Molecular Ecology (2014) 23, 1473–1496 doi: 10.1111/mec.

12421

SPECIAL ISSUE: NATURE’S MICROBIOME

INVITED REVIEWS AND META-ANALYSES
The impact of microbial symbionts on host plant
utilization by herbivorous insects
A L L I S O N K . H A N S E N and N A N C Y A . M O R A N
Yale University, West Haven, CT 06516-7388, USA

Abstract
Herbivory, defined as feeding on live plant tissues, is characteristic of highly successful
and diverse groups of insects and represents an evolutionarily derived mode of feeding.
Plants present various nutritional and defensive barriers against herbivory; neverthe-
less, insects have evolved a diverse array of mechanisms that enable them to feed and
develop on live plant tissues. For decades, it has been suggested that insect-associated
microbes may facilitate host plant use, and new molecular methodologies offer the pos-
sibility to elucidate such roles. Based on genomic data, specialized feeding on phloem
and xylem sap is highly dependent on nutrient provisioning by intracellular symbionts,
as exemplified by Buchnera in aphids, although it is unclear whether such symbionts
play a substantive role in host plant specificity of their hosts. Microorganisms present
in the gut or outside the insect body could provide more functions including digestion
of plant polymers and detoxification of plant-produced toxins. However, the extent of
such contributions to insect herbivory remains unclear. We propose that the potential
functions of microbial symbionts in facilitating or restricting the use of host plants are
constrained by their location (intracellular, gut or environmental), and by the fidelity of
their associations with insect host lineages. Studies in the next decade, using molecular
methods from environmental microbiology and genomics, will provide a more compre-
hensive picture of the role of microbial symbionts in insect herbivory.
Keywords: Buchnera, coevolution, gut microbiota, insect nutrition, insect–plant–microbe inter-
action, intracellular symbiont

Received 15 April 2013; revision received 2 June 2013; accepted 12 June 2013

herbivory is a successful feeding mode, but only after

Introduction
Herbivory, defined here as feeding on live plant tissues,
is an evolutionarily derived mode of feeding in insects
(Strong & Lawton 1984; Labandeira & Sepkoski 1993).
Evolutionary transitions to herbivory, specifically
herbivory on angiosperms, have been followed by accel-
erated rates of insect species diversification (Mitter et al.
1988; Farrell & Farrell 1998). Herbivorous insects are
indeed extraordinarily diverse, as measured both by
species numbers and by their varied lifestyles (Strong &
Lawton 1984; May 1988; Farrell 1998; Futuyma & Agra-
wal 2009). This macroevolutionary pattern implies that
Correspondence: Allison K. Hansen, Fax: 203-737-3109;
E-mail: akh@illinois.edu; and Nancy A. Moran,
Fax: 203-737-3109; E-mail: nancy.moran@austin.utexas.edu
key hurdles are overcome.
Obvious obstacles to herbivory include the low nutri-
ent content, indigestibility and toxicity of many plant
tissues. Although plants represent the largest source of
biomass in terrestrial systems, their tissues are largely
composed of recalcitrant molecules such as cellulose,
hemicellulose, pectin and lignin that are difficult or
impossible to digest for most animals (Watanabe &
Tokuda 2010). Furthermore, the levels of nitrogen rela-
tive to carbon tend to be low when compared to the
nutritional needs of animals and exhibit substantial var-
iation among different plant tissues, growth stages and
species (Mattson 1980). Besides being generally nitrogen
poor, some plant tissues present very unbalanced pro-
files of amino acids, so that growth may be severely
limited by shortages of particular essential amino acids,

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1474 A . K . H A N S E N and N . A . M O R A N
or of vitamins (e.g. Sandstr€ om & Moran 1999). Nutritive
quality is often lowered further because many plants
contain compounds such as protease inhibitors that
inactivate digestive enzymes in the insect gut, prevent-
ing digestion and absorption (Ryan 1990). Finally, plant
tissues are laced with a wide variety of other toxic allel-
ochemicals that vary extensively among plant taxa and
in mode of action on their target. There is a vast litera-
ture on the many morphological, metabolic and
behavioural adaptations of insects that enable herbivory
and on the adaptations of plants for avoiding negative
impacts of insect herbivory (Schoonhoven et al. 2005).
Recently, more researchers have begun to appreciate
the potential for associations with symbiotic microbes as
a means of expanding ecological range of eukaryotic
hosts and of animal hosts in particular (Moran 2007;
McFall-Ngai et al. 2013). Symbionts, defined as microbes
that form persistent, noninvasive associations with hosts,
are common in insects generally and in herbivorous
insects in particular. Microbes offer capabilities for syn-
thesizing nutrients, digesting recalcitrant plant polymers
and neutralizing plant toxins. The possibility that symbi-
otic associations could shape interactions with host plants
has been proposed by numerous researchers (reviewed
in Berenbaum 1988; Barbosa et al. 1991; Bernays & Chap-
man 1994; Felton & Tumlinson 2008; Janson et al. 2008;
Clark et al. 2010; Feldhaar 2011; Frago et al. 2012). How-
ever, the extent to which insect symbionts impact their
insect host’s ability to feed on plant tissues or shape spe-
cific interactions between herbivorous insects and partic-
ular plant species remains an open question.
In this review, we evaluate the impact of symbionts
on whether insects can use plants as food and on which
plants are used, and how. The primary reason for
expecting symbionts to be able to assist insects in
exploiting plant hosts is that microorganisms, including
bacteria and fungi, possess many functional capabilities
entirely absent from animals. These include biosynthetic
capabilities for universally required metabolites such as
vitamins and some amino acids, catabolic capabilities
such as cellulolytic activity and production of many
toxic molecules. As such, they present a vast new tool
kit with potential to facilitate herbivory as a lifestyle.
On the other hand, symbiont acquisition by hosts may
be unreliable, or tissue localization of symbionts may
limit their contributions to specific host functions.
Studies of herbivore–plant interactions span different
levels of analysis, from mechanistic studies of how par-
ticular insects digest specific plant tissues to evolution-
ary and population analyses. We consider all of these,
with emphasis on the use of new genomic data and
molecular techniques to infer symbiont roles in deter-
mining abilities to overcome obstacles to feeding on
particular plant tissues or species. This evaluation nec-
essarily involves considering specific mechanisms
through which symbiotic microorganisms might enable
use of particular plant tissues or plant species.
Broad categories of symbionts
For purposes of this review, we define symbiont
broadly to include any microbes that have persistent
associations with their hosts and that do not cause overt
pathogenic symptoms. Insect symbionts can be divided
into three general categories based on their location
relative to the insect body (Fig. 1). First, some symbio-
nts are intracellular or housed within the body cavity
and are passed between generations through eggs,
resulting in maternal inheritance. Heritable, usually
intracellular, symbionts are found in a variety of insect
groups, which often evolve specialized cells (bacterio-
cytes) for housing them. Second, microbes inhabit the
Fig. 1 Potential contributions of microor-
ganisms to host ability to feed on live
plants are dependent on the symbiont
location inside or outside the insect
body.
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 E V A L U A T I N G S Y M B I O N T E F F E C T S O N H O S T P L A N T U S E 1475
guts of most animals including most insects, living
within the gut lumen where they form communities of
varying complexity in different insect species. Third,
microbes regularly colonize food before it is ingested
and may impact its composition. These environmental
symbionts most likely are important in insects that feed
on a stored food resource within a nest or localized
feeding area such as a gall or gallery.
The potential functions of microbial symbionts in
facilitating or restricting the use of host plants are con-
strained by their location and by the persistence of
their associations with host lineages. For example, heri-
table symbionts form long-term associations with host
lineages and are thus ideally positioned to contribute
evolutionarily significant capabilities, but they are often
confined to the cytoplasm of specialized host cells and
thus unable to secrete gene products directly into the
gut lumen or into oral secretions, where they might
aid in digestion, detoxification or modification of host
plant recognition. Gut symbionts or environmental
symbionts are better positioned for contributing these
functions, but their transmission among individual
hosts may be less dependable in some insect life
cycles, so their contributions might often be too unreli-
able to facilitate the expansion of host plant range.
Intracellular symbionts can contribute small molecules
to host metabolism, but these contributions will
depend on host capabilities for transporting these
molecules across membranes and throughout the insect
body.
Finally, these three symbiont categories differ in
whether they have dynamic genomes that can acquire
new abilities as they evolve with hosts. In particular,
genomes of heritable symbionts typically undergo
extensive gene loss over their long-term evolution and
do not incorporate novel genes, leading to dwindling
metabolic capabilities (McCutcheon & Moran 2012). If
herbivorous insects need to respond to both short-term
and long-term changes in the nutritional quality, plant
recognition or toxicity of plant tissues that they ingest,
long-term obligate symbionts with reduced genomes
will be unlikely to provide the necessary novelty. In
contrast, most facultative heritable symbionts, as well as
gut and environmental symbionts show dynamic
genomes, featuring ongoing gain and loss of functional
genes, genome rearrangements and mobile elements,
processes that are typical of most bacterial lineages.
Intracellular heritable symbionts
Many insect symbionts that synthesize nutrients for
their insect hosts are intracellular and obligate for the
insect’s survival and reproduction. Intracellular symbio-
nts live in a stable environment generally protected
© 2013 John Wiley & Sons Ltd
from competition from other microbes and require only
simple host-derived carbon and nitrogen inputs for
energy. In contrast, symbionts that digest complex plant
metabolites into absorbable nitrogen or energy sources
are extracellular, either in the lumen of the gut or out-
side the host’s body, where complex macromolecule
breakdown must occur before absorption. In this
section, we will discuss current knowledge of how
intracellular insect symbionts provision their herbivore
hosts with required nutrients lacking in the diet and the
implications for how these symbionts allow insect
herbivores to exploit living plants as food.
Intracellular nutritional symbionts that are inherited
maternally are found in a variety of insect taxa, includ-
ing many herbivorous groups. For example, two of the
most diverse insect groups, the leaf beetles (Chrysome-
lidae) and the weevils (Curculionoidea), include species
containing bacterial symbionts that are transmitted
through eggs and believed to be critical for nutrition of
hosts (Conord et al. 2008; K€ olsch and Pedersen 2010).
Symbioses of sap-feeding insects include the most
intensively studied cases, so we focus on those in this
section.
Obligate intracellular symbioses are universal in
hemipteran insects that feed on phloem or xylem sap;
these include most members of the suborders Stern-
orrhyncha (aphids, whiteflies, psyllids, scale insects)
and of the Auchenorrhyncha (leafhoppers, cicadas, spit-
tlebugs and planthoppers) (Buchner 1965; Douglas 1998;
Moran et al. 2008; Urban & Cryan 2012). Obligate
symbionts are also widespread in hemipteran bugs
feeding on plant cell contents, such as stink bugs and
plant bugs (suborder Heteroptera, including Coreidae,
Alydidae, Pentatomidae, Plataspidae) (e.g. (Hosokawa
et al. 2006)). In many of these systems, phylogenetic
studies have revealed an evolutionary history of long-
term codiversification of insect hosts with bacterial sym-
bionts, with the associations dating to the origins of
major groups such as aphids and psyllids and in some
cases dating to the earliest stages of terrestrial herbiv-
ory, on Permian vascular plants at least 270 million
years ago (Munson et al. 1991; Thao et al. 2000a,b; Bau-
mann 2005; Downie & Gullan 2005; Moran et al. 2005;
Moran 2007). In most cases, obligate intracellular symbi-
onts are bacteria, and the symbiotic lifestyle has arisen
multiple times within several different bacterial groups,
including Gammaproteobacteria, Betaproteobacteria, Alpha-
proteobacteria and Bacteroidetes (Fig. 2, Moran et al. 2008).
Widespread herbivore–symbiont expansions into
sap-feeding niches
The central basis to these obligate symbioses is nutri-
ent provisioning, as revealed by both genomic and
1476 A . K . H A N S E N and N . A . M O R A N

Genome Val
Insect species Specialization Nutritional symbiont size (kb) Orn Arg His Lys Thr BCA Ile Leu Chorismate Trp Phe Met Sul Cys
Heteropsylla texana Ph γ Carsonella ruddii HT 158
γ Carsonella ruddii HC & 166
Heteropsylla cubana Ph γ SS HC 1,122

Ctenarytaina spatulata Ph γ Carsonella ruddii CS 163

γ Carsonella ruddii CE & 163

Ctenarytaina eucalypti Ph γ SS CE 1,441

Pachypsylla venusta Ph γ Carsonella ruddii PV 160

Pachypsylla celtidis Ph γ Carsonella ruddii PC 160

Bemisia tabaci Ph γ Portiera aleyrodidarum 358

Acyrthosiphon pisum Ph γ Buchnera aphidicola APS 656

Acyrthosiphon kondoi Ph γ Buchnera aphidicola AK 653

Uroleucon ambrosiae Ph γ Buchnera aphidicola UA 628

Schizaphis graminum Ph γ Buchnera aphidicola Sg 653

Ph
Hemiptera

Baizongia pistaciae γ Buchnera aphidicola Bp 618

γ Buchnera aphidicola Cc & 425

Cinara cedri Ph γ Serratia symbiotica Cc 1,763

Cinara tujafilina Ph γ Buchnera aphidicola Ct 453

Aspidiotus nerii Me F Uzinura diaspidicola 263

β Tremblaya princeps & 139

Planococcus citri Ph γ Moranella endobia 538

Homalodisca vitripennis Xy F Sulcia muelleri Hc & 246

γ Baumannia cicadellinicola 686

F Sulcia muelleri DSEM & 277

Xy
*
Diceroprocta semicincta α Hodgkinia cicadicola 144
F Sulcia muelleri CARI & 277
Clastoptera arizonana Xy
β Zinderia insecticola 209

Megacopta punctatissima Ph γ Ishikawaella capsulata 746

= long-term obligate symbiont encodes gene = Gene not encoded

= obligate co-symbiont encodes gene = Gene not required because
alternative pathway encoded
= putative insect homolog complements
pathway

Fig. 2 Amino acid biosynthesis capabilities of long-term nutritional symbionts with fully sequenced genomes for herbivorous insects
in the Hemiptera. Evolutionary relationships are based on Cryan & Urban (2012). Blue branches represent Sternorrhyncha, orange
branches represent Auchenorrhyncha and the purple branch represents Heteroptera. Specialization refers to the plant tissue eaten
(Ph, phloem; Me, mesophyll and Xy, xylem). Bacterial symbiont taxa symbols represent c, Gammaproteobacteria; b, Betaproteobacte-
ria; a, Alphaproteobacteria; F, Flavobacteriales. Genes from essential amino acid pathways and pathways of their intermediates are
presented as rectangles to the right of the insect tree. Orn, ornithine; Arg, Arginine; His, histidine; Lys, lysine; Thr, threonine; BCA,
shared branched-chain amino acid pathway; Val, valine; Ile, isoleucine; Leu, leucine; chorismate, shared pathway for aromatic amino
acids; Trp, tryptophan; Phe, phenylalanine; Met, methionine pathway (starting from the homoserine intermediate); Sul, sulphur
reduction and Cys, cysteine. *alternative methionine synthetase (MetH) requires additional genes for biosynthesis of a B12-like cofac-
tor; these are encoded in the Hodgkinia genome but not shown. Information for gene presence is based on GenBank data, and rele-
vant papers are cited in the main text.

experimental studies (Douglas 1998; Moran et al. 2008; with intracellular, heritable bacteria that provision
Hansen & Moran 2011). Animals feeding on plants are essential amino acids (Mittler 1971; Douglas 1988;
generally limited by available nitrogen (Schoonhoven Douglas & Prosser 1992; Febvay et al. 1995; Shigenobu
et al. 2005), and nitrogen concentrations are typically et al. 2000; Baumann 2005). Given the nutritional
two to ten times lower in phloem and xylem, respec- requirements of insects, the metabolic capabilities of
tively, compared with leaves (Mattson 1980). Further- bacteria and the restricted nutrient content of xylem
more, xylem and phloem sap contain unbalanced and phloem sap, it is no surprise that nutrition-provi-
profiles of amino acids and are especially deficient in sioning symbionts are prevalent among sap-feeding
the so-called essential amino acids, that is, those that insect taxa (Fig. 2).
cannot be synthesized by animals (Slansky & Scriber In these ancient obligate symbioses, a consequence of
1985; Andersen et al. 1989; Sandstr€ om & Pettersson the long-term maternal transmission and resulting
1994; Sandstr€ om & Moran 2001). Sap-feeding insects clonality of symbionts is that their genomes are drasti-
overcome these nutritional limitations by associating cally reduced in size and gene number and display

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 E V A L U A T I N G S Y M B I O N T E F F E C T S O N H O S T P L A N T U S E 1477
rapid sequence evolution and an A+T bias in nucleotide
base composition (McCutcheon & Moran 2012). Despite
loss of up to 90% of ancestral genes, all of these obligate
symbiont genomes retain genes for essential amino acid
pathways but have lost most genes for nonessential
amino acid biosynthesis, consistent with their nutri-
tional roles (Shigenobu et al. 2000; Moran et al. 2008;
McCutcheon & Moran 2012; Fig. 2). While a few genes
encoding enzymes in certain essential amino acid path-
ways are sometimes missing (Fig. 2), genes present in
the insect host and/or other co-occurring obligate sym-
bionts appear to provide these missing enzymes
(Fig. 2). For example, co-occurring obligate symbionts
in six independently evolved symbiont systems encode
enzymes that complement enzymes or pathways miss-
ing from their partner symbiont (Perez-Brocal et al.
2006; Wu et al. 2006; Gosalbes et al. 2008; McCutcheon
& Moran 2007; McCutcheon et al. 2009; McCutcheon &
Moran 2010; McCutcheon & von Dohlen 2011; Sloan &
Moran 2012a; Fig. 2).
In addition to these genomic data, experimental
results available for the aphid-Buchnera system also sup-
port a role of obligate symbionts in amino acid provi-
sioning (Douglas 1998; Akman G€ und€ uz & Douglas
2009; Douglas et al. 2011; Macdonald et al. 2011). Buch-
nera uses nonessential amino acids as substrates for the
production of essential amino acids. Specifically, Buch-
nera may efficiently use glutamate synthesized by insect
enzymatic machinery (GOGAT cycle), which recycles
waste ammonia, to sustain this nitrogen-limited mutual-
ism (Hansen & Moran 2011), although the extent of
such recycling is not yet clear (Macdonald et al. 2012).
Although amino acids generally appear to be the
primary nutrients provided by obligate symbionts in
sap-feeding insects, genome sequencing suggests that
these symbionts likely also provision cofactors and
other potentially beneficial compounds to hosts. In pea
aphids (Acyrthosiphon pisum), Buchnera encodes genes
involved in riboflavin production (Shigenobu et al.
2000). Baumannia, one of the cosymbionts of the sharp-
shooter Homalodisca vitripennis (Cicadellidae: Cicadelli-
nae), retains 84 genes predicted to enable the
production of several B-vitamins (Wu et al. 2006).
Baumannia was the first obligate symbiont sequenced
from a xylem-feeding host, and the presence of these
genes was initially hypothesized to reflect the absence
of these compounds from the xylem sap diet of sharp-
shooters. However, subsequent genome sequences of
symbionts of xylem feeders lack many of these genes
(McCutcheon & Moran 2012). Carotenoids also appear
to be beneficial or required nutrients for phloem-feed-
ing insects, and phloem lacks these compounds, which
are fat-soluble. Recently, it has become apparent that
phloem-feeding insects have independently evolved
© 2013 John Wiley & Sons Ltd
mechanisms for generating their own carotenoids, some-
times through incorporation of foreign genes directly into
the insect genome (see Box 2). In the case of whiteflies,
carotenoid biosynthetic genes are intact in the genome of
their obligate symbiont (Portiera), despite its extremely
reduced genome (358 kb) (Sloan & Moran 2012b).
In sum, heritable, intracellular symbionts have been
critical in enabling insects to occupy and dominate
ecological niches in which essential amino acids are
limiting, such as dietary specialization on xylem and
phloem sap (Buchner 1965; Baumann 2005; Moran et al.
2005; Moran 2007).
Obligate symbiont genomes and their influence on host
plant breadth
Insect adaptations involving morphology, behaviour
and physiology have enabled and restricted insect use
of particular host plants (Bernays & Chapman 1994;
Schoonhoven et al. 2005). As discussed above, heritable
symbionts enable insects to utilize phloem and xylem
sap as food; however, it is not clear whether these sym-
bionts are major determinants of host plant range. Sym-
biont-driven host plant specificity would depend on
differences among plants in nutrient profiles, because
the main role of obligate symbionts is to synthesize and
provision essential nutrients, particularly amino acids
and vitamins. Potentially, obligate symbionts can con-
tribute to host plant specificity by provisioning nutri-
ents lacking in specific plant species, thereby allowing
an insect to develop and reproduce on a plant other-
wise nutritionally unsuitable. Alternatively, in addition
to the nutritional capacity of the obligate symbiont,
acquisition of an additional cosymbiont could buffer the
insect from host plant-derived nutrient deficiencies,
expanding its host plant range. Conversely, symbionts
that lose particular biosynthetic pathways may con-
strain insect hosts to plant species or tissue types that
are enriched in these missing nutrients (Fig. 3).
Comparative analyses of genomes from different obli-
gate symbiont lineages of diverse hemipteran taxa
reveal convergent patterns in gene loss and retention
(Moran et al. 2008; McCutcheon & Moran 2012). When
examining symbionts across sap-feeding hemipteran
species, with single or partner symbionts, the same
overall pattern is evident: symbionts are able to provi-
sion most or all essential amino acids in each insect
host species (Fig. 2). This similarity reflects the known
deficiency of essential amino acids in both phloem sap
and xylem sap across diverse plants (Andersen et al.
1989; Andersen et al. 1992; Brodbeck et al. 1990; Sands-
tr€
om & Pettersson 1994). Because the single or paired
symbionts encode the majority of essential amino acid
pathways (Fig. 2) and because plants are generally
1478 A . K . H A N S E N and N . A . M O R A N

Gain of divergent nutritional essential amino acid co-symbiont amino acid

symbionts enabling sap feeding pathway gene loss complementation

gain of obligate symbiont A long-term obligate symbiont encodes gene gene lost
gain of obligate symbiont B
obligate co-symbiont encodes gene essential amino acid
gain of obligate co-symbiont C

Nutritional symbiont encoded factors

Influence insect nutrition Enable, maintain, and/or constrain host plant use

Insect encoded factors

host controlled amino acid host control of essential host control of symbiont
transport surrounding symbiont cells amino acid pathways cell density
outside

inside
amino acid/poly- amine/organocation
(APC) transporter
host genes regulating the symbiont’s essential host killed symbiont cell
amino acid pathways
live symbiont cell
obligate symbiont’s essential amino acid pathways
essential amino acid

Fig. 3 Mechanisms mediated by intracellular symbionts and insect hosts that potentially influence plant use by host insects. Proposed
mechanisms can either expand or contract insect host plant breadth. Upper panel: left, macroevolutionary patterns of gain and loss
of obligate symbionts; centre and right, complementarity of metabolic capabilities of cosymbionts. Lower panel: left, host transporter
structure generated by Pyre2 (Kelley & Sternberg 2009); centre, metabolism in the aphid bacteriocyte, redrawn from Hansen & Moran
(2011); right, host control of symbiont numbers, redrawn from Nishikori et al. (2009).

similar in amino acids available in phloem and xylem,
symbionts for the most part appear to have limited
influence on host plant breadth. In some insect species,
multiple strains of heritable symbionts have been
sequenced; these cases include Buchnera of aphids,
Carsonella ruddii of psyllids and Sulcia muelleri of auc-
henorrhynchan sap feeders. In each of these cases, simi-
lar gene content and order have been maintained
among strains, especially for essential amino acid bio-
synthesis, despite millions of years of divergence in
separate insect lineages of insect hosts feeding on diver-
gent host plants (Tamas 2002; Degnan et al. 2011; Lam-
elas et al. 2011; Sloan & Moran 2012a; McCutcheon &
Moran 2012, van Ham et al. 2003).

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 E V A L U A T I N G S Y M B I O N T E F F E C T S O N H O S T P L A N T U S E 1479
Although there are broad similarities across plants in
sap composition and across heritable symbionts in
nutrient-provisioning capabilities, plant species do show
some variation in sap composition, and several symbio-
nts lack genes for multiple steps in amino acid biosyn-
thesis. This variation raises the possibility that symbiont
biosynthetic capabilities partially constrain host plant
breadth. For several cases, genes that encode the major-
ity of the methionine, tryptophan, histidine or arginine
pathways have been lost (Fig. 2).
In particular, pathways for the sulphur-containing
amino acids methionine and cysteine, and for sulphur
assimilation through sulphate reduction, are not
encoded in 70% of symbionts from phloem feeders
(Fig. 2) and are also absent from the small genome of
the sole symbiont of a mesophyll-feeding scale insect
(Sabree et al. 2013). These losses present a conundrum
because animals do not encode enzymes to reduce
sulphur for incorporation into the essential amino acids
methionine and cysteine, in addition to other essential
sulphur-containing metabolites. In contrast, most
xylem-feeding insects studied to date possess cos-
ymbionts that retain most steps in the pathways for
methionine biosynthesis and sulphur assimilation.
Potentially, xylem sap lacks sufficient reduced sulphur
or sulphur-containing organic molecules and thus
imposes a more stringent requirement that obligate
symbionts assimilate inorganic sulphur. This difference
between xylem and phloem feeders might reflect the
fact that a major form of reduced sulphur, S-methyl-
methionine, is transported primarily in the phloem not
xylem, especially when sulphur is reduced in mature
leaves. But the site of sulphur reduction varies depend-
ing on environmental conditions and plant species
(Bourgis et al. 1999; Tan et al. 2010). For example, in
legumes such as pea (Pisum sativum), sulphur reduction
occurs in the roots, and S-methyl-methionine is then
transported from xylem to phloem (Buchner et al. 2004).
In sum, it is not yet clear why certain obligate symbio-
nts retain or lose pathways for methionine, sulphur
reduction and cysteine. We hypothesize that the reten-
tion and loss of these pathways are associated with the
mode of reduced sulphur translocation in the plant and
with the insect host’s ability to assimilate this reduced
sulphur into methionine and cysteine.
In addition to the widespread loss of the sulphate
assimilation pathway from phloem feeders, several
other amino acid biosynthetic pathways are occasionally
absent. For example, pathways for tryptophan, histidine
and arginine biosynthesis are absent from Carsonella,
the nutritional symbiont of psyllids (Nakabachi et al.
2006; Sloan & Moran 2012a; Fig. 2). In some psyllid
species, such as Heteropsylla cubana and Cternarytaina
eucalypti, the tryptophan and/or arginine pathways are
© 2013 John Wiley & Sons Ltd
encoded in genomes of cosymbionts (Sloan & Moran
2012a; Fig. 2). However, cosymbionts are lacking in
some psyllid species, and how these psyllids acquire
histidine and tryptophan is not clear (Nakabachi et al.
2006; Sloan & Moran 2012a). Potentially some psyllids,
such as gall-forming species of Pachypsylla, obtain
enhanced nutrition through manipulation of the host
plant. As mentioned above, a majority of sequenced
genomes of symbionts of phloem-feeding insects also
lack genes for arginine biosynthesis, specifically for the
intermediate metabolite ornithine; nevertheless, most
retain genes encoding the enzymes that catalyse the
reactions for arginine synthesis from ornithine (Fig. 2),
suggesting that insect- and/or plant-derived ornithine
is required for arginine biosynthesis.
In conclusion, obligate symbionts appear to lose some
genes within essential amino acid pathways if these
amino acids or their intermediates can be provisioned
by the insect or cosymbiont or directly from the diet.
Loss of symbiont genes is irreversible, because long-
term symbiont genomes do not obtain genes through
horizontal transfer (Tamas 2002; Degnan et al. 2011;
Lamelas et al. 2011). Ultimately, such gene loss in sym-
bionts may constrain insects to a specific host plant that
is enriched in particular nutrients, unless these can be
obtained from cosymbionts and/or insect-encoded
enzymes (Fig. 3). Potentially this can lead to increasing
numbers of obligate associations with symbionts that
provision different required nutrients, as seen in the
cosymbionts of Auchenorrhyncha (McCutcheon &
Moran 2012). It is unclear whether symbiont gain or
replacements can increase host plant breadth.
Dynamic potential of the integrated insect–microbe
amino acid metabolism
The insect’s own biosynthetic capabilities, and their
regulation, must be integrated with those of nutrient-
provisioning symbionts. This integration of insect and
symbiont metabolic capabilities could affect dietary
nutritional requirements and thus insect host plant
range. For example, in one pea aphid–Buchnera lineage,
a frameshift mutation was detected in argC, one of the
five genes required for biosynthesis of ornithine, an
intermediate in arginine biosynthesis (Vogel & Moran
2010). This aphid clone incurred fitness costs when fed
on artificial diet lacking arginine (Vogel & Moran 2010).
This arginine dependence was attributed not only to
Buchnera’s frameshift in argC, but also to insect host-
encoded factors based on genetic crosses (Vogel &
Moran 2010). This observation fits with the fact that
ornithine may be either present in the phloem sap or
produced by the host insect, for example, through the
enzyme ornithine aminotransferase, which interconverts
1480 A . K . H A N S E N and N . A . M O R A N

ornithine and glutamate and which is upregulated
in pea aphid bacteriocytes (Hansen & Moran 2011).
Therefore, in this example, the transport of ornithine
into Buchnera cells may be critical, and this transport is
likely to be dependent on host-encoded products, as
transporter genes are largely absent from Buchnera ge-
nomes (Shigenobu et al. 2000) and are present in the
aphid genome and upregulated in bacteriocytes (Han-
sen & Moran 2011; Poliakov et al. 2011; Price et al.
2011). Thus, despite little variation in which amino acid
pathways are encoded by symbionts, essential amino
acid requirements may vary due to variation in the
insect host genotype. For example, arginine dependence
may be impacted by variation among pea aphid geno-
types affecting capabilities for efficiently transporting
ornithine into Buchnera cells, whereas in aphid species
in which Buchnera has lost the ornithine pathway
(Fig. 4), the aphid may be able to efficiently transport
ornithine to Buchnera.
Potentially, symbiont and/or insect regulation of
amino acid biosynthetic pathways plays an important
role in host plant use. For example, if essential amino
acid pathways can be regulated based on insect nutri-
tional demands, this may increase the suitability of
plants presenting different nutrient compositions in
different plant parts, times of year or plant taxa. Alter-
natively, if certain essential amino acids are not regu-
lated efficiently for host demands, this may limit the
insect’s survival and therefore persistence on particular
host plants. Currently, only limited evidence, primarily
in aphids, has been found for an ability of obligate sym-
bionts to regulate essential amino acid pathways in
response to insect nutritional demands (Moran et al.
2003, 2005; Charles et al., 2006; Reymond et al., 2006).
Most symbiont genes regulating biosynthetic pathways
have been lost in the course of genome reduction
(Shigenobu et al. 2000; McCutcheon & Moran 2012). The
majority of genes encoding transcriptional regulators
for essential amino acid pathways are repressors, and
these are eliminated from symbiont genomes, possibly
as an adaptation for overproducing the needed nutri-
ents (Moran et al. 2003). But metR, a transcription acti-
vator, has been retained in genomes of some Buchnera
and of Baumannia. In the aphid Schizaphis graminum, the
Buchnera metR responds moderately to the demand for
methionine resulting in increased transcription of metE,
encoding methionine synthase (Moran et al. 2003). In
Buchnera, essential amino acid genes are located on the
main chromosome, except for leuABCD and trpEG,
which are located on plasmids in some species (Shige-
nobu et al. 2000; Moran 2007). Interestingly, genes on
the leucine plasmid are regulated to enable synthesis of
leucine according to aphid demand (Vi~ nuelas et al.
2011). This study also found that leucine is a limiting

Ornithine-(Arg) Ile BCAA Phe AroAA

Suborder Superfamily Family Species Nutritional symbiont(s) argA, argAG argB argC argD argE ilvA ilvE aspC aroE
Auchenorrhynycha Cercopoidea Clastopteridae Clastoptera arizonana Sulcia and Zinderia

Cicadoidea Cicadidae Diceroprocta semicincta Sulcia and Hodgkinia

Membracoidea Cicadellidae Homalodisca vitripennis Sulcia and Baumannia

Sternorrhynycha Aphidoidea Aphididae Acyrthosiphon pisum Buchnera APS

Acyrthosiphon kondoi Buchnera AK

Uroleucon ambrosiae Buchnera Uamb

Schizaphis graminum Buchnera Sgram

Pemphigidae Baizongia pistaciae Buchnera Bp

Lachnidae Cinara cedri Buchnera Cc and Serratia symbiotica

Cinara tujafilina Buchnera Ct

Aleyrodoidea Aleyrodidae Bemisia tabaci Portiera

Psylloidea Aphalaridae Pachypsylla venusta Carsonella PV

Pachypsylla celtidis Carsonella PC
Ctenarytaina eucalypti Carsonella CE and SS
Ctenarytaina spatulata Carsonella CS
Psyllidae Heteropsylla texana Carsonella HT
Heteropsylla cubana Carsonella HC and SS

Coccoidea Pseudococcidae Planococcus citri Tremblaya and Moranella endobia

Heteroptera Pentatomoidea Plataspidae Megacopta punctatissima Ishikawaella

Fig. 4 Missing genes in essential amino acid pathways of intracellular nutritional symbionts. Missing ornithine biosynthesis genes
include argA, argAG, argB, argC, argD, argE. ilvA is an isoleucine biosynthesis gene. All branched-chain amino acid biosynthesis path-
ways include ilvE. aspC is a phenylalanine biosynthesis gene. Aromatic amino acid pathway genes include aroE. Blue, gene present
in symbiont; white, gene absent from symbiont and putative insect homolog may complement pathway.

© 2013 John Wiley & Sons Ltd

 E V A L U A T I N G S Y M B I O N T E F F E C T S O N H O S T P L A N T U S E 1481
factor for aphid growth and that higher leucine concen-
trations increase the insect’s preference for a diet
(Vi~nuelas et al. 2011). Plasmid-associated regulation of
essential amino acid production in obligate symbionts is
rare. Among systems for which genomes of symbionts
of sap-sucking insects are sequenced, only Buchnera and
Ishikawaella from the stink bug Megacopta punctatissima
possess plasmids.
Self-regulation of symbiont cell density may be
another mechanism that regulates essential amino acid
concentrations. In the unique nested symbioses of the
mealybug Planococcus citri, the coresident symbiont
(Moranella) resides within cells of the nutritional symbi-
ont (Tremblaya). During different insect life stages,
Tremblaya was initially predicted to regulate Moranella’s
cell densities (Kono et al. 2008). However, after both
symbiont genomes were sequenced, no genes were
identified that would be capable of bacterial symbiont-
meditated regulation. Instead, the insect host may
regulate symbiont cell numbers and organization
(McCutcheon & von Dohlen 2011). In general, insect
hosts are hypothesized to regulate symbiont cell num-
bers, which fluctuate according to the insect life stages
and morphs (Douglas & Dixon 1987; Braendle et al.
2003; Wolschin et al. 2004; Rio et al. 2006; Nishikori et al.
2009; Stoll et al. 2010).
The insect host may regulate symbiont-provisioned
essential amino acid concentrations by regulating sym-
biont cell density and/or by changing the activity of
insect genes and transporters involved in the inte-
grated metabolism with the symbiont (Wilson et al.
2010; Hansen & Moran 2011; Price et al. 2011). If
insects can regulate symbiont production of essential
amino acids in response to nutritional demands, then
the underlying insect genetic factors could be gained,
modified or lost, potentially influencing insect host
plant use (Fig. 3). Overall, we predict that the insect’s
genome and not that of the intracellular symbiont may
be more important in influencing specific patterns of
host plant use.
Recent evidence for the pea aphid–Buchnera system
suggests that insect regulation of the integrated metabo-
lism may be key in responding to variable amino acid
availability in plant sap. More specifically, insect-
encoded enzymes are hypothesized to collaborate with
the obligate symbiont in the biosynthesis of essential
amino acids. For example, three enzymes integral to six
essential amino acid pathways are missing from the
genome of Buchnera of pea aphids (Shigenobu et al.
2000). These missing enzymes may be complemented
by aphid-encoded homologs, based on aphid genomic
data (Wilson et al. 2010), and supported by RNAseq
and proteomics data (Hansen & Moran 2011; Poliakov
et al. 2011). For example, an aphid-encoded homolog of
© 2013 John Wiley & Sons Ltd
threonine ammonia-lyase (IlvA, EC 4.3.1.19) was
hypothesized to convert threonine into 2-oxobutanoate,
thus providing this metabolite to Buchnera as the sub-
strate for isoleucine biosynthesis. Second, the pea aphid
homolog for a branched-chain amino acid transaminase
(IlvE, EC 2.6.1.42) was hypothesized to catalyse the final
step of isoleucine, leucine and valine production. Third,
the aphid homolog for aspartate transaminase (AspC,
EC 2.6.1.1) was predicted to catalyse the final step of
phenylalanine biosynthesis (Wilson et al. 2010). Phenyl-
alanine could then be converted into tyrosine via
another aphid enzyme (1.14.16.1). Transcriptome data
from Hansen & Moran (2011) strongly supported all of
these predicted hypotheses as all three aphid homologs
were significantly upregulated in aphid bacteriocytes
(Hansen & Moran 2011); however, ilvA was upregulated
less than twofold (Hansen & Moran 2011), and an alter-
native enzyme may produce 2-oxobutanoate (Poliakov
et al. 2011).
Based on gene contents of symbionts, host and/or
diet complementation appears widespread in phloem
feeders in Sternorrhyncha and Heteroptera (Figs 3 and
4). Curiously, nutritional symbionts of xylem feeders
from three separate superfamilies in Auchenorrhyncha
do not appear to require insect genes to complement
any of their essential amino acid pathways (Fig. 4). As
mentioned above, the first half of the arginine pathway
is missing in 75% of phloem-feeding symbionts (12 of
16), yet genes are retained for the steps of this pathway
subsequent to ornithine biosynthesis (Fig. 4). In con-
trast, the entire ornithine and arginine pathway is intact
in nutritional symbionts of xylem-feeding insects.
Second, most phloem feeders (14 of 16) have lost the
enzyme ilvA in the isoleucine pathway; however, this
gene is retained by symbionts of all xylem feeders
(Fig. 4). Third, symbionts of all phloem feeders except
psyllids have lost ilvE, the final reaction synthesizing
each of the three branched-chain amino acids. Fourth,
the terminal reaction of phenylalanine biosynthesis
(aspC) converting phenylpyruvate to phenylalanine is
missing in all symbionts of phloem feeders but is pres-
ent in xylem-feeding symbionts (Fig. 4).
This prevalence of insect host complementation in
phloem feeders may emerge because host genes are
important in regulating arginine, phenylalanine and
branched-chain amino acids primarily in phloem feed-
ers and not xylem feeders. Conversely, insects within
Auchenorrhyncha may have lost host homologs for
complementing genes. Currently all Auchenorrhyncha
for which symbiont sequences are available are xylem
feeders, and it will be of interest to see whether symbio-
nts of phloem-feeding Auchenorrhyncha show different
genetic repertoires. Only after more symbiont and insect
genomes have been sequenced from phloem- and
1482 A . K . H A N S E N and N . A . M O R A N
xylem-feeding Auchenorrhyncha can this hypothesis be
adequately explored and tested.
In summary, obligate intracellular symbionts of sap
feeders show little variation in amino acid biosynthetic
capabilities: they typically retain most essential amino
acid pathways, and when particular genes are missing,
the missing genes are present in genomes of a cosymbi-
ont and/or the insect host (Fig. 2). Only in the pea
aphid has the insect host complementation of symbiont
provisioning been studied directly, so it is not yet clear
whether there are general patterns with respect to the
dynamics of essential amino acid regulation and trans-
port in nutrient-provisioning symbioses. Based on the
current information from complete genome sequences
of symbionts, insect enzymes appear to complement
those of symbionts only in phloem feeders. Potentially,
insect hosts are able to regulate symbiont provisioning
through control of nutrient transport and of symbiont
cell densities (Fig. 3). Although there is little empirical
evidence to date that heritable symbionts directly affect
host plant specificity (see Box 1), the insect’s integrative
metabolism in conjunction with nutritional symbiont(s)
genotypes do have potential to influence host plant
utilization.
Gut symbionts
As in animals generally, almost all insects house
microbes in the gut lumen. Most insect gut microbiomes
are dominated by a small number of bacterial species,
with wide variation among species and higher taxa and
few clear patterns based on surveys to date (Colman
et al. 2012). Much higher levels of community diversity
have been recorded in some insect species, particularly
species feeding on detritus or dead wood (e.g. Egert
et al. 2003; Warnecke et al. 2007; Andert et al. 2010; Reid
et al. 2011; Schauer et al. 2012). Although surveys of gut
communities in herbivorous insects are still somewhat
limited, a few species have been studied in depth. As
summarized below, rather few direct demonstrations of
symbiont roles in herbivory have been published to
date.
Among the simplest insect gut microbiomes are those
of species in a number of herbivorous families of
Heteroptera, including Plataspidae, Pentatomidae, Bery-
tidae, Coreidae, Lygaeidae and Alydidae. Most species
in these groups have guts with specialized midgut cae-
ca, which harbour a single bacterial type in each insect
species (Fukatsu & Hosokawa 2002; Kikuchi et al. 2005,
2010; Prado et al. 2009; Hosokawa et al. 2012; Matsuura
et al. 2012). In some cases, these are transmitted mater-
nally via symbiont capsules that are deposited with
eggs and then ingested by new hatchlings. In the
plataspid bug Megacopta punctatissima, the maternally
transmitted gut symbiont Ishikawaella capsulata has
undergone genomic reduction comparable to that of
bacteriocyte-associated symbionts such as Buchnera in
aphids, and Ishikawaella retains a similar set of genes for
the biosynthesis of essential amino acids, strongly sug-
gesting this function (Hosokawa et al. 2006; Nikoh et al.
2011). In addition, Ishikawaella contains a plasmid
encoding a gene for oxalate decarboxylase, raising the
possibility that it provides the host with defence to the
widespread plant toxin oxalic acid.
In some other herbivorous Heteroptera, gut symbio-
nts are acquired from the environment every genera-
tion, and this acquisition is sometimes highly selective
for a particular bacterial strain (Kikuchi et al. 2005,
2010, 2011; Hosokawa et al. 2012; Matsuura et al. 2012).
In one such case, the bean bug Riptortus pedestris
(Heteroptera: Alydidae) is host to a specific Burkholderia
strain (Kikuchi et al. 2005). When this insect occurs in
fields heavily treated with the insecticide fenitrothion,
its Burkholderia symbiont shows capability to degrade
fenitrothion, possibly due to gene uptake from nearby
environmental bacteria, which show high levels of feni-
trothion degrading activity at such sites (Kikuchi et al.
2012). This raises the possibility that a gut symbiont
with a dynamic genome is able to acquire novel capa-
bilities that enhance fitness of the host, a case that
parallels that of gut bacteria in humans acquiring genes
for the degradation of specific foods consumed in spe-
cific populations (Hehemann et al. 2012).
Bees feed exclusively on nectar, which is sugar rich,
and pollen, their sole source of protein. The gut micro-
biota of adult worker honey bees Apis mellifera (Hyme-
noptera: Apidae) is somewhat unusual in being
transmitted through social contact within the hive and
in consisting of a distinctive set of bacterial species spe-
cific to honey bees and to the related bumble bees
(Bombus species) (Martinson et al. 2012; Moran et al.
2012). Based on a metagenomic study of this commu-
nity, the genomes of these bacteria show evidence of
horizontal gene transfer and possess an excess of genes
for metabolism of sugar and other carbohydrates (Engel
et al. 2012). One species, Gilliamella apicola (Kwong &
Moran 2012), includes strains that encode pectate lyase
and were demonstrated to be able to digest pectin in
laboratory cultures (Engel et al. 2012). As pectin is a
part of the cell wall that makes up one of the layers sur-
rounding the protein-rich pollen germ, this pectinase
function may assist bees in obtaining protein from their
pollen diet. Interestingly, the pectate lyase-encoding
genes in G. apicola are closely related to those from
plant pathogens and appear to have been acquired
through horizontal gene transfer.
Although most bark beetles (Scolytidae) feed on tis-
sues of dead plants, members of the genus Dendroctonus
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 E V A L U A T I N G S Y M B I O N T E F F E C T S O N H O S T P L A N T U S E 1483

Box 1
Is host plant specificity driven by symbionts in aphids?
The pea aphid is a species complex of recently diversified host races using different plant species in the legume
family (Ferrari et al. 2008). These races appear to be very recently established, possibly in association with host
plant shifts that coincided with post-Pleistocene warning and anthropogenic range expansion of several domesti-
cated legumes (Peccoud et al. 2009). Studies on sympatric populations of the races specialized to alfalfa (Medicago
sativa) and clover (Trifolium species) show that host plant specificity is determined primarily by host plant deter-
rents and assortative mating on food plants (Caillaud & Via 2000, 2012). Quantitative genetic studies, using genetic
crosses between the host plant races, showed that several aphid genes underlie differences in ability to use alfalfa
and clover (Hawthorne & Via 2001; Caillaud & Via 2012). These studies did not detect a maternal effect, as would
be expected if heritable symbionts had a substantial role in determining host plant use. Aphid genes appear to be
involved in differences among the other races of pea aphid as well: a recent genome-wide screen of divergent pea
aphid host races revealed 11 loci under divergent selection that are potentially responsible for differential host
plant usage by these two races; five of these loci may be involved in host plant adaptation and encode olfactory
and salivary proteins (Jaquiery et al. 2012).
It has been suggested that facultative symbionts of insect herbivores, particularly in the pea aphid system, may
help drive host plant adaptation resulting in specialized host-adapted races (Leonardo & Muiru 2003; Tsuchida
et al. 2004). A survey of facultative symbionts in pea aphid host races found significant associations between host
race and presence of particular symbionts (Ferrari et al. 2012). However, an association of a particular secondary
symbiont with a specific host plant race in the field does not imply that the symbiont is affecting host plant utiliza-
tion. Among the several alternative explanations are historical associations, because the secondary symbionts are
maternally transmitted and may happen to be coinherited with aphid genotypes able to use particular plants, and
also indirect effects of symbionts that impact host use. Indirect effects could include protection against natural ene-
mies (Oliver et al. 2003, 2005, 2009; Ferrari et al. 2004; Scarborough et al. 2005; Vorburger et al. 2010; Hansen et al.
2012) or thermal tolerance (Montllor et al. 2002); both are well documented and involve factors expected to differ
among plants. Direct experimental results regarding effects of facultative symbionts on plant host use are mixed.
One of the most prominent associations is between presence of the symbiont Regiella insecticola and use of clover as
a host plant (Ferrari et al. 2012). In one study, it was shown experimentally that R. insecticola increases pea aphid
performance on clover, but does not affect performance on fava bean (Vicia faba), a plant on which most pea aphid
host races perform well (Tsuchida et al. 2004). But in several other studies, using different clones of both aphid and
R. insecticola, this result has not been supported (Leonardo 2004; Ferrari et al. 2007; McLean et al. 2011).

attack living coniferous trees, feeding on the phloem
tissue. Their food is poor in nitrogen and high in plant
secondary metabolites, including abundant terpenes.
The microbial communities in the guts of Dendroctonus
species contain a rather low diversity of bacteria (Mor-
ales-Jimenez et al. 2009, 2012; Adams et al. 2013).
Whole-animal and whole-gut preparations of these bee-
tles show nitrogen fixation in laboratory assays, and iso-
lates of major members of this community are able to
fix molecular nitrogen and to recycle uric acid waste
into usable amino acids (Morales-Jimenez et al. 2009,
2012, 2013). In addition, isolates from the gut commu-
nity of Dendroctonus valens displayed cellulase activity
and thus potentially aid hosts in cellulose digestion
(Morales-Jimenez et al. 2009). A metagenomic study of
bacterial communities associated with Dendroctonus pon-
derosae, including both gut and environmental symbio-
nts, revealed an elevated number of genes involved in
terpene degradation, relative to metagenomic data sets
from other bacterial communities that degrade plant
biomass (Adams et al. 2013). Together these findings
suggest that gut microbial associates in bark beetles
probably contribute to nutrient provisioning, digestion
of plant polymers and detoxification of plant secondary
metabolites.
Besides the relatively specialized cases of gut symbio-
ses of heteropterans, honey bees and Dendroctonus bark
beetles, studies of gut microbiome composition in
herbivorous insects are available for lepidopteran larvae
and grasshoppers (Dillon & Charnley 2002; Sittenfeld
et al. 2002; Broderick et al. 2009; Robinson et al. 2010;
Tang et al. 2012). Generally, these communities are
highly variable even within a species, but they do con-
tain somewhat distinctive sets of bacterial species,
although relative abundances vary with the develop-
mental stage and diet of the host (e.g. Broderick et al.
2004; Robinson et al. 2010; Tang et al. 2012).
A major challenge to insects that ingest whole plant
cells, including most foliage feeders with chewing
mouthparts, such as many lepidopteran larvae and bee-

© 2013 John Wiley & Sons Ltd

1484 A . K . H A N S E N and N . A . M O R A N
tles, is the digestion of plant cell walls. These contain
polysaccharides such as cellulose, xylan, pectin and
mannan. The needed enzymatic activities were once
considered to be generally lacking in animals, and genes
encoding such enzymes are not present in Drosophila,
mosquitoes or Bombyx mori (Watanabe & Tokuda 2010).
However, some insects do possess endogenous genes
that encode these enzymes: this was first discovered in
termites (Watanabe et al. 1998) and later found in other
groups such as some beetles and crickets (Watanabe &
Tokuda 2010). The role of gut microbes in digestion is
more intensively researched in insects such as termites
and scarab beetles that feed on dead plant tissues (e.g.
Warnecke et al. 2007; Hongoh et al. 2008; Andert et al.
2010), but similar roles of gut microbes are plausible in
insects that feed on live plants. Some wood-feeding
beetles (Cerambycidae) contain fungal gut symbionts
that have critical roles in digestion and that are some-
times transmitted by maternal smearing of eggs
(Gr€unwald et al. 2010); as these beetles are feeding on
dead tissues, they are perhaps not technically herbi-
vores, but they can damage living plants.
Some herbivorous insects also appear capable of pro-
ducing cellulases and pectinases encoded in their own
genomes; for example, the leaf beetle Phaedon cochleariae
(Chrysomelidae) possesses several enzymes capable of
breakdown of plant polysaccharides (Kirsch et al. 2012).
In some cases, insect genes encoding these enzymes
may have been acquired through horizontal gene trans-
fer from microbes, possibly from gut symbionts (See
Box 2); in other cases, they appear to be ancestral
within large groups of insects. The current expansion in
complete genome sequencing in diverse insect species
will help to clarify which sets of these genes are ances-
tral in insects (and animals generally) and which are
acquired through horizontal gene transfer to specific
groups.
Gut microbes in herbivorous insects could also play a
role in detoxifying plant compounds. As for the case of
digestive contributions, there is very little information
about this for herbivorous insects. Studies on velvet-
bean caterpillars, Anticarsia gemmatalis (Lepidoptera:
Noctuidae) showed that survivorship and growth were
improved when bacterial gut communities were intact,
suggesting some beneficial role (Vis^ otto et al. 2009).
These insects feed on leguminous plants that are rela-
tively nitrogen rich but that produce protease inhibitors
able to prevent digestion of proteins. However, bacterial
species isolated from the A. gemmatalis gut secreted pro-
teases that were relatively unaffected by the protease
inhibitors present in the plant tissues (Pilon et al. 2013).
These observations fall short of proving that gut bacte-
ria are crucial to overcoming these plant defences, but
they do provide suggestive evidence for such a role.
Earlier studies in cigarette beetles, Lasioderma serricorne
(Coleoptera: Anobiidae), suggested that gut fungi
neutralized toxins that were experimentally added to
laboratory diets. Specifically, some fungal isolates from
the guts were able to degrade the compounds, and
insect performance was decreased upon exposure to
fungicides and dietary toxins together (Dowd & Shen
2011). Experiments aimed at testing a role of gut fungi
in breakdown of caffeine in the coffee berry borer
Hypothenemus hampei (Coleoptera: Curculionidae) pro-
duced negative results, as the isolated fungi were inhib-
ited themselves by caffeine (Vega et al. 2003). Earlier
experiments on degradation of dietary terpenes in the
Douglas-fir tussock moth, Orgyia pseudotsugata, weighed
against a regular role of gut bacteria (Andrews &
Spence 1980). A similar proposal has been made for
gypsy moths: their gut communities sometimes contain
Rhodococcus strains, some of which are known to break
down plant-derived monoterpene toxins (Broderick
et al. 2004). However, direct demonstration that this
activity occurs within the herbivore guts has not been
reported. Overall, a role of gut microbial species in
detoxification appears to have been proposed more
often than directly tested.
In summary, although a number of researchers have
suggested that herbivorous insects might depend on
gut microorganisms for provisioning nutrients, diges-
tion of plant tissues or neutralization of plant toxins,
surprisingly few clear cases have been reported. Possi-
bly the lack of examples reflects a paucity of experimen-
tal studies; however, roles of gut microbes in these
processes have been proposed for decades (Berenbaum
1988; Barbosa et al. 1991). More broadly, there is a very
large literature on mechanisms by which herbivorous
insects digest plant tissues or overcome plant chemical
defences. Possibly, gut microbes are simply not central
players in many instances.
Environmental symbionts
Microbes living outside the insect’s body may directly
influence insect–host plant utilization by providing the
insect with nutrients, modulating host plant recognition
and response or detoxifying secondary plant com-
pounds. A large diversity of microbes inhabit the leaf
phyllosphere, and the taxonomic profiles of these
communities can be host plant-specific regardless of
geographical location (Redford et al. 2010). Environmen-
tal bacteria can function as symbionts of herbivores and
potentially contribute to insect–host plant use only if
symbionts are transmitted by the insects themselves or
are persistently maintained in or around the insect’s
host plant tissue. We refer to these collectively as ‘envi-
ronmental symbionts’. Currently, only a few examples
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 E V A L U A T I N G S Y M B I O N T E F F E C T S O N H O S T P L A N T U S E 1485

Box 2
The role of horizontal gene transfer in enabling insect herbivory
Although it was once thought to be almost absent as a process affecting the evolution of animal genomes, acquisi-
tion of functional genes from foreign sources has now been found in a variety of cases (Moran & Jarvik 2010;
Acu~ na et al. 2012). A large proportion of these cases involved genes that might enable plant-feeding animals to be
more efficient as herbivores; for example, plant parasitic nematodes appear to have acquired vitamin biosynthesis
pathways and enzymes for cell wall degradation through incorporation of foreign genes into their own genomes
(Craig et al. 2008; Danchin et al. 2010; Paganini et al. 2012; Schuster & Sommer 2012). A well-supported case rele-
vant to the herbivorous lifestyle in insects is the acquisition of a mannanase-encoding gene by the coffee berry
borer (Coleoptera: Curculionidae) (Acu~ na et al. 2012). Mannan is the dominant polysaccharide in the coffee berries
that are the sole diet of these beetles, and the beetle genome has incorporated bacterial genes encoding mannanase
demonstrated to be functional in digestion of these molecules. Phylogenetic analysis indicated that the genes were
derived from a Bacillus species, potentially a bacterium in the gut or associated with the plant.
The complete genome sequence of the spider mite Tetranychus urticae contains several laterally transferred genes
that may facilitate feeding on the wide variety of plants used by this herbivorous arthropod (Grbic et al. 2011).
Among the genes for which the evidence of transfer was strong were genes encoding dioxygenases proposed to
function in breakdown of toxic plant aromatic compounds, cyanate lyase that might function to detoxify cyano-
genic glycosides produced by some plants, exo-fructosidases that are upregulated on certain host plants and cobal-
amin-independent methionine synthase, which could provide a source for this essential amino acid. Other genes
for which there is strong support for horizontal transfer to genomes of herbivorous arthropods include several
independent cases of carotenoid biosynthetic genes transferred from fungi: recipients include aphids that retain the
genes from their transfer to an aphid ancestor (Moran & Jarvik 2010), spider mites (Grbic et al. 2011; Altincicek
et al. 2012) and gall midges (Cobbs et al. 2013). Analysis of sequenced genomes for several lepidopteran species
(Bombyx mori, Heliconius melpomene and Danaus plexippus) revealed about 12 putative horizontal transfer events for
each genome, with transferred genes arriving from bacterial or fungal donors and at least some of the transfers
appearing to be ancient based on phylogenetic analyses of their distributions (Sun et al. 2013). Functions of genes
transferred to lepidopteran genomes mostly involve sugar, starch or amino acid metabolism and potential detoxifi-
cation enzymes (Li et al. 2011).
Numerous other studies using genomic or transcriptomic data have provided suggestive evidence for possible
microbe-to-insect horizontal transfers of genes encoding potential cell wall-degrading enzymes; the recipients have
been herbivorous insects ranging from beetles to moths to lygaeid bugs (e.g. Shen 2003; Kirsch et al. 2012; Sun et al.
2013). Some of these polysaccharide-degrading genes may result from relatively recent acquisition of microbial
genes facilitating herbivory, but others likely represent ancient and widespread animal genes that appear to be hor-
izontally transferred due to sampling artefacts and their absence from some of the initially sequenced insect ge-
nomes such as that of Drosophila (Pauchet et al. 2010; Calder on-Cortes et al. 2012). The extent to which the
acquisition of herbivorous lifestyles has depended on acquisition of foreign genes is thus still unclear.

of such associations have been described for herbivo-
rous insects; these primarily involve fungal symbionts
that are reliably transmitted and subsequently localized
to insect galls, nests or galleries.
In one such association, basidiomycete fungi and leaf-
cutter ants (species in the genera Acromyrmex and Atta)
have an obligatory symbiosis together, in which ants
culture the fungus for food in specialized gardens.
Worker ants tend the fungus garden and provide fresh
plant material for the fungal garden’s nutrients. In
return, the symbiotic fungus provides the ant colony
with its primary food source by producing specialized
hyphal tips (gongylidia) (H€ olldobler & Wilson 2010;
Aylward et al. 2012b; Mueller 2012). Leaf-cutter ants
may have specific nitrogen requirements because of this
long-term obligate symbiosis feeding from specialized
hyphal tips. For example, complete genomes from two
leaf-cutter ant species, Acromyrmex echinatior and Atta
cephalotes, reveal that the arginine biosynthesis pathway
is not encoded; however, this pathway is encoded in
other insects with complete metamorphosis, including
other ant species (Nygaard et al. 2011; Suen et al. 2011).
These results suggest that leaf-cutter ant genomes may
have lost genes encoding enzymes for arginine biosyn-
thesis, because the obligate fungal cultivars may
provide a reliable supply of this essential free amino
acid, analogous to aphids and their obligate symbiont,
Buchnera.
The symbiotic fungus garden converts plant biomass
into nutrients for ants by breaking down a wide variety

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1486 A . K . H A N S E N and N . A . M O R A N
of plant polymers, potentially from a diverse array of
host plant polymer types (Gomes De Siqueira et al.
1998; D’Ettorre et al.2002; Richard et al. 2005; Silva
et al.2006a,b; Schiøtt et al. 2008; Erthal et al. 2009; De
Fine Licht et al. 2010; Kooij et al. 2011). Some plant
biomass degradation enzymes are upregulated in the
specialized gongylidia located in the middle of the gar-
den, where ants feed. Ants then deposit their faecal
fluid containing active fungal-derived enzymes on top
of the fresh leaf pulp substrate, aiding in leaf cell wall
digestion (Schiøtt et al. 2010; Moller et al. 2011). Plant
digestive enzymes are also encoded and expressed in
bacterial microbes associated with fungal gardens (Suen
et al. 2010; Aylward et al. 2012a). However, it is
currently unclear whether any of these bacterial taxa
contribute nutrients to the ant.
The live plant material leaf cutters collect may con-
tain secondary metabolites that are toxic not only to the
ant but also to their fungal cultivars (Berenbaum 1988).
As a result, leaf cutters select host plant leaves that are
nontoxic towards themselves or their symbiotic fungi;
nonhost plant leaves can have fungicidal activities
towards the symbiotic fungi (Hubbell et al. 1983, 1984;
Howard 1988). In both the field and laboratory, high
concentrations of tannins in leaves deter foraging leaf
cutters, and these concentrations inhibit fungal cultivar
growth (Nichols-Orians 1991a,b). Nevertheless, the ant’s
fungal cultivar can broaden the ant’s host plant breadth
by producing enzymes that are important in detoxifying
plant secondary metabolites. For example, the fungal
cultivar of leafcutters produces a specific laccase
enzyme that is expressed highly in specialized gongyli-
dia that ants feed upon (De Fine Licht et al. 2013). Ants
then ingest but do not digest this carbohydrate-active
enzyme, and the enzyme is then passed on through the
ant’s faecal fluid directly onto fresh leaf pulp on top of
the garden where it displays a high level of laccase
activity and oxidizes phenolics. Orthologous genes
encoding this laccase enzyme from symbiotic and free-
living fungi reveal that this enzyme has been positively
selected in fungal cultivars that produce gongylidia, the
common ancestor of higher attine ants and leaf cutters
(De Fine Licht et al. 2013). It is assumed that this
enzyme has been modified for detoxification of phenols,
potentially leading to the major transition to active
herbivory 8–12 million years ago in the leaf-cutter line-
age (De Fine Licht et al. 2013).
Analogous to leaf-cutting ants, some fungal symbio-
nts of bark beetles provide their hosts with nutrients
and potentially detoxify secondary compounds. Most
bark beetles feed on dead or stressed trees; however,
several species in the genus Dendroctonus can feed on
living or recently killed plant tissue (Raffa & Berryman
1983). Bark beetles are universally associated with
fungi, which are transported tree to tree by their beetle
vector and can grow within larval galleries. A variety
of Dendroctonus bark beetle species that feed on fungus-
colonized phloem compared to noncolonized phloem
display higher larval survival, and their larval galleries
are shorter, indicating that beetles do not have to
compensate for a low nutrient diet by consuming more
plant material (Six 2012). These results indicate that
some bark beetles benefit from their stable fungal asso-
ciates, because fungal symbionts can provide necessary
nutrients, such as nitrogen and sterols, which are
limited in phloem tissue (Six 2012).
Fungal associates of bark beetles benefit by colonizing
an ephemeral resource early, such as phloem tissue,
before competitive fungal saprophytes arrive (Paine
et al. 1997). Stable fungal associates of tree-killing bark
beetles generally do not overwhelm tree defences or
cause tree death for the insect host (Six & Wingfield
2011); however, both the insect and fungus need to
tolerate and/or detoxify their host plant’s allelochemi-
cals to develop and compete with other fungal taxa on
phloem tissue (Paine et al. 1997). The genome sequence
of the beneficial fungal symbiont Grosmannia clavigera of
the mountain pine beetle (Dendroctonus ponderosae)
reveals that G. clavigera may detoxify a variety of host
plant defences, including terpenoids using secondary
plant compounds as a carbon source (DiGuistini et al.
2011). Also, as mentioned above in the section on gut
symbionts, a recent metagenomic study revealed that
bacterial communities that associate with the same host
plants and phloem tissue as the bark beetle, D. pondero-
sae, encode genes for terpene and monoterpene degra-
dation (Adams et al. 2013). Whether or not these
environmental microbes reliably help the bark beetle
detoxify host tree defences remains to be tested.
In another environmental fungal symbiont system,
some lineages of ambrosia gall midge (Diptera:
Cecidomyiidae, tribes Lasiopterini and Asphondyliini)
symbiotically associate with a monophyletic clade of
fungi called Botryosphaeria dothidea (Janson et al. 2010).
This fungus species is also known as a widespread
plant pathogen that infects a wide range of host plant
families (Heath & Stireman 2010; Janson et al. 2010).
Gall midge lineages that develop symbiotic associations
with B. dothidea deposit both fungal conidia and eggs
into host plant tissues, which subsequently develop into
galls that are internally lined with the symbiotic fungi.
These fungal symbionts provide midges with nutrients
such as sterols (Bissett & Borkent 1988; Rohfritsch 2008;
Janson et al. 2009) and may provide protection against
natural enemies (Weis 1982; Heath & Stireman 2010;
Janson et al. 2010).
A recent phylogenetic study reveals that gall midge
lineages with symbiotic fungi have broader host plant
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Box 3
Insect endosymbionts that influence host plant metabolism and defences
Microbes that associate with plants are generally viewed from the plant’s perspective and are classified as plant
pathogens, endophytes or mutualists, depending on the microbe’s relationship with the host plant. When microbial
associations are shared between an insect and plant partner, these categories become less defined and depend lar-
gely on the host organism of interest. A plant pathogen or endophyte that is vectored by an insect is generally
called a plant pathogen or endophyte respectively regardless of the impact on the insect host. But some of these
plant microbes closely associate with the insect in which they can be inherited and/or be regularly maintained,
and on which they have important effects. For example, when insects vector microbes to host plants, positive
effects on insect fitness tend to occur for efficient and highly specialized insect vector systems (reviewed in Wein-
traub & Beanland 2006; Stout et al. 2006; Kluth et al. 2002; Belliure et al. 2004). Some plant-associated microbes
(pathogenic or endophytic) can also be viewed as facultative symbionts of insect hosts, because some of these
microbes are vertically transmitted in insects, are highly host specific to a particular insect host and/or are persis-
tently maintained in insect populations (Hansen et al. 2008; Raddadi et al. 2011; Bressan et al. 2012; Caspi-Fluger
et al. 2012). Recently, a few studies have revealed that insect endosymbionts, including vectored plant microbes,
can influence plant signalling. For a more comprehensive review on insect–plant–microbe interactions, especially
for plant pathogens that are not vectored or intimately associated with insects, see Stout et al. (2006).
One insect–microbe system in which the microbe affects the insect–plant interface is the leafhopper Macrosteles
quadrilineatus, which vectors Aster Yellows phytoplasma, strain Witches’ Broom (AY-WB). When the leafhopper
feeds on AY-WB-infected host plants (Arabidopsis), the insect displays higher offspring production compared with
insects feeding on uninfected plants, primarily due to the phytoplasma’s modulation of plant defences (Sugio et al.
2011). More specifically, using transgenic lines of Arabidopsis, it was found that the phytoplasma’s effector protein
(SAP11) is responsible for destabilizing two Arabidopsis transcription factors, ultimately resulting in reduced expres-
sion of lipooxygenase 2 and decrease in subsequent jasmonic acid (JA) production, which is involved in the major
signalling pathway for upregulating defences against herbivores (Sugio et al. 2011).
In another specialized insect–plant pathogen system, the tomato psyllid Bactericera cockerelli vectors the alphapro-
teobacterium, Liberibacter psyllaurous, into its solanaceous host plants potato and tomato (Hansen et al. 2008). Liberib-
acter psyllaurous is associated with the plant disease called psyllid yellows but is also vertically transmitted at
moderate to high frequencies in psyllid eggs and maintained throughout psyllid development, increasing in titre as
the psyllid develops (Hansen et al. 2008; Casteel et al. 2012). When L. psyllaurous is inoculated into tomato alone
without the insect, tomato genes induced by jasmonic acid and salicylic acid signalling are suppressed, especially
transcripts of defence genes that are known to target insect pests (Casteel et al. 2012). Additionally, when both
psyllids and L. psyllaurous simultaneously infect tomato plants, dampened defensive host plant responses similar to
treatments of L. psyllaurous alone occur, especially for psyllid life stages that harbour the highest concentrations of
the bacterium. Collectively these results suggest that the bacterium may play a role in suppressing insect-related
host plant defences when L. psyllaurous is vectored into tomato by the psyllid. Other Liberibacter species may also
be important in psyllid–host plant interactions. For example, other Liberibacter species are vectored only by psyllids
into phloem tissue and are known as plant pathogens and endophytes (McClean & Oberholzer 1965; Halbert &
Manjunath 2004; Teixeira et al. 2005; Hansen et al. 2008; Raddadi et al. 2011).
The leaf-mining moth Phyllonorycter blancardella and its intracellular alphaproteobacterial endosymbiont Wolbachia
represent another insect–endosymbiont system that has been hypothesized to interact with the host plant; Wolbachia
is not known to infect plant tissue. The leaf-mining moth remains active in senescing apple leaves by feeding
within photosynthetically active green patches on the leaf. The green patches contain increased concentrations of
cytokinins and nutrients compared with senescing leaf margins (Giron et al. 2007). Cytokinins are plant hormones
involved in nutrient mobilization and inhibition of senescence and are thought to be involved in a variety of
insect–plant and microbe–plant interactions (Giron et al. 2007; Robert-Seilaniantz et al. 2007; Walters et al. 2008).
After treatment with antibiotics, the leaf miner cannot produce green patches in senescing leaves, and cytokinin
concentrations decline; in contrast, untreated leaf miners maintain Wolbachia infections, produce green patches and
display higher fitness (Kaiser et al. 2010). Therefore, symbiont presence in the leaf miner corresponds to green
patches and higher cytokinin concentrations in senescing apple leaves. In this system, it is unclear how Wolbachia
may be involved in green-patch production and if the moth, plant or symbiont produces this cytokinin (Kaiser
et al. 2010). Alternatively, Wolbachia may not be responsible; potentially the phenomenon is caused by a different
microbe present at a lower density and also eliminated during antibiotic treatments.

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1488 A . K . H A N S E N and N . A . M O R A N

Box 3 Continued
In a second lepidopteran system, when the western corn rootworm, Diabrotica virgifera, feeds on maize and is
infected with Wolbachia, plant defence-related genes are downregulated compared with plants fed on by antibiotic-
treated rootworms (Barr et al. 2010). As in the leaf-mining P. blancardella system above, it is unclear whether or
how symbionts directly influence the host plant, whether Wolbachia and not another symbiont is involved, and
whether microbes influence insect-related factors that in turn impact host plant defence signalling.
Finally, another indirect route by which insect endosymbionts may influence insect–host plant interactions is by
protecting insect hosts from natural enemies that would otherwise make a plant unavailable as a host. Protection
of herbivorous insects by endosymbionts has been well established, particularly for cases in aphids (reviewed in
Oliver et al. 2010). Such effects may be widespread for facultative symbionts, including symbionts from Enterobac-
teriaceae such as Hamiltonella defensa (Oliver et al. 2003), but also Wolbachia (Hedges et al. 2008; Teixeira et al. 2008)
and Spiroplasma species (Jaenike & Brekke 2011).

ranges than those of nonsymbiotic gall midges (Joy
2013). In addition, midge lineages with fungal symbio-
nts are more species-rich at the tribe and genus levels
compared with related lineages lacking fungal symbio-
nts (Joy 2013). Collectively these results suggest that
fungal symbionts may facilitate niche expansions and
diversification of their gall-forming insect hosts. But the
underlying mechanism by which fungal symbionts
facilitate radiation of gall midges are not yet clear;
potentially multiple mechanisms are involved (e.g.
nutritional, behavioural preference, protection against
natural enemies and/or modulating plant defences).
In addition to gall midges, fungal plant pathogens
that are also insect symbionts may facilitate host plant
exploitation, as suggested by studies of the grape berry
moth Lobesia botrana. In this system, the phytopathogen
Botrytis cinerea is vectored by the grape berry moth
(Fermaud & Le Menn 1992) and tunnelling moth larvae
facilitate fungal penetration into grape berries (Fermaud
& Le Menn1989). Female grape berry moths preferen-
tially lay eggs on fungus-infected grape berries and dis-
play higher fitness on berries infected with fungi than
on uninfected berries (Mondy et al. 1998). Fitness bene-
fits from fungi may be attributed to fungus-derived
sterols provided to the insect (Mondy & Corio-Costet
2000).
In summary, many microbes persist in the herbivore’s
environment including the phyllosphere and potentially
interact with insects and plants. Experimental studies
demonstrating environmental symbionts of insects that
are persistently maintained and that reliably influence
insect–host plant use and evolution are extremely rare.
Perhaps environmental bacterial symbionts that are ben-
eficial and influence insect–host plant use rapidly transi-
tion to an and insect location that can be maintained
more efficiently such as the gut and/or inside the body
and salivary glands. Complex insect behavioural and/or
morphological adaptations may be required for insects
to maintain transmission and localization of beneficial
microbes in the environment to their food plants, as seen
in a few fungal symbionts. Otherwise, host plant-specific
microbes may be maintained more effectively by the
host plant itself for its own benefit against natural
enemies. In some cases, however, plant microbes may
be plant pathogens that can influence insect host plant
use if they are reliably vectored by the insect and main-
tained as insect bacterial endosymbionts (See Box 3).
Concluding remarks
Insects have evolved many adaptations to overcome the
nutritional, digestive and defensive obstacles encoun-
tered during herbivory. Major evolutionary transitions
towards insect herbivory potentially have also been
dependent on the aid of microbial symbionts. Microbes
associate with all animals including insects, and they
encode an enormous range of metabolic capabilities that
dwarf and complement those of animal genomes. The
ability of microbial symbionts to impact insect host
plant use has been proposed for decades, but experi-
mental evidence for most of these proposals has lagged
behind. Whether this is due to experimental and tech-
nological limitations, especially in the past, or to a
limited role of symbionts, is not yet clear.
The possible roles played by symbionts in facilitating
herbivory largely depend on the location of the symbi-
ont relative to the insect body. The location, whether
intracellular, in the gut or outside the body, heavily
influences the fidelity of transmission, persistence of the
association during development, insect host control and
microbe genome evolution, and these factors shape the
potential contributions of symbionts. Intracellular sym-
bionts, especially of sap feeders, have been extensively
studied, and many of these symbiont genomes have
been sequenced. Genome sequence analyses combined
with experimental results show that intracellular symbi-
onts contribute to insect nutrition by supplementing
nutrients lacking in plant diets. Compared with most

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 E V A L U A T I N G S Y M B I O N T E F F E C T S O N H O S T P L A N T U S E 1489

Box 4
Outstanding questions regarding the role of symbiotic microorganisms in the use of plant tissues or plant
species by herbivorous insects
To what extent has horizontal gene transfer, from microbial sources to the insect genome, played a role in enabling
herbivorous lifestyles?
How frequently do gut microorganisms of herbivorous insects aid in digestion of plant tissues or in detoxification
of plant-derived compounds?
Are environmental symbionts (living outside the insect body) continuously maintained in most herbivorous insect
lineages or are they primarily associated with the host plant/phyllosphere? In either case, how often do they influence
host plant use?
To what extent are gut symbionts reliably transmitted between individuals of herbivorous insect species or reliably
acquired from environmental sources?
How often has the acquisition of a herbivorous lifestyle, or the ability to use particular plant tissues or species,
depended on the acquisition of foreign genes?
Overall, how often is adaptation for utilizing particular plants based on variation and innovation in genetic capabili-
ties of the insect versus variation in symbiont genotype and cosymbiont associations?
How common are vertically transmitted insect endosymbionts that play dual roles as plant pathogens/endophytes
in herbivorous insects?

gut symbionts and most environmental symbionts,
intracellular symbionts are relatively reliable in facilitat-
ing in host plant use over evolutionary time because
they are heritable, and the host most likely controls
most metabolic interactions. Despite high fidelity
in transmission, evolutionary novelty is limited in
intracellular symbionts, which undergo extensive gene
loss and do not acquire foreign genes. The gain of other
symbionts and/or host regulation and control may
enable hosts to maintain or expand abilities to feed on
host plants. Gut symbionts offer the potential advanta-
ges of strain diversity and the dynamic genomes able to
acquire new genes and functions, but they are probably
less reliably transmitted between hosts, except in cases
with specific behavioural adaptations on the part of
hosts. Their location could enable gut symbionts to play
major roles in digestion of plant macromolecules and in
detoxification, but despite this potential, empirical stud-
ies supporting these roles are limited. Environmental
symbionts have similar genome dynamics as gut symbi-
onts; however, their association with the insect and its
host plant may be less reliable unless complex insect
behaviour and/or morphological adaptations maintain
the symbiosis. Only a handful of insect systems are
known in which environmental symbionts, primarily
fungi, aid in host plant use by supplementing nutrients
and/or breaking down large plant polysaccharides or
toxins.
Although a general role of symbionts in enabling
herbivory is clear, especially for sap feeders, there is
limited experimental evidence for an effect of symbionts
on specificity of insect host plant use. In particular, gut
symbionts, although present in almost all insects, have
rarely been found to affect host plant range. Perhaps
transient, study system-specific effects of symbionts on
insect–plant interactions will be documented; however,
symbiont-facilitated expansions of host plant range may
be rare events.
In order for a microbial symbiont to have a central
role in facilitating herbivory in general or the use
of particular plant species in particular, it must be
consistently associated with an insect host. For micro-
organisms in the gut and outside the insect body, the
consistency and persistence of associations of particular
microbes with particular host lineages are still being
explored and appear to vary enormously among insect
species (e.g. Colman et al. 2012; Mueller 2012). More
work is needed, and many of the outstanding
questions (Box 4) can now be addressed in studies
exploiting molecular methodologies developed for
environmental microbiology, genomics and metage-
nomics.
Acknowledgements
The authors thank Gordon Bennett and Patrick Degnan
for helpful comments on the manuscript. We also thank
three anonymous reviewers for their helpful comments
and suggestions. Funding was provided by the US
Department of Agriculture [2011-67012-30707] to A.H
and by Yale University.
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N.A.M.’s research is on genome evolution and on
evolutionary and ecological associations between insects
and microorganisms. A.K.H. is interested in evolution-
ary and ecological genomics and the molecular mecha-
nisms that underpin insect–microbe coevolution.
© 2013 John Wiley & Sons Ltd