperspect.a016113Cold Spring Harb Perspect Biol Angela E. Douglas Symbiosis as a General Principle in Eukaryotic Evolution

Subject Collection The Origin and Evolution of Eukaryotes

FunctionEukaryotic Gen(om)e Architecture and Cellular The Persistent Contributions of RNA to

Jürgen Brosius

Mitochondrion Acquired?Eukaryotic Origins: How and When Was the

Anthony M. Poole and Simonetta Gribaldo

the Plant KingdomGreen Algae and the Origins of Multicellularity in

James G. Umen

Bacterial Influences on Animal OriginsRosanna A. Alegado and Nicole King

Phylogenomic PerspectiveThe Archaeal Legacy of Eukaryotes: A

Lionel Guy, Jimmy H. Saw and Thijs J.G. Ettemathe Eukaryotic Membrane-Trafficking SystemMissing Pieces of an Ancient Puzzle: Evolution of

Klute, et al.Alexander Schlacht, Emily K. Herman, Mary J.

OrganellesCytoskeleton in the Network of Eukaryotic Origin and Evolution of the Self-Organizing

Gáspár Jékely LifeIntracellular Coevolution and a Revised Tree ofOrigin of Eukaryotes and Cilia in the Light of The Neomuran Revolution and Phagotrophic

Thomas Cavalier-Smith

from Fossils and Molecular ClocksOn the Age of Eukaryotes: Evaluating Evidence

et al.Laura Eme, Susan C. Sharpe, Matthew W. Brown,

Consequence of Increased Cellular ComplexityProtein Targeting and Transport as a Necessary

Maik S. Sommer and Enrico Schleiff

SplicingOrigin of Spliceosomal Introns and Alternative

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How Natural a Kind Is ''Eukaryote?''W. Ford Doolittle

Eukaryotic Epigeneticsand Geochemistry on the Provenance ofImprints of Bacterial Biochemical Diversification Protein and DNA Modifications: Evolutionary

et al.L. Aravind, A. Maxwell Burroughs, Dapeng Zhang,

Insights from Photosynthetic EukaryotesEndosymbionts to the Eukaryotic Nucleus? What Was the Real Contribution of

David Moreira and Philippe Deschamps

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Phylogenomic PerspectiveThe Eukaryotic Tree of Life from a Global

Fabien BurkiComplex LifeBioenergetic Constraints on the Evolution of

Nick Lane

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