Bacteriocyte-AssociatedSymbionts of Insects

A variety of insect groups harbor ancientprokaryotic endosymbionts

Nancy A. Moran and Aparna Telang

Endosymbiosis has been pro-posed as an evolutionary in-novation that underlies the

success and diversification of somegroups of organisms by enablingthem to exploit new ecological niches(e.g., Margulis and Fester 1991,Maynard Smith and Szathmary1995). Three kinds of informationare needed to evaluate this proposal.First, it is necessary to know some-thing of the role played by symbiontsin the lives of hosts: Are their effectsones that allow use of novel ecologi-cal niches? Second, information isneeded on the evolutionary historyof endosymbioses: How old are theyand, in particular, do endosymbioticinfections predate diversification ofmajor host groups? Finally, the evo-lutionary consequences for hosts ofacquiring new capabilities in the formof symbiotic associations must beevaluated: Are these novel traitsstable? Do they impose constraintsas well as new capabilities on hosts?

Recent studies have begun to shedlight on these questions for bac-teriocyte-associated endosymbiosesin insects. Bacteriocytes (or myce-tocytes) are cells of animal hosts thatare positioned at characteristic loca-tions in the body and appear to bespecialized for housing bacteria (Fig-ure 1; Buchner 1965, Dasch et al.

Nancy A. Moran (e-mail: nmoran@u.arizona.edu) is a professor in the De-partment of Ecology and EvolutionaryBiology, and Aparna Telang is a gradu-ate student in the Interdisciplinary Pro-gram in Insect Science, at the Universityof Arizona, Tucson, AZ 85721. © 1998American Institute of Biological Sciences.

Endosymbiosis appears tofacilitate diversificationwithin ecological niches

that would otherwisebe inadequate, butdependence on anendosymbiont may

simultaneously enforcesome restrictions on

host evolution

1984). Bacteriocyte associates arefound in many insect orders and fami-lies and have been estimated to occurin 10% of insect species (Douglas1989).

Before the past decade, much ofthe knowledge of bacteriocyte asso-ciations was based on extensive mi-croscopical studies by Paul Buchnerand his associates. His fascinatingcompilation of this work (Buchner1965) details astonishing diversityin animal-microbe associations.Most of this diversity is still unex-plored, in large part because intra-cellular symbionts typically cannotbe cultured outside of their hosts.Since 1990, however, molecular stud-ies on a handful of endosymbioseshave yielded considerable insight intotheir evolutionary histories and intothe specific adaptations to symbiosis

that are found in the bacteria them-selves. Some of the most revealing ofthese studies concern the bacterio-cyte-associated, mutualistic endo-symbionts of insects.

The microscopical explorationsperformed by Buchner and his asso-ciates gave only hints regarding therelationships of insect endosymbiontsto other bacterial groups and to otherendosymbionts. The characterizationof these bacteria and their phyloge-netic placement relative to free-liv-ing prokaryotes remained almost acomplete mystery until the develop-ment of molecular methods, in par-ticular methods for determiningnucleotide sequences. These meth-odological advances have revolution-ized understanding of prokaryoteevolution in general by providing asurplus of relatively reliable charac-ters for phylogeny reconstruction. Amajor additional advantage of DNAsequences as phylogenetic charac-ters is that they can be obtained fornoncultivable organisms. In particu-lar, sequences of the 16S ribosomalRNA (rRNA) genes have been usedfor the phylogenetic and taxonomicplacement of many bacteria, includ-ing endosymbionts, that were previ-ously unclassifiable (Maidak et al.1996).

In this article, we provide a briefoverview of recent work on the na-ture ofthe relationships between in-sect hosts and bacteriocyte-associ-ated organisms (see Dasch et al. 1984,Dadd 1985, Douglas 1989 for de-tailed reviews). We then consider inmore detail the emerging picture ofendosymbiont evolution presented

April 1998295

by molecular phylogenetic findings.Finally, we consider how such stud-ies bear on the following sets of ques-tions: First, what is the distributionof endosymbiotic bacteria on theprokaryotic tree of life? Which bac-terial groups have produced lineagesthat are endosymhiotic in insects?Are these related to one another asmembers of one or a few monophyl-etic groups specialized for endosym-biotic living? Or do endosymbiontsof different insect lineages representindependent origins of endosymbi-otic bacteria? Second, do the bacte-ria show long-term patterns ofcospeciation with their hosts, as re-

Figure 1. Electron mi-crographs oiBuchnerain aphid bacteriocytes.(a) Bacteriocytes adja-cent to the midgut ofthe aphid Uroleuconsonchi. Bar = 10 |im.(b) Bacteriocytes adja-cent to the ovarioles ofU. sonchi. Bar = 5 |im.(c) Bacteriocytes adja-cent to secondary en-dosymbionts housed inseparate cells withinthe aphid Uroleucon ja-ceae; arrowhead pointsto host cell plasmamembrane. Bar = 2 |im.b, Buchnera; e, midgutepithelium; 1, midgutlumen; m, mitochon-dria; n, host cell nucle-us; ov, ovariole; s, sec-ondary endosymbiont.

fleeted in parallelphylogenies? Whatages can we infer forthe endosymbiosesof insects? Third,what adaptations ofendosymbiotic bac-teria function in themutualistic interac-tion with hosts?Fourth, what are thelong-term evolution-ary consequences ofendosymbiosis, asreflected in geneticchanges in the sym-biotic bacteria?

In the arena ofmolecular studies,the most intensiveresearch on insectendosymbioses has

been on aphids, small insects thatfeed by tapping phloem sap of vascu-lar plants, and on their primary en-dosymbionts, Buchnera aphidicola.Molecular studies of this associationhave been reviewed recently in moredetail (P. Baumann et al. 1997).

The mutualistic nature ofbacteriocyte associationsBuchner considered bacteriocyte as-sociates to be mutualists that benefitinsect hosts by providing limitingnutrients. His views were based onmyriad observations of specializedmechanisms for the transmission and

housing of endosymbionts by hosts.For many insect symbioses, benefi-cial effects of the bacteria on theirhosts are supported by experimentalevidence: Antibiotic or heat treat-ments causing loss of symbionts areaccompanied by reductions in per-formance, including lowered growthand survivorship and loss of repro-ductive abilities (e.g., Nogge 1976,Ishikawa and Yamaji 1985, Ohtakaand Ishikawa 1991, Prosser andDouglas 1991, Sasaki et al. 1991,Costa et al. 1993, Heddi et al. 1993;additional studies cited in Koch 1967and Dasch et al. 1984). Many hosts,including aphids and other insectgroups that feed exclusively on plantsap, can grow or reproduce only withintact symbionts. Others, includingSitophilus weevils, which feed ongrain, can survive and reproducewithout the symbionts, but they growmore slowly and are smaller thansymbiotic hosts (Heddi et al. 1993).Although interpreting studies of sym-biont-free hosts is complicated bythe possibility that antibiotic or heattreatment may have direct effects onhost metabolism, the preponderanceof evidence points to a beneficialrole of bacteriocyte associates forhost insects.

A nutritional dependence of hostson their symbionts presumably ex-plains why many insect taxa possess-ing bacteriocytes use narrow andnutritionally unbalanced diets, suchas the phloem sap of plants or theblood of vertebrates. Because ani-mals lack biosynthetic pathways thatare typically present in prokaryotes,most animals must ingest a largenumber of essential nutrients. Alter-natively, a microbial associate canprovide the missing nutrients, allow-ing animal hosts to exploit resourcesthat would otherwise be nutrition-ally deficient. Examples includemembers of the Hemiptera that usetheir sucking mouthparts to feedsolely on plant phloem or xylem sap(Dasch et al. 1984}. Plant phloemsap contains free amino acids butlacks some amino acids that are es-sential nutrients for animals. An-other dietary habit associated withendosymbiosis is restriction to verte-brate blood, which is lacking in somevitamins. Endosymbiosis connectedto blood feeding throughout the lifecycle has evolved independently in a

296 BioScience Vol. 48 No. 4

number of insect orders, includingDiptera, Hemiptera, and Mallophaga(Buchner 1965, Dasch et al. 1984).Some insects with more generalizeddiets also have endosymbionts; theseinclude cockroaches, some ants, andsome weevils. In ants, however, en-dosymbiosis may be associated withdependence on nutritionally deficientplant fluids.

The general conclusion of a nutri-tional role for symbionts has beenaccepted by almost all researcherswho have studied bacteriocyte asso-ciates of insects (e.g., Buchner 1965,Koch 1967, Dasch et al. 1984, Dadd1985). Yet definitive evidence thatendosymbionts provide essentialnutrients to insect hosts is rare. Thebest evidence pertains to the provi-sion of tryptophan by Buchnera, theendosymbionts of aphids (Douglasand Prosser 1992, Lai et al. 1994).Also, some evidence suggests thattsetse symbionts provision hosts withB vitamins (Nogge 1981).

How are endosymbiontstransmitted?Bacteriocyte-associated endosym-bionts are typically maternally in-herited, often through complex de-velopmental events that ensuretransovariole transfer from themother to the developing egg orembryo (Buchner 1965, Dasch et al.1984). In the case of aphids andBucbnera, the bacteria are housedwithin bacteriocytes that are groupedinto a loose organ witbin the bodycavity, in the vicinity of the develop-ing ovarioles (Figure lb; Buchner1965, Hinde 1971a, 1971b). Thematernal bacteriocyte adjacent to anembryo near tbe blastoderm stageforms a small opening through whicba bacterial inoculum passes. Tbe in-oculum then moves through tbe in-tervening hemolymph and enters anearby opening on tbe oocyte sur-face. During early embryonic devel-opment, the presumptive bacterio-cytes form, and the inoculatingbacteria migrate into tbese cells.

This apparent fine tuning of tbeinfection process is typical ofbacteriocyte-associated symbionts ininsects and suggests a long bistory ofselection favoring bost adaptationsthat help to maintain tbe associa-tion. However, the particulars of tbe

infection process vary tremendouslyamong insect groups (e.g., Sacchi etal. 1988,Hypsal993).Forexample,in wbiteflies, an entire maternal bac-teriocyte (or, in some species, severalbacteriocytes) is transferred into eachegg, witb the maternal nucleus laterdegenerating (Bucbner 1965, Costaetal. 1993).

Secondary endosymbiontsSome insects harbor so-called "sec-ondary" endosymbionts that coexistin the same individual hosts with thebacteriocyte-inbabiting primary en-dosymbionts. These secondary sym-bionts are maternally inherited butappear not to share a long evolution-ary bistory witb their hosts. Tbey areusually not present in the bacterio-cytes tbat bouse primary endosym-bionts (Figure lc; Buchner 1965).Tsetse fly secondary symbionts oc-cur in midgut cells, by contrast to tbeprimary symbionts, wbich are foundin specialized bacteriocytes in tbeanterior gut (Pinnock and Hess1974). Aphid secondary endosym-bionts occur mostly in a sbeatblikesyncytium bordering the bacterio-cytes tbat house Buchnera (Bucbner1965, Fukatsu and Isbikawa 1993,Chen and Purcell 1997). Whiteflysecondary endosymbionts do inhabitthe bacteriocytes in wbich primaryendosymbionts are found but areclumped irregularly within vacuoles(Costa etal. 1993,1996).

Effects of secondary endosym-bionts on host fitness are not clear.Bucbner (1965) described secondaryendosymbionts of apbids as "recentlyacquired guests wbich are still inneed of adaptation." They are moresporadic in tbeir numbers and pres-ence among species and even in theirdistribution among individuals of thesame species, as bas been sbown foraphids (Fukatsu and Ishikawa 1993,Chen and Purcell 1997), whiteflies(Costa et al. 1993), and tsetse flies(Pinnock and Hess 1974). Cben andPurcell (1997) found tbat secondaryendosymbionts are present in onlysome strains of pea apbid {Aeyrtho-siphon pisum) and are identical in16S rDNA sequence to secondaryendosymbionts of rose aphid (Mijcro-siphum rosfle). Vertical transmissionfrom the time of tbe common ances-tor of tbese two bosts would almost

certainly have resulted in some se-quence divergence; for example, theBuchnera 16S rDNA sequences forsimilarly closely related genera withinAphididae all differ by more tban2% (Munsonet al. 1991).

The observed sequence identity ofthe secondary symbionts implies theoccurrence of either horizontal trans-fer between species or recent, inde-pendent infections by a bacteriumthat is widely distributed in tbe envi-ronment. Furthermore, the sym-bionts could be experimentally trans-ferred by injecting hemolympb frominfected clones into uninfected clonesof the same or different species. Thus,although maternal transmission wasobserved (Chen and Purcell 1997)and is presumably the usual route ofinfection, tbese secondary endosym-bionts must also undergo some trans-fer among bost lineages, includingtransfer between species. Their spo-radic distribution within host spe-cies implies that unlike most primaryendosymbionts, they are not requiredfor bost development or reproduc-tion. Currently, there is almost noevidence indicating whether tbeireffects on bost fitness are positive,negative, or neutral.

The phylogenetic distributionof insect endosymbiontsMolecular pbylogenetic results basedon 16S rDNA sequences show thatwithin each of tbe insect groups ex-amined so far, the primary endosym-bionts descended from a single an-cestor, as indicated by their forminga well-supported clade. This resulthas been obtained for the primaryendosymbionts of aphids (Munsonet al. 1991, Moran et al. 1993), mea-lybugs (Munson et al. 1993), wbite-flies (Clark et al. 1992), carpenter ants[Candidatus camponotii; Schroder etal. 1996),5;top/7/7M5 weevils (Campbelletal. 1992), tsetse flies (Aksoy 1995),and cockroaches plus termites (Bandiet al. 1994, 1995). Tbe endosym-bionts of each of these insect groupsform a distinct monophyletic group.The most obvious explanation forthese findings is that the commonancestor to each of these ciades wasalso endosymbiotic in an ancestor tothe same group of hosts.

The positions of these endosym-biont clades on the bacterial phylo-

Aprill998297

TJ U U

Proteobacteria

eubacteriaFieure 2. Phylogenetic placement of endosymhionts. (a) Phylogenetic relationships of major euhacterial groups (Woese 1987),showing the position of the Proteohacteria, which includes most hacteriocyte associates of insects, and the bacteroides-flavohacteria group, which includes the hacteriocyte associates of cockroaches and primitive termites, (b) Phylogeneticrelationships of bacteriocyte-associated symhionts of insects and representative free-living bacteria withm the Proteobacteriabased on published analyses of 16S ribosomal DNA (see text for references).

genetic tree show that insect endo-symbionts have arisen a number oftimes independently from free-livingbacterial groups (Figure 2). Mostendosymbionts have originated fromwithin the Proteobacteria, particu-larly the gamma and beta subdivi-sions. These two subdivisions arelarge sister clades within the Proteo-bacteria, each containing a wide va-riety of ecological types.

A number of insect endosymbiontsfall within the gamma-3 subdivisionofthe Proteobacteria, a diverse groupthat includes Escberichia coU andother enteric bacteria. There are twomain groups of insect symbiontswithin this subdivision. Clusteredwithin the Enterobacteriaceae near£. coli are the secondary endosym-bionts of pea aphids (Unterman et al.1989), the secondary endosymbionts

of tsetse flies (Aksoy et al. 1995),and the primary endosymbionts ofSitopbilus weevils (Campbell et al.1992). Together, these endosym-bionts may form a clade, althoughvariation in 16S rDNA sequences isnot sufficient to resolve relationshipswithin the gamma subdivision (Fig-ure 2).

Also within the gamma-3 subdivi-sion, but outside of the Enterobacte-

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riaceae, is a clade consisting of pri-niary endosymbionts of aphids, car-penter ants, and tsetse flies, witheach of the three symbiont groupsforming a well-supported clade. Cur-rent phylogenetic evidence raises thepossibility that the shared ancestorfor tsetse, ant, and aphid primarysymbionts was endosymbiotic orpossessed habits causing it to readilyenter into endosymbiotic associationswith insects. However, this possibil-ity is not yet firmly established; in-deed, it wonld be surprising in viewof major differences among the hosttaxa and among the developmentalpatterns of the different symbioses.The 16S rDNA sequences of theseendosymbionts have a low G+C con-tent compared with free-living mem-bers of the gamma-3 subdivision(Table 1), raising the possibility thattheir apparent close phylogeneticrelationship results in part from con-vergent evolution at some nucleotidepositions. Also, undiscovered free-living bacteria may belong to thesame clade. Thus, phylogenetic evi-dence from 16S rDNA is inconclu-sive but consistent with the indepen-dent derivation of aphid, ant, andtsetse endosymbionts from free-liv-ing ancestors.

The best-known exception to theconcentration of insect bacteriocyteassociates within the Proteobacteriaare the intracellular symbionts ofcockroaches and primitive termites(Bandietal. 1994,1995). These sym-bionts form a clade within thebacteroides-flavobacteria group andare, thus, distantly related to otherknown bacteriocyte associates.

Ages and patternsof cospeciationAnother consistent pattern that hasemerged for bacteriocyte-associatedbacteria in insects is congruence ofthe phylogenies of the bacterial andhost clades. For every case that hasbeen studied sufficiently, the phy-logeny for the bacteria matches thephylogeny for the corresponding in-sects. This congruence is strong evi-dence for parallel diversification aris-ing from faithful and long-termvertical transmission of endosym-bionts along host lineages. Supportfor congruence varies among thecases studied; it depends on the num-

Table 1. G + C composition of 16S rDNA of free-living and symbiotic bacteria.

Taxon G + Cin 16SrDNA(%)

Aeromonas trotaAlcaligenes sp.Escherichia coliHalomonas etongataHaemophilus influenzaeLegionella sp.Neisseria gonnorheaeProteus uulgarisPseudomonas aeruginosaPseudomonas testosteroniVibrio parahaemolyticusYersinia pestisAphid primary symbiont {Buchnera aphidicola)Ant primary symbiont {Candidatus componotii)Tsetse primary symbiont {Wigglesworthia glossinidia)Whitefly primary symbiontWeevil symbiontMealybug symbiontPea apbid secondary symbiontWbitefly secondary symbiont

55.254.954.456.152.253.155.052.854.154.653.854.149.347.747.947.854.055.754.154.4

ber of taxa included and on the ro-bustness of the phylogenies. Varyinglevels of positive support have beenfound for aphids (Munson et al.1991, Moran et al. 1993), tsetse flies(Aksoy et al. 1995), carpenter ants(Schroder et al. 1996), and cock-roaches (Bandi et al. 1995). Evenmore important, no study has yetproduced evidence contradicting thehypothesis of parallel phylogenesisfor bacteriocyte-associated endosym-bionts in insects. Thus, the syndromeof long-term associations that arestrictly maternally inherited may betypical of these associations.

The support for long-term codiversi-fication of insects and their endosym-bionts is consistent with microscopicaland experimental observations ofmaternal transmission by Buchner(1965) and others. However, thephylogenetic studies extend theseresults by demonstrating that evenrare instances of horizontal trans-mission are absent, a fact that couldnever be established based on ex-perimental observations of transmis-sion events alone. Even infrequenthorizontal transmission events wouldscramble patterns of congruence ofhost-symbiont phylogenies whensuch phylogenies extend millions ofyears into the past.

For each insect bacteriocyte asso-ciation, congruence of host and sym-biont phylogenies provides strongevidence that the original infectionpredates the basal ancestor of thesymbiont and of the correspondinghosts. In addition, congruence im-

phes that those ancestors lived at thesame time. Consequently, minimumages of the infections can be inferredfrom the fossil records of the insects(Table 2). The general conclusionfrom thus incorporating paleonto-logical evidence is that these infec-tions are very old, dating back toperhaps 300 million years in the caseof the clade containing cockroachesplus termites and to 200 million yearsin the case of the clade representedby aphids (Table 2). Schroder et al.(1996) suggest that the ancestor ofall ants may have possessed an endo-symbiont that was subsequently lostin most lineages but retained in car-penter ants and certain other speciesin the subfamily Formicinae. Thishypothesis remains to be testedthrough examination of otherformicines. If it is correct, the age ofthe infection would exceed 80 mil-lion years. In addition to the evi-dence based on congruent phylog-enies and insect fossil dating, theantiquity of the infections is sup-ported by levels of 16S rDNA se-quence divergence within clades ofendosymbionts.

The phylogenetic evidence forbacteriocyte associates of insectscontrasts with that for some otherendosymbioses. In Wolbachia pipi-entis, a bacterium that infects insectgerm line cells and causes variousreproductive abnormalities, molecu-lar phylogenies give a clear indica-tion of noncongruence, indicatinghorizontal movement of bacteriaamong lineages (Werren 1997,

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Table 2. Insect groups for which molecular phylogenetic studies support parallelcladogenesis of bacteriocyte-associated endosymhionts and hosts, implying ancientinfection of a common ancestor of the host clade. Approximate minimum ages arebased mainly on fossil evidence.

Host cladeMinimum age of symbiotic association(i.e., age of common ancestor of hosts) Reference(s)

Aphids (Hemiptera: 150-250 million yearsAphidoidea)

Cockroaches + 135-300 million yearstermites (Isoptera +Blattaria)

Tsetse flies (Diptera: 40 million yearsGlossinidae)

Carpenter ants{Hymenopetera:Formicidae; genusCamponotus)

More than 50 million years?

Munson etal. 1991,Moran etal. 1993

Bandi et al. 1995

Aksoy et al. 1995

Schroder et al. 1996

Bourtzis and O'Neill 1998). Like-wise, in some marine invertebrates,host and endosymbiont phylogeniesalso appear not to be congruent(Dubilier et al. 1995, Krueger andCavanaugh 1997, Distel 1998).

What is the basis for this differ-ence? That is, why might bacteriocyteassociates of insects lack the capac-ity for horizontal transfer? Whensymbionts are maternally transmit-ted and beneficial to hosts, hosts areselected to provide a continually fa-vorable environment to ensure thepersistence of the association. Con-sequently, selection on bacteria forthe ability to withstand conditionsoutside bacteriocytes will be relaxed.The resulting intolerance of condi-tions outside hosts could effectivelyeliminate horizontal transfer, facili-tating long-term cospeciation of hostand symbiont.

In the case of Wolbacbia^ by con-trast, the typical infection route ismaternal but the interaction appearsto be more parasitic than mutualistic(Werren 1997, Bourtzis and O'Neill1998). The bacteria do not reside inspecialized bacteriocytes, and theirmaintenance and transmission do notappear to be facilitated by host ad-aptations. Rather, they inhabit avariety of cell types and invade hosteggs using their own devices (Werren1997). As a result, they may retain amore generalized ability to live un-der different conditions and thus havegreater propensity for rare horizon-tal transmission. In some marine in-vertebrates, such as bivalves andmarine annelids, the associations are

mutualistic, and hosts have evolvedspecialized cell types for housing sym-biotic bacteria (Conway et al. 1992,Distel 1998). But long-term cospeci-ation may be prevented by the modeof infection: In at least some suchassociations, infecting stages are notmaternally transferred but are water-borne, providing more opportunityfor cross-infection of host lineages.

Adaptations ofendosymbiotic bacteriaBecause bacteriocyte associates ofinsects cannot be cultured outside ofhosts, it has been difficult to showhow they differ from free-living bac-teria. However, genetic character-ization of Bucbnera, the primaryendosymbiont of aphids, has dem-onstrated some striking adaptationsto endosymbiotic hfe (P. Baumann etal. 1997).

The best-documented contribu-tion of aphid endosymbionts to theirhosts is the provision of requiredamino acids that are rare or absentfrom the phloem sap diet. In particu-lar, several lines of evidence supportthe role of Buchnera in provisioninghosts with tryptophan, an amino acidthat is required by animals but is rarein phloem sap (Douglas and Prosser1992, Lai et al. 1994). Tryptophanbiosynthesis is regulated by feedbackinhibition acting on anthranilate syn-thase, which is encoded by trpEG. InBuchnera that are endosymbioticwith members of the Aphididae,trpEG is excised from the chromo-some and amplified on plasmids (Lai

et al. 1994). This amplification,which involves both the tandem du-plication of the operon on each plas-mid and the presence of multiple plas-mids, appears to function in theoverproduction of tryptophan throughenhanced production of the limitingbiosynthetic enzyme. Comparisonsof phylogenies based on plasmid-borne and chromosomal genes indi-cate that trpEG on plasmids origi-nated from the chromosomal geneswithin the same bacterium and thatthe plasmids are transmitted strictlyvertically (Rouhbaksh et al. 1996,1997). The trpEG plasmid presum-ably was fixed through favorableselection at the host level; that is,host insects bearing endosymbiontswith this feature experienced greatergrowth rates and became establishedat the expense of other aphid lin-eages of the same species in whichendosymbionts possessed only achromosomal trpEG copy.

A similar instance of plasmid am-plification enabling the overproduc-tion by Buchnera of an essential aphidnutrient has been discovered for leu-cine production (Bracho etal. 1995).The four genes that underlie produc-tion of leucine are excised from thechromosome and transferred to plas-mids in some Buchnera species. How-ever, only a single set of the leucinegenes is present on each plasmid.The presence of the replicating unitof the plasmid appears to be ances-tral in Buchnera of all aphids, but itsacquisition of the leucine biosyn-thetic genes appears to have occurredindependently in different lineages(van Ham etal. 1997).

Other unusual aspects of theBuchnera genome may representadaptations to endosymbiotic life.Because symbionts are confined tohost cells, their growth rates must bedepressed and coordinated with thedevelopment of the host. ForBucbnera, there is a tight coupling ofbacterial cell number and aphidgrowth, with Buchnera showing adoubling time of approximately twodays, a growth rate that is muchlower than the maximum exhibitedby many free-living bacteria (Bau-mann and Baumann 1994). Prob-ably in connection with this lowmaximal growth rate, both Bucbneraand Wiggleswortbia, the bacteriocyteassociate of tsetse flies, possess only

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a single copy of the rRNA genes, incontrast to free-living bacteria, whichtypically have several copies (Aksoy1995, Baumann et al. 1995). Reduc-tion in the number of copies of rRNAgenes in prokaryotes is associatedwith lowered maximum growth rates.

Endosymbiont adaptations forimproved function as mutualistspresent the clear possibility of con-flict between selection on individualcell lineages within a host aphid andselection on individual aphids. Forexample, the excess production oftryptophan must be beneficial tohosts but is probably detrimental tothe bacterial cell lineage producingthe nutrient. In related free-livingbacteria, production of anthranilatesynthase has been shown to be costly;indeed, it lowers fitness of bacteriawhen tryptophan is not limiting(Dykhuisen 1978}, suggesting that amutant Buchnera lacking the excesscopies would enjoy a short-term ad-vantage. However, host lineages inwhich all symbionts were lacking theexcess copies would suffer a nutri-tional handicap and would thus beat a selective disadvantage withinaphid populations. The high muta-tion rate for loss of tandem repeatsin bacteria (10^-10"^ per generation;Roth et al. 1996), in combinationwith the immediate selective advan-tage for mutant cells, would be ex-pected to destabilize these host-leveladaptations. In the long term, how-ever, selection at the host level wouldfavor adaptations that stabilize ben-eficial gene amplification.

Some findings suggest that suchhost-level selection has resulted inreduced homologous recombinationin Buchnera. Curiously, in certainBucbnera of the family Aphididae,reduction in plasmid-borne func-tional trpEG has occurred throughthe transformation of some of therepeats into pseudogenes (Lai et al.1996, L. Baumann et al. 1997). Typi-cally, however, such unneeded re-peats would instead be eliminatedthrough recombination. A possibleexplanation for the transformationinto pseudogenes is selection on hoststo stabilize beneficial gene amplifi-cation, resulting in loss of some re-combination capabilities (P. Bau-mann et al. 1997). If certain aphidlineages subsequently evolved feed-ing habits or life cycles that reduced

their need for tryptophan provisionby Buchnera, selection then mighthave favored reduced trpEG expres-sion due to the energy expense ofmaking unneeded anthranilate syn-thase. If recombinational mecha-nisms for eliminating repeat copieshad been lost earlier, gene silencingwould have had to occur through thefixation of stop codons and frame-shifts in the DNA sequence, result-ing in transformation of the func-tional genes into pseudogenes. If thisscenario is correct, then the loss ofcertain recombination functionswould appear to be an adaptation tostabilize mutualistic traits of the en-dosymbiont that might otherwise beeliminated through a combinationof high mutation rates and selectionat the bacterial cell level.

Evolutionary consequencesof endosymbiosisEndosymbiosis is sometimes consid-ered to be an initial step in the evolu-tion of organelles (e.g., Margulis1993). However, organelles havearisen only a few times from free-living prokaryotes, either once ortwice each for mitochondria andchloroplasts (Cavalier-Smith 1992,Cray 1992}. By contrast, intracellu-lar symbionts have arisen far moreoften, as a glimpse at Buchner's(1965} volume confirms. Thus, or-ganelle characteristics are not an in-evitable consequence of endosym-biosis combined with maternaltransmission.

One feature of mitochondrial andchloroplast genomes is the loss ofmost genes present in the ancestralprokaryotic genome and the depen-dence on host gene products for manyof the basic functions required forreplication. The only endosymbiontfor which substantial genome char-acterization has been carried out isBuchnera, for which 85 kb has beensequenced in the Baumann labora-tory (summary in P. Baumann et al.1997). Despite substantial overallsequence divergence from relatedfree-living bacteria, Buchnera cod-ing gene sequences indicate selectiveconstraint of the functioning poly-peptides; the reading frame is main-tained and nucleotide base substitu-tions are concentrated at silent, orsynonymous, sites (i.e., sites that do

not affect amino acid identity}. Ceneswith a wide range of "housekeep-ing" and biosynthetic functions havebeen identified in Buchnera (sum-maries in Baumann et al. 1995, P.Baumann et al. 1997, Clark and Bau-mann 1997, Clark et al. 1997}. Thiswork provides strong evidence thatBuchnera contains a complement ofgenes enabling a wide range of pro-cesses involved in cell growth andreproduction. This endosymbiotic as-sociation arose at least 200 millionyears ago, so the retention of mostgenes is not readily explained as theresult of the association being tooyoung for Buchnera to have devel-oped characteristics of organelles. Amore important limitation on theloss of genes might be that endosym-bionts in animals reside primarily insomatic cells (the bacteriocytes},eliminating most opportunities forgene transfer between symbiont andhost nuclear genome.

One apparent long-term conse-quence of endosymbiosis is increasedrates of substitution in DNA se-quences of endosymbionts relativeto non-endosymbiotic relatives(Moran 1996}. This increase is seenin the 16S rDNA of endos.ymbiontsof aphids, whiteflies, mealybugs,tsetse flies, and carpenter ants(Moran 1996, Lambert and Moranin press}. In Buchnera., in which manymore genes have been sequenced thanin any other endosymbiont, the in-creased rate extends to all genes ex-amined (Moran 1996, unpublishedanalyses}.

Positive natural selection for basesubstitutions seems unlikely to ex-plain the faster evolution at all loci,because the effects of positive selec-tion are expected to be locus and sitespecific within genes. In fact, it ap-pears that the base substitutions aredeleterious. Evidence for this hypoth-esis is provided by the changes inprotein-coding genes in Buchnera.The net effect of the faster evolutionin Buchnera is the accumulation,throughout the genome, of A and Tat the expense of representation of Gand C. This shift in nucleotide basecomposition is strong enough thatmost amino acid differences betweenpolypeptides of Buchnera and thoseof E. coli are ones that allow in-creased A -H T in the correspondingcodon and thus in the DNA sequence.

April 1998301

It is hard to imagine that the accu-mulation, in all polypeptides, ofamino acids with A + T-rich codonfamilies is adaptive at the level ofprotein function. A more plausibleexplanation is mutational bias fa-voring A + T combined with an in-creased rate of base substitutionwithin lineages.

If positive selection is excluded asthe basis for the faster substitutionrates of endosymbionts, we are leftwith two other categories of expla-nation. One is that the increased rateof substitution (fixed changes occur-ring in a lineage) is due to an in-creased rate of new mutations. Thisincreased mutation rate could resultfrom either an increased rate pergeneration or a faster average gen-eration time. But if increased inci-dence of mutation were the sole ex-planation, the increase would haveequivalent effects on substitutionrates at sites that are subject to selec-tion and at sites that are effectivelyneutral with respect to selection, andcomparative sequence analysis indi-cates stronger effects on selected sites.A convenient way to categorize sitesaccording to the intensity of selec-tion is to separate silent (or synony-mous) from replacement (or non-synonymous) sites in protein codingsequences. At silent sites, wherenucleotide changes do not affect thecorresponding amino acid and thusdo not affect the gene product, basesubstitutions have little consequencefor fitness. At replacement sites,where a nucleotide change results ina change in the amino acid and thusin the selected phenotype, base sub-stitutions are more likely to affectfitness. The increase in substitutionrate in coding genes of Buchnera isheavily concentrated at replacementsites (Moran 1996). This observa-tion rules out increased mutation asthe primary basis for the accelera-tion in substitution rates.

This result instead supports thesecond explanation, that the excesschanges represent deleterious substi-tutions. According to this view, pu-rifying selection is less effective atpurging deleterious mutations. Thisreduction in the effects of selectioncould arise in either of two ways:from relaxed selection or from lowereffectiveness of selection as a resultof population structure. The first pos-

sibility, that selection is uniformly re-laxed at all genes of Buchnera, regard-less of function, is doubtful. Becauseendosymbionts show reduced effec-tive population sizes than free-livingbacteria, with a consequently greatersusceptibility to genetic drift, thesecond possibility may apply. Thisproposal resembles the argumentsmade by Lynch (1996, 1997) for theaccumulation of deleterious muta-tions in mitochondria and other or-ganelles. Organelles have similarpopulation structure to endosym-bionts because they are asexual, in-tracellular, and maternally inherited.

Current mysteries andfuture problemsThe recent molecular studies of in-sect bacteriocyte-associated endo-symbioses have given glimpses intothe evolution of some of these asso-ciations. But most remain entirelyunstudied, particularly the complexmultiple infections found in someinsects, such as the planthoppers andtreehoppers (Hemiptera; Buchner1965). Furthermore, this work hasraised some new questions about theevolution of bacteriocyte associatesin insects. For example, both Buch-nera and tsetse endosymbionts ex-hibit unusually high levels of GroELprotein (Ishikawa 1989, Aksoy 1995,Baumann et al. 1996), a chaperoninthat normally functions in refoldingother polypeptides. The reason forthis overexpression is not yet clear,although speculative explanationshave been put forward (Ishikawa1989, Moran 1996, P. Baumann etal. 1997). The finding of plasmid-borne pseudogenes in some Buchnerapresents another mystery: Why arenonfunctional copies not lost by re-combination, as is typical for bacte-ria? Some basic difference involvingrecombination pathways may, as al-ready discussed, distinguish Buch-nera from free-living bacteria.Whether other endosymbionts alsohave atypical recombination path-ways is not yet known. The basis forthe nucleotide base compositionalbias against G + C, a distinctive fea-ture of numerous bacteriocyte asso-ciates (Table 1), is also unknown.

Endosymbiotic interactions areshaped by forces acting at multiplelevels, primarily that of the host in-

dividual and the bacterial cell lin-eage. Some mutations in endosym-bionts will favor the individual sym-biont lineage at the expense of thehost. Such "selfish" mutations wouldinclude any that result in higher rep-lication of the symbiont at the host'sexpense. If higher replication resultsin disproportionate transmission toprogeny, these selfish symbiontscould increase in frequency within ahost population, at least in the shortterm. Michod (1997) presents a theo-retical framework for evaluating thisvery situation that characterizesbacteriocyte associations in insects:a balance between frequent muta-tion in the direction of "selfish" traitsfavoring individual cell lineages andselection on the overall group (inthis case, the host individual and itsresident bacterial population).Buchner (1965) mentions cases inwhiteflies in which symbionts spreadbeyond their usual limits within in-dividual host insects, indicating thepossibility of such selfish behaviorthat harms the host.

Of course, selection favoring bet-ter-nourished (and faster-growing)host individuals will counter thespread of selfish bacterial lineages.One host adaptation that would limitthe spread of selfish symbionts wouldbe the sequestration of "germ line"symbionts from more rapidly divid-ing "somatic" symbionts that func-tion in provisioning hosts with nu-trients. Early microscopical studiessuggest this kind of system in cicadas(Koch 1967). The infection of prog-eny with entire maternal bacterio-cytes, as in whiteflies (Buchner 1965,Costa et al. 1996), would also havethe effect of limiting the spread of"selfish" bacteria to single host prog-eny, provided that bacteria are con-fined to single bacteriocytes early inthe development of the host. Frank(1996) proposes thatthe segregation

of germ line symbionts may be a"policing" mechanism for the con-tainment of selfish cell lineages withinhosts of endosymbionts.

Role of endosymbiosis inhost evolutionEndosymbiosis has been argued tobe a route for evolutionary innova-tion that underlies the origin anddiversification of many groups of

302 BioScience Vol. 48 No. 4

organisms that would otherwise notexist. Within the realm of the in-sects, this hypothesis appears to betrue: Essentially all strict phloem-feeders, strict blood-feeders, andother groups with restricted dietspossess endosymbionts. Recent mo-lecular phylogenetic evidence indi-cates that endosymbiotic infectionsdate to the origins of a variety ofmajor insect clades, reinforcing theidea that the origin of endosymbiosiswas an integral factor spurring di-versification of some insect taxa.Because some of these groups arelarge adaptive radiations, endosym-biosis has probably had a massiveeffect on patterns of insect diversity.

Although endosymbiosis appearsto facilitate diversification withinecological niches that would other-wise be inadequate, dependence onan endosymbiont may simulta-neously enforce some restrictions onhost evolution. Cenes encoded withinthe endosymbiont are subject to adifferent set of population geneticparameters than genes encoded inthe insect nuclear genome. As notedabove, endosymbionts are subject towithin-host selection, which often maycounter selection between hosts. Inaddition, mutation rates and genera-tion times differ between endosym-hiont and host genomes. The distinctpopulation genetics of endosymbiontgenes may affect the speed of theresponse to selection. For example,adaptation may occur faster in sym-biont genes than host genes due tothe shorter generation time and con-sequent greater supply of mutationsof the bacteria. Finally, symbiontgenes are subject to a populationstructure in which genetic drift ismore likely to limit the effectivenessof selection, resulting in increasedfixation of mildly deleterious muta-tions. This increased rate of accumu-lation of deleterious mutations willbe countered, to some extent, byselection at the level of host indi-viduals. The adequacy of this coun-terbalancing will depend on the im-pact of selection at the host level,which is dependent in turn on theeffective population size of the host.

It is not yet clear what factors aremost important in determining thenet effect of these conflicting evolu-tionary forces. One intriguing possi-bility is that the accumulation of

deleterious mutations would over-run purifying selection at the hostlevel, except in cases in which thehost population size is large. Thispossibility may help to explain whymaternally transmitted endosym-bionts are rare in animals such asvertebrates, which have small popu-lations, as compared with insects,many of which have unusually largepopulations.

AcknowledgmentsResearch on insect endosymbiontsin the Moran lab is supported bygrants from the National ScienceFoundation (DEB-9527635) and theUS Department of Agriculture, Na-tional Research Initiative (96-35302-3817). Aparna Telang was supportedby a fellowship from the Plant-InsectInteractions Training Grant to theUniversity of Arizona from NSF,USDA, and the US Department ofEnergy. We thank Paul Baumann forcomments on an early draft of thisreview and Leah Kenaga and DavidBentley of the Imaging Facility (Ari-zona Research Laboratories) for as-sistance with electron microscopy.

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