Download Bacteriocyte-Associated Symbionts of Insects

Bacteriocyte-Associated
Symbionts of Insects
A variety of insect groups harbor ancient
prokaryotic endosymbionts
Nancy A. Moran and Aparna Telang
E
ndosymbiosis has been proposed as an evolutionary innovation that underlies the Endosymbiosis appears to
success and diversification of some
facilitate diversification
groups of organisms by enabling
them to exploit new ecological niches
within ecological niches
(e.g., Margulis and Fester 1991,
Maynard Smith and Szathmary
that would otherwise
1995). Three kinds of information
are needed to evaluate this proposal.
be inadequate, but
First, it is necessary to know somedependence on an
thing of the role played by symbionts
in the lives of hosts: Are their effects
endosymbiont may
ones that allow use of novel ecological niches? Second, information is
simultaneously enforce
needed on the evolutionary history
of endosymbioses: How old are they
some restrictions on
and, in particular, do endosymbiotic
infections predate diversification of
host evolution
major host groups? Finally, the evolutionary consequences for hosts of
acquiring new capabilities in the form 1984). Bacteriocyte associates are
of symbiotic associations must be found in many insect orders and famievaluated: Are these novel traits lies and have been estimated to occur
stable? Do they impose constraints in 10% of insect species (Douglas
as well as new capabilities on hosts?
Recent studies have begun to shed 1989).
Before the past decade, much of
light on these questions for bacthe
knowledge of bacteriocyte assoteriocyte-associated endosymbioses
ciations
was based on extensive miin insects. Bacteriocytes (or mycecroscopical
studies by Paul Buchner
tocytes) are cells of animal hosts that
and
his
associates.
His fascinating
are positioned at characteristic locacompilation
of
this
work (Buchner
tions in the body and appear to be
1965)
details
astonishing
diversity
specialized for housing bacteria (Figin
animal-microbe
associations.
ure 1; Buchner 1965, Dasch et al.
Most of this diversity is still unexplored, in large part because intraNancy A. Moran (e-mail: nmoran@ cellular symbionts typically cannot
u.arizona.edu) is a professor in the De- be cultured outside of their hosts.
partment of Ecology and Evolutionary Since 1990, however, molecular studBiology, and Aparna Telang is a gradu- ies on a handful of endosymbioses
ate student in the Interdisciplinary Pro- have yielded considerable insight into
gram in Insect Science, at the University their evolutionary histories and into
of Arizona, Tucson, AZ 85721. © 1998 the specific adaptations to symbiosis
American Institute of Biological Sciences.
April 1998
that are found in the bacteria themselves. Some of the most revealing of
these studies concern the bacteriocyte-associated, mutualistic endosymbionts of insects.
The microscopical explorations
performed by Buchner and his associates gave only hints regarding the
relationships of insect endosymbionts
to other bacterial groups and to other
endosymbionts. The characterization
of these bacteria and their phylogenetic placement relative to free-living prokaryotes remained almost a
complete mystery until the development of molecular methods, in particular methods for determining
nucleotide sequences. These methodological advances have revolutionized understanding of prokaryote
evolution in general by providing a
surplus of relatively reliable characters for phylogeny reconstruction. A
major additional advantage of DNA
sequences as phylogenetic characters is that they can be obtained for
noncultivable organisms. In particular, sequences of the 16S ribosomal
RNA (rRNA) genes have been used
for the phylogenetic and taxonomic
placement of many bacteria, including endosymbionts, that were previously unclassifiable (Maidak et al.
1996).
In this article, we provide a brief
overview of recent work on the nature ofthe relationships between insect hosts and bacteriocyte-associated organisms (see Dasch et al. 1984,
Dadd 1985, Douglas 1989 for detailed reviews). We then consider in
more detail the emerging picture of
endosymbiont evolution presented
295
housing of endosymbionts by hosts.
For many insect symbioses, beneficial effects of the bacteria on their
hosts are supported by experimental
evidence: Antibiotic or heat treatments causing loss of symbionts are
accompanied by reductions in performance, including lowered growth
and survivorship and loss of reproductive abilities (e.g., Nogge 1976,
Ishikawa and Yamaji 1985, Ohtaka
and Ishikawa 1991, Prosser and
Douglas 1991, Sasaki et al. 1991,
the aphid Uroleucon ja- Costa et al. 1993, Heddi et al. 1993;
ceae; arrowhead points additional studies cited in Koch 1967
to host cell plasma and Dasch et al. 1984). Many hosts,
membrane. Bar = 2 |im. including aphids and other insect
b, Buchnera; e, midgut groups that feed exclusively on plant
epithelium; 1, midgut sap, can grow or reproduce only with
lumen; m, mitochon- intact symbionts. Others, including
dria; n, host cell nucle- Sitophilus weevils, which feed on
us; ov, ovariole; s, sec- grain, can survive and reproduce
ondary endosymbiont. without the symbionts, but they grow
more slowly and are smaller than
fleeted in parallel symbiotic hosts (Heddi et al. 1993).
phylogenies? What Although interpreting studies of symages can we infer for biont-free hosts is complicated by
the endosymbioses the possibility that antibiotic or heat
of insects? Third, treatment may have direct effects on
what adaptations of host metabolism, the preponderance
endosymbiotic bac- of evidence points to a beneficial
teria function in the role of bacteriocyte associates for
mutualistic interac- host insects.
tion with hosts?
Fourth, what are the
A nutritional dependence of hosts
long-term evolution- on their symbionts presumably exary consequences of plains why many insect taxa possessendosymbiosis, as ing bacteriocytes use narrow and
reflected in genetic nutritionally unbalanced diets, such
changes in the sym- as the phloem sap of plants or the
biotic bacteria?
blood of vertebrates. Because animals
lack biosynthetic pathways that
In the arena of
molecular studies, are typically present in prokaryotes,
the most intensive most animals must ingest a large
research on insect number of essential nutrients. Alterendosymbioses has natively, a microbial associate can
been on aphids, small insects that provide the missing nutrients, allowfeed by tapping phloem sap of vascu- ing animal hosts to exploit resources
lar plants, and on their primary en- that would otherwise be nutritiondosymbionts, Buchnera aphidicola. ally deficient. Examples include
Molecular studies of this association members of the Hemiptera that use
have been reviewed recently in more their sucking mouthparts to feed
solely on plant phloem or xylem sap
detail (P. Baumann et al. 1997).
(Dasch et al. 1984}. Plant phloem
sap contains free amino acids but
The mutualistic nature of
lacks some amino acids that are essential nutrients for animals. Anbacteriocyte associations
other dietary habit associated with
Buchner considered bacteriocyte as- endosymbiosis is restriction to vertesociates to be mutualists that benefit brate blood, which is lacking in some
insect hosts by providing limiting vitamins. Endosymbiosis connected
nutrients. His views were based on to blood feeding throughout the life
myriad observations of specialized cycle has evolved independently in a
mechanisms for the transmission and
Figure 1. Electron mi-
crographs oiBuchnera
in aphid bacteriocytes.
(a) Bacteriocytes adjacent to the midgut of
the aphid Uroleucon
sonchi. Bar = 10 |im.
(b) Bacteriocytes adjacent to the ovarioles of
U. sonchi. Bar = 5 |im.
(c) Bacteriocytes adjacent to secondary endosymbionts housed in
separate cells within
by molecular phylogenetic findings.
Finally, we consider how such studies bear on the following sets of questions: First, what is the distribution
of endosymbiotic bacteria on the
prokaryotic tree of life? Which bacterial groups have produced lineages
that are endosymhiotic in insects?
Are these related to one another as
members of one or a few monophyletic groups specialized for endosymbiotic living? Or do endosymbionts
of different insect lineages represent
independent origins of endosymbiotic bacteria? Second, do the bacteria show long-term patterns of
cospeciation with their hosts, as re296
BioScience Vol. 48 No. 4
number of insect orders, including
Diptera, Hemiptera, and Mallophaga
(Buchner 1965, Dasch et al. 1984).
Some insects with more generalized
diets also have endosymbionts; these
include cockroaches, some ants, and
some weevils. In ants, however, endosymbiosis may be associated with
dependence on nutritionally deficient
plant fluids.
The general conclusion of a nutritional role for symbionts has been
accepted by almost all researchers
who have studied bacteriocyte associates of insects (e.g., Buchner 1965,
Koch 1967, Dasch et al. 1984, Dadd
1985). Yet definitive evidence that
endosymbionts provide essential
nutrients to insect hosts is rare. The
best evidence pertains to the provision of tryptophan by Buchnera, the
endosymbionts of aphids (Douglas
and Prosser 1992, Lai et al. 1994).
Also, some evidence suggests that
tsetse symbionts provision hosts with
B vitamins (Nogge 1981).
How are endosymbionts
transmitted?
infection process vary tremendously
among insect groups (e.g., Sacchi et
al. 1988,Hypsal993).Forexample,
in wbiteflies, an entire maternal bacteriocyte (or, in some species, several
bacteriocytes) is transferred into each
egg, witb the maternal nucleus later
degenerating (Bucbner 1965, Costa
etal. 1993).
Secondary endosymbionts
Some insects harbor so-called "secondary" endosymbionts that coexist
in the same individual hosts with the
bacteriocyte-inbabiting primary endosymbionts. These secondary symbionts are maternally inherited but
appear not to share a long evolutionary bistory witb their hosts. Tbey are
usually not present in the bacteriocytes tbat bouse primary endosymbionts (Figure lc; Buchner 1965).
Tsetse fly secondary symbionts occur in midgut cells, by contrast to tbe
primary symbionts, wbich are found
in specialized bacteriocytes in tbe
anterior gut (Pinnock and Hess
1974). Aphid secondary endosymbionts occur mostly in a sbeatblike
syncytium bordering the bacteriocytes tbat house Buchnera (Bucbner
1965, Fukatsu and Isbikawa 1993,
Chen and Purcell 1997). Whitefly
secondary endosymbionts do inhabit
the bacteriocytes in wbich primary
endosymbionts are found but are
clumped irregularly within vacuoles
(Costa etal. 1993,1996).
Bacteriocyte-associated endosymbionts are typically maternally inherited, often through complex developmental events that ensure
transovariole transfer from the
mother to the developing egg or
embryo (Buchner 1965, Dasch et al.
1984). In the case of aphids and
Bucbnera, the bacteria are housed
within bacteriocytes that are grouped
Effects of secondary endosyminto a loose organ witbin the body bionts on host fitness are not clear.
cavity, in the vicinity of the develop- Bucbner (1965) described secondary
ing ovarioles (Figure lb; Buchner endosymbionts of apbids as "recently
1965, Hinde 1971a, 1971b). The acquired guests wbich are still in
maternal bacteriocyte adjacent to an need of adaptation." They are more
embryo near tbe blastoderm stage sporadic in tbeir numbers and presforms a small opening through whicb ence among species and even in their
a bacterial inoculum passes. Tbe in- distribution among individuals of the
oculum then moves through tbe in- same species, as bas been sbown for
tervening hemolymph and enters a aphids (Fukatsu and Ishikawa 1993,
nearby opening on tbe oocyte sur- Chen and Purcell 1997), whiteflies
face. During early embryonic devel- (Costa et al. 1993), and tsetse flies
opment, the presumptive bacterio- (Pinnock and Hess 1974). Cben and
cytes form, and the inoculating Purcell (1997) found tbat secondary
bacteria migrate into tbese cells.
endosymbionts are present in only
some strains of pea apbid {AeyrthoThis apparent fine tuning of tbe siphon pisum) and are identical in
infection process is typical of 16S rDNA sequence to secondary
bacteriocyte-associated symbionts in endosymbionts of rose aphid (Mijcroinsects and suggests a long bistory of siphum rosfle). Vertical transmission
selection favoring bost adaptations from the time of tbe common ancesthat help to maintain tbe associa- tor of tbese two bosts would almost
tion. However, the particulars of tbe
Aprill998
certainly have resulted in some sequence divergence; for example, the
Buchnera 16S rDNA sequences for
similarly closely related genera within
Aphididae all differ by more tban
2% (Munsonet al. 1991).
The observed sequence identity of
the secondary symbionts implies the
occurrence of either horizontal transfer between species or recent, independent infections by a bacterium
that is widely distributed in tbe environment. Furthermore, the symbionts could be experimentally transferred by injecting hemolympb from
infected clones into uninfected clones
of the same or different species. Thus,
although maternal transmission was
observed (Chen and Purcell 1997)
and is presumably the usual route of
infection, tbese secondary endosymbionts must also undergo some transfer among bost lineages, including
transfer between species. Their sporadic distribution within host species implies that unlike most primary
endosymbionts, they are not required
for bost development or reproduction. Currently, there is almost no
evidence indicating whether tbeir
effects on bost fitness are positive,
negative, or neutral.
The phylogenetic distribution
of insect endosymbionts
Molecular pbylogenetic results based
on 16S rDNA sequences show that
within each of tbe insect groups examined so far, the primary endosymbionts descended from a single ancestor, as indicated by their forming
a well-supported clade. This result
has been obtained for the primary
endosymbionts of aphids (Munson
et al. 1991, Moran et al. 1993), mealybugs (Munson et al. 1993), wbiteflies (Clark et al. 1992), carpenter ants
[Candidatus camponotii; Schroder et
al. 1996),5;top/7/7M5 weevils (Campbell
etal. 1992), tsetse flies (Aksoy 1995),
and cockroaches plus termites (Bandi
et al. 1994, 1995). Tbe endosymbionts of each of these insect groups
form a distinct monophyletic group.
The most obvious explanation for
these findings is that the common
ancestor to each of these ciades was
also endosymbiotic in an ancestor to
the same group of hosts.
The positions of these endosymbiont clades on the bacterial phylo-
297
TJ U U
Proteobacteria
eubacteria
Fieure 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 bacteroidesflavohacteria group, which includes the hacteriocyte associates of cockroaches and primitive termites, (b) Phylogenetic
relationships of bacteriocyte-associated symhionts of insects and representative free-living bacteria withm the Proteobacteria
based on published analyses of 16S ribosomal DNA (see text for references).
genetic tree show that insect endosymbionts have arisen a number of
times independently from free-living
bacterial groups (Figure 2). Most
endosymbionts have originated from
within the Proteobacteria, particularly the gamma and beta subdivisions. These two subdivisions are
large sister clades within the Proteobacteria, each containing a wide variety of ecological types.
298
A number of insect endosymbionts
fall within the gamma-3 subdivision
ofthe Proteobacteria, a diverse group
that includes Escberichia coU and
other enteric bacteria. There are two
main groups of insect symbionts
within this subdivision. Clustered
within the Enterobacteriaceae near
£. coli are the secondary endosymbionts of pea aphids (Unterman et al.
1989), the secondary endosymbionts
of tsetse flies (Aksoy et al. 1995),
and the primary endosymbionts of
Sitopbilus weevils (Campbell et al.
1992). Together, these endosymbionts may form a clade, although
variation in 16S rDNA sequences is
not sufficient to resolve relationships
within the gamma subdivision (Figure 2).
Also within the gamma-3 subdivision, but outside of the EnterobacteBioScience VoL 48 No. 4
riaceae, is a clade consisting of pri- Table 1. G + C composition of 16S rDNA of free-living and symbiotic bacteria.
niary endosymbionts of aphids, carG + Cin 16SrDNA(%)
penter ants, and tsetse flies, with Taxon
each of the three symbiont groups Aeromonas trota
55.2
forming a well-supported clade. Cur- Alcaligenes sp.
54.9
54.4
rent phylogenetic evidence raises the Escherichia coli
56.1
possibility that the shared ancestor Halomonas etongata
52.2
Haemophilus
influenzae
for tsetse, ant, and aphid primary
53.1
Legionella sp.
symbionts was endosymbiotic or Neisseria gonnorheae
55.0
possessed habits causing it to readily Proteus uulgaris
52.8
54.1
enter into endosymbiotic associations Pseudomonas aeruginosa
54.6
with insects. However, this possibil- Pseudomonas testosteroni
53.8
ity is not yet firmly established; in- Vibrio parahaemolyticus
54.1
Yersinia
pestis
deed, it wonld be surprising in view Aphid primary symbiont {Buchnera aphidicola)
49.3
of major differences among the host Ant primary symbiont {Candidatus componotii)
47.7
47.9
taxa and among the developmental Tsetse primary symbiont {Wigglesworthia glossinidia)
47.8
patterns of the different symbioses. Whitefly primary symbiont
54.0
Weevil
symbiont
The 16S rDNA sequences of these
55.7
Mealybug
symbiont
endosymbionts have a low G+C con- Pea apbid secondary symbiont
54.1
tent compared with free-living mem- Wbitefly secondary symbiont
54.4
bers of the gamma-3 subdivision
(Table 1), raising the possibility that
phes that those ancestors lived at the
their apparent close phylogenetic ber of taxa included and on the ro- same time. Consequently, minimum
bustness
of
the
phylogenies.
Varying
relationship results in part from conages of the infections can be inferred
vergent evolution at some nucleotide levels of positive support have been from the fossil records of the insects
found
for
aphids
(Munson
et
al.
positions. Also, undiscovered free(Table 2). The general conclusion
living bacteria may belong to the 1991, Moran et al. 1993), tsetse flies from thus incorporating paleonto(Aksoy
et
al.
1995),
carpenter
ants
same clade. Thus, phylogenetic evilogical evidence is that these infecdence from 16S rDNA is inconclu- (Schroder et al. 1996), and cock- tions are very old, dating back to
roaches
(Bandi
et
al.
1995).
Even
sive but consistent with the indepenperhaps 300 million years in the case
dent derivation of aphid, ant, and more important, no study has yet of the clade containing cockroaches
produced
evidence
contradicting
the
tsetse endosymbionts from free-livhypothesis of parallel phylogenesis plus termites and to 200 million years
ing ancestors.
for bacteriocyte-associated endosym- in the case of the clade represented
by aphids (Table 2). Schroder et al.
The best-known exception to the bionts in insects. Thus, the syndrome (1996) suggest that the ancestor of
of
long-term
associations
that
are
concentration of insect bacteriocyte
all ants may have possessed an endoassociates within the Proteobacteria strictly maternally inherited may be symbiont that was subsequently lost
typical
of
these
associations.
are the intracellular symbionts of
in most lineages but retained in carThe support for long-term codiversi- penter ants and certain other species
cockroaches and primitive termites
(Bandietal. 1994,1995). These sym- fication of insects and their endosym- in the subfamily Formicinae. This
bionts form a clade within the bionts is consistent with microscopical hypothesis remains to be tested
bacteroides-flavobacteria group and and experimental observations of through examination of other
are, thus, distantly related to other maternal transmission by Buchner formicines. If it is correct, the age of
(1965) and others. However, the the infection would exceed 80 milknown bacteriocyte associates.
phylogenetic studies extend these lion years. In addition to the eviresults by demonstrating that even dence based on congruent phylogAges and patterns
rare instances of horizontal trans- enies and insect fossil dating, the
of cospeciation
mission are absent, a fact that could antiquity of the infections is supnever be established based on exAnother consistent pattern that has perimental observations of transmis- ported by levels of 16S rDNA seemerged for bacteriocyte-associated sion events alone. Even infrequent quence divergence within clades of
bacteria in insects is congruence of horizontal transmission events would endosymbionts.
the phylogenies of the bacterial and scramble patterns of congruence of
The phylogenetic evidence for
host clades. For every case that has host-symbiont phylogenies when bacteriocyte associates of insects
been studied sufficiently, the phy- such phylogenies extend millions of contrasts with that for some other
logeny for the bacteria matches the years into the past.
endosymbioses. In Wolbachia pipiphylogeny for the corresponding inentis, a bacterium that infects insect
For
each
insect
bacteriocyte
assosects. This congruence is strong evigerm line cells and causes various
dence for parallel diversification aris- ciation, congruence of host and sym- reproductive abnormalities, molecuing from faithful and long-term biont phylogenies provides strong lar phylogenies give a clear indicavertical transmission of endosym- evidence that the original infection tion of noncongruence, indicating
bionts along host lineages. Support predates the basal ancestor of the horizontal movement of bacteria
for congruence varies among the symbiont and of the corresponding among lineages (Werren 1997,
cases studied; it depends on the num- hosts. In addition, congruence imApril 1998
199
Table 2. Insect groups for which molecular phylogenetic studies support parallel
cladogenesis of bacteriocyte-associated endosymhionts and hosts, implying ancient
infection of a common ancestor of the host clade. Approximate minimum ages are
based mainly on fossil evidence.
Minimum age of symbiotic association
(i.e., age of common ancestor of hosts)
Reference(s)
Aphids (Hemiptera:
Aphidoidea)
150-250 million years
Munson etal. 1991,
Moran etal. 1993
Cockroaches +
termites (Isoptera +
Blattaria)
135-300 million years
Bandi et al. 1995
Tsetse flies (Diptera:
Glossinidae)
40 million years
Aksoy et al. 1995
Carpenter ants
{Hymenopetera:
Formicidae; genus
Camponotus)
More than 50 million years?
Schroder et al. 1996
Host clade
Bourtzis and O'Neill 1998). Likewise, in some marine invertebrates,
host and endosymbiont phylogenies
also appear not to be congruent
(Dubilier et al. 1995, Krueger and
Cavanaugh 1997, Distel 1998).
What is the basis for this difference? That is, why might bacteriocyte
associates of insects lack the capacity for horizontal transfer? When
symbionts are maternally transmitted and beneficial to hosts, hosts are
selected to provide a continually favorable environment to ensure the
persistence of the association. Consequently, selection on bacteria for
the ability to withstand conditions
outside bacteriocytes will be relaxed.
The resulting intolerance of conditions outside hosts could effectively
eliminate horizontal transfer, facilitating long-term cospeciation of host
and symbiont.
In the case of Wolbacbia^ by contrast, the typical infection route is
maternal but the interaction appears
to be more parasitic than mutualistic
(Werren 1997, Bourtzis and O'Neill
1998). The bacteria do not reside in
specialized bacteriocytes, and their
maintenance and transmission do not
appear to be facilitated by host adaptations. Rather, they inhabit a
variety of cell types and invade host
eggs using their own devices (Werren
1997). As a result, they may retain a
more generalized ability to live under different conditions and thus have
greater propensity for rare horizontal transmission. In some marine invertebrates, such as bivalves and
marine annelids, the associations are
300
mutualistic, and hosts have evolved
specialized cell types for housing symbiotic bacteria (Conway et al. 1992,
Distel 1998). But long-term cospeciation may be prevented by the mode
of infection: In at least some such
associations, infecting stages are not
maternally transferred but are waterborne, providing more opportunity
for cross-infection of host lineages.
Adaptations of
endosymbiotic bacteria
Because bacteriocyte associates of
insects cannot be cultured outside of
hosts, it has been difficult to show
how they differ from free-living bacteria. However, genetic characterization of Bucbnera, the primary
endosymbiont of aphids, has demonstrated some striking adaptations
to endosymbiotic hfe (P. Baumann et
al. 1997).
The best-documented contribution of aphid endosymbionts to their
hosts is the provision of required
amino acids that are rare or absent
from the phloem sap diet. In particular, several lines of evidence support
the role of Buchnera in provisioning
hosts with tryptophan, an amino acid
that is required by animals but is rare
in phloem sap (Douglas and Prosser
1992, Lai et al. 1994). Tryptophan
biosynthesis is regulated by feedback
inhibition acting on anthranilate synthase, which is encoded by trpEG. In
Buchnera that are endosymbiotic
with members of the Aphididae,
trpEG is excised from the chromosome and amplified on plasmids (Lai
et al. 1994). This amplification,
which involves both the tandem duplication of the operon on each plasmid and the presence of multiple plasmids, appears to function in the
overproduction of tryptophan through
enhanced production of the limiting
biosynthetic enzyme. Comparisons
of phylogenies based on plasmidborne and chromosomal genes indicate that trpEG on plasmids originated from the chromosomal genes
within the same bacterium and that
the plasmids are transmitted strictly
vertically (Rouhbaksh et al. 1996,
1997). The trpEG plasmid presumably was fixed through favorable
selection at the host level; that is,
host insects bearing endosymbionts
with this feature experienced greater
growth rates and became established
at the expense of other aphid lineages of the same species in which
endosymbionts possessed only a
chromosomal trpEG copy.
A similar instance of plasmid amplification enabling the overproduction by Buchnera of an essential aphid
nutrient has been discovered for leucine production (Bracho etal. 1995).
The four genes that underlie production of leucine are excised from the
chromosome and transferred to plasmids in some Buchnera species. However, only a single set of the leucine
genes is present on each plasmid.
The presence of the replicating unit
of the plasmid appears to be ancestral in Buchnera of all aphids, but its
acquisition of the leucine biosynthetic genes appears to have occurred
independently in different lineages
(van Ham etal. 1997).
Other unusual aspects of the
Buchnera genome may represent
adaptations to endosymbiotic life.
Because symbionts are confined to
host cells, their growth rates must be
depressed and coordinated with the
development of the host. For
Bucbnera, there is a tight coupling of
bacterial cell number and aphid
growth, with Buchnera showing a
doubling time of approximately two
days, a growth rate that is much
lower than the maximum exhibited
by many free-living bacteria (Baumann and Baumann 1994). Probably in connection with this low
maximal growth rate, both Bucbnera
and Wiggleswortbia, the bacteriocyte
associate of tsetse flies, possess only
BioScience VoL 48 No. 4
a single copy of the rRNA genes, in
contrast to free-living bacteria, which
typically have several copies (Aksoy
1995, Baumann et al. 1995). Reduction in the number of copies of rRNA
genes in prokaryotes is associated
with lowered maximum growth rates.
Endosymbiont adaptations for
improved function as mutualists
present the clear possibility of conflict between selection on individual
cell lineages within a host aphid and
selection on individual aphids. For
example, the excess production of
tryptophan must be beneficial to
hosts but is probably detrimental to
the bacterial cell lineage producing
the nutrient. In related free-living
bacteria, production of anthranilate
synthase has been shown to be costly;
indeed, it lowers fitness of bacteria
when tryptophan is not limiting
(Dykhuisen 1978}, suggesting that a
mutant Buchnera lacking the excess
copies would enjoy a short-term advantage. However, host lineages in
which all symbionts were lacking the
excess copies would suffer a nutritional handicap and would thus be
at a selective disadvantage within
aphid populations. The high mutation rate for loss of tandem repeats
in bacteria (10^-10"^ per generation;
Roth et al. 1996), in combination
with the immediate selective advantage for mutant cells, would be expected to destabilize these host-level
adaptations. In the long term, however, selection at the host level would
favor adaptations that stabilize beneficial gene amplification.
Some findings suggest that such
host-level selection has resulted in
reduced homologous recombination
in Buchnera. Curiously, in certain
Bucbnera of the family Aphididae,
reduction in plasmid-borne functional trpEG has occurred through
the transformation of some of the
repeats into pseudogenes (Lai et al.
1996, L. Baumann et al. 1997). Typically, however, such unneeded repeats would instead be eliminated
through recombination. A possible
explanation for the transformation
into pseudogenes is selection on hosts
to stabilize beneficial gene amplification, resulting in loss of some recombination capabilities (P. Baumann et al. 1997). If certain aphid
lineages subsequently evolved feeding habits or life cycles that reduced
April 1998
their need for tryptophan provision
by Buchnera, selection then might
have favored reduced trpEG expression due to the energy expense of
making unneeded anthranilate synthase. If recombinational mechanisms for eliminating repeat copies
had been lost earlier, gene silencing
would have had to occur through the
fixation of stop codons and frameshifts in the DNA sequence, resulting in transformation of the functional genes into pseudogenes. If this
scenario is correct, then the loss of
certain recombination functions
would appear to be an adaptation to
stabilize mutualistic traits of the endosymbiont that might otherwise be
eliminated through a combination
of high mutation rates and selection
at the bacterial cell level.
Evolutionary consequences
of endosymbiosis
Endosymbiosis is sometimes considered to be an initial step in the evolution of organelles (e.g., Margulis
1993). However, organelles have
arisen only a few times from freeliving prokaryotes, either once or
twice each for mitochondria and
chloroplasts (Cavalier-Smith 1992,
Cray 1992}. By contrast, intracellular symbionts have arisen far more
often, as a glimpse at Buchner's
(1965} volume confirms. Thus, organelle characteristics are not an inevitable consequence of endosymbiosis combined with maternal
transmission.
One feature of mitochondrial and
chloroplast genomes is the loss of
most genes present in the ancestral
prokaryotic genome and the dependence on host gene products for many
of the basic functions required for
replication. The only endosymbiont
for which substantial genome characterization has been carried out is
Buchnera, for which 85 kb has been
sequenced in the Baumann laboratory (summary in P. Baumann et al.
1997). Despite substantial overall
sequence divergence from related
free-living bacteria, Buchnera coding gene sequences indicate selective
constraint of the functioning polypeptides; the reading frame is maintained and nucleotide base substitutions are concentrated at silent, or
synonymous, sites (i.e., sites that do
not affect amino acid identity}. Cenes
with a wide range of "housekeeping" and biosynthetic functions have
been identified in Buchnera (summaries in Baumann et al. 1995, P.
Baumann et al. 1997, Clark and Baumann 1997, Clark et al. 1997}. This
work provides strong evidence that
Buchnera contains a complement of
genes enabling a wide range of processes involved in cell growth and
reproduction. This endosymbiotic association arose at least 200 million
years ago, so the retention of most
genes is not readily explained as the
result of the association being too
young for Buchnera to have developed characteristics of organelles. A
more important limitation on the
loss of genes might be that endosymbionts in animals reside primarily in
somatic cells (the bacteriocytes},
eliminating most opportunities for
gene transfer between symbiont and
host nuclear genome.
One apparent long-term consequence of endosymbiosis is increased
rates of substitution in DNA sequences of endosymbionts relative
to non-endosymbiotic relatives
(Moran 1996}. This increase is seen
in the 16S rDNA of endos.ymbionts
of aphids, whiteflies, mealybugs,
tsetse flies, and carpenter ants
(Moran 1996, Lambert and Moran
in press}. In Buchnera., in which many
more genes have been sequenced than
in any other endosymbiont, the increased rate extends to all genes examined (Moran 1996, unpublished
analyses}.
Positive natural selection for base
substitutions seems unlikely to explain the faster evolution at all loci,
because the effects of positive selection are expected to be locus and site
specific within genes. In fact, it appears that the base substitutions are
deleterious. Evidence for this hypothesis is provided by the changes in
protein-coding genes in Buchnera.
The net effect of the faster evolution
in Buchnera is the accumulation,
throughout the genome, of A and T
at the expense of representation of G
and C. This shift in nucleotide base
composition is strong enough that
most amino acid differences between
polypeptides of Buchnera and those
of E. coli are ones that allow increased A -H T in the corresponding
codon and thus in the DNA sequence.
301
It is hard to imagine that the accumulation, in all polypeptides, of
amino acids with A + T-rich codon
families is adaptive at the level of
protein function. A more plausible
explanation is mutational bias favoring A + T combined with an increased rate of base substitution
within lineages.
If positive selection is excluded as
the basis for the faster substitution
rates of endosymbionts, we are left
with two other categories of explanation. One is that the increased rate
of substitution (fixed changes occurring in a lineage) is due to an increased rate of new mutations. This
increased mutation rate could result
from either an increased rate per
generation or a faster average generation time. But if increased incidence of mutation were the sole explanation, the increase would have
equivalent effects on substitution
rates at sites that are subject to selection and at sites that are effectively
neutral with respect to selection, and
comparative sequence analysis indicates stronger effects on selected sites.
A convenient way to categorize sites
according to the intensity of selection is to separate silent (or synonymous) from replacement (or nonsynonymous) sites in protein coding
sequences. At silent sites, where
nucleotide changes do not affect the
corresponding amino acid and thus
do not affect the gene product, base
substitutions have little consequence
for fitness. At replacement sites,
where a nucleotide change results in
a change in the amino acid and thus
in the selected phenotype, base substitutions are more likely to affect
fitness. The increase in substitution
rate in coding genes of Buchnera is
heavily concentrated at replacement
sites (Moran 1996). This observation rules out increased mutation as
the primary basis for the acceleration in substitution rates.
This result instead supports the
second explanation, that the excess
changes represent deleterious substitutions. According to this view, purifying selection is less effective at
purging deleterious mutations. This
reduction in the effects of selection
could arise in either of two ways:
from relaxed selection or from lower
effectiveness of selection as a result
of population structure. The first pos302
sibility, that selection is uniformly relaxed at all genes of Buchnera, regardless of function, is doubtful. Because
endosymbionts show reduced effective population sizes than free-living
bacteria, with a consequently greater
susceptibility to genetic drift, the
second possibility may apply. This
proposal resembles the arguments
made by Lynch (1996, 1997) for the
accumulation of deleterious mutations in mitochondria and other organelles. Organelles have similar
population structure to endosymbionts because they are asexual, intracellular, and maternally inherited.
Current mysteries and
future problems
The recent molecular studies of insect bacteriocyte-associated endosymbioses have given glimpses into
the evolution of some of these associations. But most remain entirely
unstudied, particularly the complex
multiple infections found in some
insects, such as the planthoppers and
treehoppers (Hemiptera; Buchner
1965). Furthermore, this work has
raised some new questions about the
evolution of bacteriocyte associates
in insects. For example, both Buchnera and tsetse endosymbionts exhibit unusually high levels of GroEL
protein (Ishikawa 1989, Aksoy 1995,
Baumann et al. 1996), a chaperonin
that normally functions in refolding
other polypeptides. The reason for
this overexpression is not yet clear,
although speculative explanations
have been put forward (Ishikawa
1989, Moran 1996, P. Baumann et
al. 1997). The finding of plasmidborne pseudogenes in some Buchnera
presents another mystery: Why are
nonfunctional copies not lost by recombination, as is typical for bacteria? Some basic difference involving
recombination pathways may, as already discussed, distinguish Buchnera from free-living bacteria.
Whether other endosymbionts also
have atypical recombination pathways is not yet known. The basis for
the nucleotide base compositional
bias against G + C, a distinctive feature of numerous bacteriocyte associates (Table 1), is also unknown.
Endosymbiotic interactions are
shaped by forces acting at multiple
levels, primarily that of the host in-
dividual and the bacterial cell lineage. Some mutations in endosymbionts will favor the individual symbiont lineage at the expense of the
host. Such "selfish" mutations would
include any that result in higher replication of the symbiont at the host's
expense. If higher replication results
in disproportionate transmission to
progeny, these selfish symbionts
could increase in frequency within a
host population, at least in the short
term. Michod (1997) presents a theoretical framework for evaluating this
very situation that characterizes
bacteriocyte associations in insects:
a balance between frequent mutation in the direction of "selfish" traits
favoring individual cell lineages and
selection on the overall group (in
this case, the host individual and its
resident bacterial population).
Buchner (1965) mentions cases in
whiteflies in which symbionts spread
beyond their usual limits within individual host insects, indicating the
possibility of such selfish behavior
that harms the host.
Of course, selection favoring better-nourished (and faster-growing)
host individuals will counter the
spread of selfish bacterial lineages.
One host adaptation that would limit
the spread of selfish symbionts would
be the sequestration of "germ line"
symbionts from more rapidly dividing "somatic" symbionts that function in provisioning hosts with nutrients. Early microscopical studies
suggest this kind of system in cicadas
(Koch 1967). The infection of progeny with entire maternal bacteriocytes, as in whiteflies (Buchner 1965,
Costa et al. 1996), would also have
the effect of limiting the spread of
"selfish" bacteria to single host progeny, provided that bacteria are confined to single bacteriocytes early in
the development of the host. Frank
(1996) proposes thatthe segregation
of germ line symbionts may be a
"policing" mechanism for the containment of selfish cell lineages within
hosts of endosymbionts.
Role of endosymbiosis in
host evolution
Endosymbiosis has been argued to
be a route for evolutionary innovation that underlies the origin and
diversification of many groups of
BioScience Vol. 48 No. 4
organisms that would otherwise not
exist. Within the realm of the insects, this hypothesis appears to be
true: Essentially all strict phloemfeeders, strict blood-feeders, and
other groups with restricted diets
possess endosymbionts. Recent molecular phylogenetic evidence indicates that endosymbiotic infections
date to the origins of a variety of
major insect clades, reinforcing the
idea that the origin of endosymbiosis
was an integral factor spurring diversification of some insect taxa.
Because some of these groups are
large adaptive radiations, endosymbiosis has probably had a massive
effect on patterns of insect diversity.
Although endosymbiosis appears
to facilitate diversification within
ecological niches that would otherwise be inadequate, dependence on
an endosymbiont may simultaneously enforce some restrictions on
host evolution. Cenes encoded within
the endosymbiont are subject to a
different set of population genetic
parameters than genes encoded in
the insect nuclear genome. As noted
above, endosymbionts are subject to
within-host selection, which often may
counter selection between hosts. In
addition, mutation rates and generation times differ between endosymhiont and host genomes. The distinct
population genetics of endosymbiont
genes may affect the speed of the
response to selection. For example,
adaptation may occur faster in symbiont genes than host genes due to
the shorter generation time and consequent greater supply of mutations
of the bacteria. Finally, symbiont
genes are subject to a population
structure in which genetic drift is
more likely to limit the effectiveness
of selection, resulting in increased
fixation of mildly deleterious mutations. This increased rate of accumulation of deleterious mutations will
be countered, to some extent, by
selection at the level of host individuals. The adequacy of this counterbalancing will depend on the impact of selection at the host level,
which is dependent in turn on the
effective population size of the host.
It is not yet clear what factors are
most important in determining the
net effect of these conflicting evolutionary forces. One intriguing possibility is that the accumulation of
April 1998
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characterization of a plasmid localized in
Buchnera sp., bacterial endosymbiont of
Research on insect endosymbionts
tbe aphid Rhopalosiphum padi. Journal of
in the Moran lab is supported by
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witb Plant Microorganisms. New York:
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Interscience,
US Department of Agriculture, NaBC, Bragg TS, Turner CE. 1992.
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by a fellowship from the Plant-Insect
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Interactions Training Grant to the
Insect Biochemistry and Molecular Biology
22: 415^21.
University of Arizona from NSF,
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