Impact of intraspecific variation in insect microbiomes on host phenotype and evolution

Abstract

Microbes can be an important source of phenotypic plasticity in insects. Insect physiology, behaviour, and ecology are influenced by individual variation in the microbial communities held within the insect gut, reproductive organs, bacteriome, and other tissues. It is becoming increasingly clear how important the insect microbiome is for insect fitness, expansion into novel ecological niches, and novel environments. These investigations have garnered heightened interest recently, yet a comprehensive understanding of how intraspecific variation in the assembly and function of these insect-associated microbial communities can shape the plasticity of insects is still lacking. Most research focuses on the core microbiome associated with a species of interest and ignores intraspecific variation. We argue that microbiome variation among insects can be an important driver of evolution, and we provide examples showing how such variation can influence fitness and health of insects, insect invasions, their persistence in new environments, and their responses to global environmental changes.

Full text

PERSPECTIVE OPEN
Impact of intraspecific variation in insect microbiomes on host
phenotype and evolution
Claudia Lange
1
✉, Stéphane Boyer
2
, T. Martijn Bezemer
3
, Marie-Caroline Lefort
4
, Manpreet K. Dhami
1
, Eva Biggs
1
,
Ronny Groenteman
1
, Simon V. Fowler
1
, Quentin Paynter
5
, Arletys M. Verdecia Mogena
6
and Martin Kaltenpoth
7
© The Author(s) 2023
Microbes can be an important source of phenotypic plasticity in insects. Insect physiology, behaviour, and ecology are influenced
by individual variation in the microbial communities held within the insect gut, reproductive organs, bacteriome, and other tissues.
It is becoming increasingly clear how important the insect microbiome is for insect fitness, expansion into novel ecological niches,
and novel environments. These investigations have garnered heightened interest recently, yet a comprehensive understanding of
how intraspecific variation in the assembly and function of these insect-associated microbial communities can shape the plasticity
of insects is still lacking. Most research focuses on the core microbiome associated with a species of interest and ignores
intraspecific variation. We argue that microbiome variation among insects can be an important driver of evolution, and we provide
examples showing how such variation can influence fitness and health of insects, insect invasions, their persistence in new
environments, and their responses to global environmental changes.
The ISME Journal; https://doi.org/10.1038/s41396-023-01500-2
INTRODUCTION
Insects are associated with a range of microbes that influence their
biology and life history traits. The insect-associated microbial
community (the microbiome) can vary between, but also within
species [1]. Meta-analyses of factors contributing to insect
microbiome structure and diversity across insect orders are rare.
Host species and diet/trophy appear to be the most relevant
drivers, but sex, life stage, and sample origin/habitat also have
some impact, while the abundance of endosymbionts and
phylogeny only have weak influence [2–6]. Intraspecific micro-
biome variation can be driven by several factors, such as the host
itself, the diet, and the environment [7–9]. Microbe transmission
routes, recruitment, maintenance, and interactions further shape
these variations [10–12]. Insect symbionts can be separated into
obligate (primary) and facultative (secondary) symbionts. While
obligate symbionts are essential for their hosts’survival and
reproduction and usually have an ancient stable host association
through vertical transmission, facultative symbionts are not
required for growth or reproduction but can also affect adaptive
host traits and can be horizontally transmitted [13]. In the context
of intraspecific variation, facultative symbionts are of particular
interest, and we focus this perspective on the importance of
facultative insect symbionts that often vary in prevalence and
abundance within and between insect populations. Intraspecific
microbiome variation does not only have consequences for
individual insects by impacting their behaviour, metabolism, and
defence against antagonists, but also affects insect populations
through changing reproduction, host range expansion, and host
race formation [14]. Such population-level adaptations can have
significant implications for insect invasions and population
resilience and may ultimately drive evolution.
This perspective synthesises recent insights into how microbes
control insect physiology and behaviour and describes the
consequences of microbiome changes on insect invasions and
persistence in novel ecosystems. We discuss the latest literature
on the drivers and consequences of microbiome variation in
insects with focus on herbivorous species, due to the available
literature, and propose future research directions that are needed
to improve our understanding of how intraspecific microbiome
variation impacts host ecology and evolution. While there are
many descriptive studies, experimental and field studies often
ignore intraspecific microbiome variation, so its effect on host
phenotypic traits, performance, and population dynamics remains
poorly understood. This is particularly concerning given the
potential of microbes to enable their insect hosts to rapidly adapt
to changing environments, a topic that is highly relevant in the
context of insect invasions and to understand the susceptibility or
resilience of insect populations in the face of global environmental
changes.
KEY DRIVERS OF INTRASPECIFIC VARIATION IN MICROBIAL
COMMUNITIES
Insect host physiology
The host plays a key role in determining its microbial diversity,
especially during development from immature to adult life stages,
Received: 10 June 2023 Revised: 20 August 2023 Accepted: 22 August 2023
1
Manaaki Whenua Landcare Research, Lincoln, New Zealand.
2
Institut de Recherche sur la Biologie de l’Insecte, UMR 7261 CNRS - Université de Tours, Tours, France.
3
Above-
Belowground Interactions Group, Institute of Biology, Leiden University, Leiden, The Netherlands.
4
UMR 7324 CITERES, Université de Tours, Tours, France.
5
Manaaki Whenua
Landcare Research, Auckland, New Zealand.
6
Institute of Plant Sciences, University of Bern, Bern, Switzerland.
7
Department of Insect Symbiosis, Max Planck Institute for Chemical
Ecology, Jena, Germany. ✉email: LangeC@landcareresearch.co.nz
www.nature.com/ismej
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when insects undergo considerable morphological and physiolo-
gical changes. This leads to diversification of ecological niches of
larvae and adults, thereby reducing intraspecific competition [15].
Invertebrates can either preserve beneficial symbionts through life
stages [16] or decouple microbial communities between larval and
adult stage [17,18]. As an example, dragonflies change their
microbiome richness when they change from aquatic life in larval
stages to terrestrial life in their adulthood [19]. A recent review
summarised that the insect gut hosts the highest diversity of
microbes across all invertebrates [7], providing a high potential for
intraspecific variation. Guts shape microbial communities via
chemical and physical conditions such as pH, nutrient availability,
immune system, oxygen levels, and compartmentalisation. For
example, the termite gut system has several compartments, and
both pH and oxygen decrease from one compartment to another
as food passes through. This enables the establishment of
specialised bacteria in different compartments that help termites
with cellulose degradation and nitrogen fixation [20]. While most
studies on intraspecific microbiome variation focus on insect guts
[1,21,22], microbes also inhabit other insect organs with specific
physical and chemical properties, that can impact the assembly
and functioning of microbiomes, such as haemolymph and
salivary glands, especially in blood sucking insects [23]. In some
species, the diversity in these organs can be higher than in the
gut. For example, bacterial diversity is much higher in reproduc-
tive organs and in saliva of a number of different mosquito species
than in the gut [24,25], suggesting that there may be more
intraspecific variation in microbiome composition in these organs
than in the gut.
External environmental factors
In addition to the host physiology, external factors, or the
environment in which the insects live, also have a major impact
on their microbial communities. When insect microbiomes shift,
the environment is a dominant source of the microbes that are
acquired. Diet (food as an external environmental factor) is often
mentioned as one of the main factors that influence the diversity
of insect microbiomes, especially among herbivorous [8,9] and
carnivorous insect taxa [26]. The diet of an insect can act as a
source of novel microbes when they are ingested with the food
[27], but nutritional properties of the food can also influence an
insect’s microbiome via its effect on the already existing microbes
[28]. However, even though diet has a major influence on the
composition or abundance of the microbes of an insect, several
studies have shown that non-food aspects of the local habitat of
the insect also act as important factors determining the
microbiomes of insects. For example, the folivorous cabbage
moth Mamestra brassicae actively acquires microbes from soil [29],
and these microbes can be beneficial to its host as they may
increase pesticide resistance [29–31]. The microbiome composi-
tion of insects can also be affected by environmental factors, such
as temperature, habitat, elevation, and human interference
[19,32–34]. For example, increases in temperature lead to
reduced microbial diversity and increased abundance of specific
taxa associated with Eastern subterranean termites, which
negatively impacts termite survival (Fig. 1).
Symbiont transmission routes
Within the environment, insect-associated microbes are acquired
and transmitted in several ways. The transmission route of
intracellular symbionts is predominantly vertical as these sym-
bionts can be present within reproductive cells or transferred
inside the developing egg. However, cases of horizontal acquisi-
tion have also been documented, for example in the whitefly
Bemisia tabaci subjected to non-lethal probing by Wolbachia-
infected parasitoids [35]. To survive and develop, individuals also
need specific gut microbial communities from the onset of their
lives [36]. Despite being extracellular, these microbes can still be
vertically transmitted, through the smearing of the egg surface
[37], the inoculation of the oviposition sites with faeces, specific
secretions [38], or more sophisticated structures that are produced
by females [39,40] and consumed by the offspring immediately
after hatching from the egg. Young individuals of gregarious and
social insects can obtain the necessary symbionts by feeding on
the faeces of their congeners (coprophagy) [41] or by direct fluid
exchange from anus to mouth (proctodeal trophallaxis) [42]. The
transmission of gut microbiota can also follow a horizontal route,
either mediated by soil or plant materials that have been
externally smeared with the faeces of other individuals [43], or
through trophic interactions with other species [11]. Transmission
may occur, for example, when two herbivores feed on the same
plant [44], when predators feed on their prey [45], or when
parasitoids feed on their hosts [46]. The recruitment of extra-
cellular gut bacteria invariably relies on their ingestion and their
colonisation of the gut lumen [20]. It has been hypothesised that
specific traits can also be acquired from transient bacteria (such as
plant-associated bacteria) that do not establish in the gut but
engage in horizontal transfer with native bacteria that are already
Fig. 1 Warming reduces microbial community diversity and survival, feeding, and cold tolerance responses in termites. Experimental
warming in Eastern subterranean termites (Reticulitermes flavipes) resulted in a reduction in gut prokaryotic diversity, especially when exposed
to elevated temperature treatment (35 °C) (p< 0.05). The community composition also exhibited significant differences with Bacteroidetes
symbionts increasing markedly under warming. Stress tolerance of termites also declined with a reduction in feeding, survival and cold
tolerance responses observed [149]. While feeding activity and dispersal of termites is expected to rise under warming [150], gut dysbiosis
due to warming may alter their survival and persistence.
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established in the gut [47]. Both pathways, horizontal and
imprecise vertical transmission of insect-associated microbes,
can promote intraspecific microbiome variation and stochasticity
[11,44,48].
Microbial assembly and maintenance
Once the transmission of microbes has occurred, different
processes affect how the community gets assembled and
maintained. The processes of microbial community assembly
and maintenance affect the function of the microbiome and host
fitness. For example, a reciprocal microbiome transplant in the
Water flea Daphnia magna found that different host genotypes
selected different microbiomes. Such selective uptake of specific
microbes with host beneficial functions as well as a high diversity
of strains with complementary gene functions increased the host’s
tolerance to toxic cyanobacteria [49]. In the whiteflyBemisia
tabaci, the acquisition of gut-associated bacteria was strongly
affected by the identity of the host plant. After switching the host
plant, host-specific microbiomes were assembled and maintained
over multiple generations, leading to improved metabolism and
survival of the host. This was largely attributed to direct effects of
available nutrients and or secondary metabolites [12]. An example
for local adaptive microbiome maintenance is the Colorado potato
beetle (Leptinotarsa decemlineata), whose microbiome adapted
along its invasion path in China. The beetle population that was
leading the invasion front had a higher abundance of microbiota
in oral secretions, a higher gut bacterial diversity, and different
relative abundance than the ancestral population 500 km away.
The adapted microbiome improved the suppression of plant-
induced defences and enabled geographic expansion [50].
Microbial interactions
Individual members of the microbiome also interact with each
other, which affects the microbiome composition and host
performance. For example, under nutritional stress, the Drosophila
symbionts Lactobacillus plantarum and Acetobacter pomorum
exchange metabolites to fulfil their own requirements. The
provisioning of lactate from L. plantarum to A. pomorum supports
this species’metabolism and results in a release of anabolic
metabolites by A. pomorum, which in turn, supports host larval
growth of individuals that are exposed to nutritional stress [51]. In
contrast, negative interactions between symbionts, such as
competition or antagonism also occur. The silk moth Bombyx
mori is protected from microsporidia pathogens by its symbiont
Enterococcus faecalis that reduces spore germination, ameliorates
gut injury, and reduces colonisation of the pathogen. Increasing
abundance of E. faecalis reduces the abundance and infection
efficiency of the pathogen [31]. However, in the honeybee gut,
closely related Lactobacillus species are able to overcome
competition for nutritional resources and to coexist because they
utilise different pollen-derived carbohydrates. This suggests that
dietary choices of the host or natural variation of the diet will
influence the gut microbiome diversity [52]. In the bean bug
Riptortus pedestris,Burkholderia symbionts improve growth and
fecundity of the host by recycling host metabolic wastes in the
midgut crypts. Upon acquisition of the symbiont from soil, the
acquisition of other symbionts is stopped by it altering the host
midgut morphology, a mechanism that proposedly supports host-
symbiont specificity in the absence of vertical transmission [53].
CONSEQUENCES OF INTRASPECIFIC VARIATION IN MICROBIAL
COMMUNITIES FOR THE HOST
Although evidence exists for intraspecific variation in insect
microbiomes and for the various drivers, the functional implica-
tions of these changes for the host and for host evolution remain
poorly understood. In the following, we discuss several examples
of consequences for the insect host.
Insect behaviour
Microbes can shape how insects respond to a stimulus [54].
Although most of the studies addressing this focus on the
implications of specific symbionts, entire microbial populations
are also linked to diverse roles in the physiology and behaviour of
insects such as Drosophila. For instance, microbial composition is
linked to the dietary preference of Drosophila melanogaster [55]. In
this study, the insects were more attracted towards the diet that
contained the microbes on which they were reared, implying that
the gustatory preference can be driven by microbial populations.
An example of a tripartite symbiosis that affects insect behaviour
is the “killer yeast”. When Drosophila spp. associated Sacchar-
omyces cerevisiae strains are infected with two complementary
viruses they turn into killer yeast strains. These strains kill
uninfected yeasts in fruit and are more attractive to insect vectors.
This interaction influences the feeding of Drosophila spp. and
promotes the dispersal of killer yeasts to new fruits [56].
Microbes are also widely associated with the capacity of insects
to find suitable egg-laying spots. This phenomenon has been
described for flies and other insects such as Encarsia pergandiella
[57]. This parasitoid wasp changes its oviposition habits when
invaded by Cardinium spp., enabling the bacterium to manipulate
host behaviour and to spread through the insect population [58].
Metabolism and detoxification
For plant feeding insects, the microbial community is often critical
for their nutritional status and survival [59]. The relevance of the
obligate symbionts for nutrient provisioning (such as essential
amino acids, vitamins, and sterols), digestion (such as plant cell
wall degrading enzymes), and detoxification has remained a focal
research theme. For example, some symbionts are involved in the
physiological mechanisms of sterol intake by different insect
species [60]. Nevertheless, studies of obligate symbionts do not
show intraspecific variation, and the contribution of facultative
symbionts to metabolism, digestion, and detoxification just
recently started to garner interest [47,61,62]. The insect digestive
system shelters a plethora of microbes, whose role in degrading
plant structural compounds, providing nutrients, and detoxifying
plant secondary metabolites has been, although less, already
acknowledged [27,47]. Gut symbionts are in contact with
environmental microbes, increasing the possibilities of genetic
material transmission or even the substitution by new microbes.
Hence, the insect host could attain new detoxifying genes or
perhaps microbes with novel metabolic capacities. Some studies,
including a field experiment, concluded that the capacity of some
insects to feed on a wide range of plants is moderately related to
the facultative microbial associates [12]. Moreover, insect gut
microbes have been reported to metabolise terpenes, flavonoids,
alkaloids, phenolics, and isothiocyanates [63–68]. Microbes
associated with saliva of herbivorous insects can influence
defence responses in the host plant [69,70], which influences
the quality of the ingested food and, in turn, the gut microbiome.
However, how microbes from saliva vary in their effects on
induced plant defence, plant interactions, and the role of
intraspecific variation in saliva microbiomes is poorly understood.
Defence against antagonists
By stimulating the host’s immune system or by directly inhibiting
or competitively excluding antagonists, microbial symbionts can
contribute to the defence of their host against antagonists,
including predators, parasitoids, parasites, and pathogens [71]. In
contrast to nutritional symbioses, defensive insect-microbe
partnerships are often dynamic and experience horizontal influx
of symbionts, resulting in intraspecific differences in microbial
communities that can significantly impact defence traits of the
insect host [71].
In aphids, facultative endosymbionts can enhance protection of
their hosts against parasitoid wasps [72–78], fungal pathogens
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[79,80], and viruses [81]. However, these defensive symbionts
incur costs for the insect in the absence of the relevant
antagonists [73,82–85]. Furthermore, the extent of protection
(at least against parasitoids) is governed by the interaction
between host, symbiont, and antagonist genotype, resulting in
complex fitness outcomes of symbiont infection for the host that
are dependent on antagonist abundance in the environment
[85,86]. As such, symbiont-mediated protection can have
cascading effects on multitrophic communities and affect species
coexistence under laboratory conditions [74,87]. Field studies
support a defensive role of hemipteran facultative symbionts
against antagonists but yield mixed results on fitness conse-
quences for the host [82,88,89]. Thus, further studies are needed
to understand how intraspecific differences in host-symbiont-
antagonist interactions affect population dynamics and species
coexistence in the field [90].
Other insects also engage in defensive symbiotic associations
with bacterial or fungal partners, but evidence for intraspecific
variation in symbiont-mediated protection and its fitness con-
sequences remains patchy. Antibiotic producing bacterial sym-
bionts of Lagria beetles [16,91,92] and beewolf wasps [93–95]
predominantly rely on vertical transmission but experience
occasional horizontal symbiont acquisition, resulting in multi-
partite defensive communities or occasional symbiont replace-
ment, respectively [94,96,97]. While this has direct consequences
for the defensive chemistry provided to the host [91,92], the
extent of this variation under field conditions and its relevance for
host fitness remain to be explored. In a rare case of combining
laboratory- and field-based investigations of a defensive symbio-
sis, it was found that Spiroplasma recently spread across
Drosophila neotestacea populations in North America due to its
role in protecting the host against the sterilising effects of a
parasitic nematode [98]. Other studies revealing a protective role
of gut bacteria against intestinal parasites [99] and cuticular
microbes against pathogenic fungi [26,100] indicate that
symbiont mediated defence is common across insects. As
intestinal and cuticular microbiomes are often variable in their
composition, these studies also suggest an impact of intraspecific
microbiome variation on host defence. However, systematic
studies are urgently needed to characterise the extent of
intraspecific variation in defensive microbial communities and
their importance for host fitness and population dynamics under
natural conditions.
Host range expansion and host race formation
Gut microbiota can be an important driving force of the speciation
process in phytophagous insects. Microbes play significant roles in
the exploitation of a novel host plant by phytophagous insects
[63,68]. Indeed, the presence of a specific set of core microbes,
capable of metabolising plant defence chemical compounds
could explain the ability to exploit new hosts [47], which
constitutes an essential first step towards host range expansion
[101,102]. However, the mechanisms underlying this phenotypic
plasticity are still to be clarified [103].
It was recently hypothesised that rapid host plant switching
might partially rely on transient associations between insects and
bacteria, the latter providing an additional flexible metabolic
“toolbox”that facilitates the effective use of a novel host plant
[12]. By influencing the ability of the insect to feed on particular
plant hosts, intraspecific variation of gut microbiota profiles can
create ecological barriers that facilitate sympatric speciation. In
addition to this symbiont-mediated niche exploitation and
behavioural change (pre-mating isolation), symbiont-mediated
intraspecific incompatibilities and coevolutionary processes (post-
mating isolation) can also contribute to sympatric speciation [104].
Hence, intraspecific variations in insect-microbe associations could
lead successively to host range expansion, host shift, host race
formation, and ultimately to sympatric speciation [14,105]. The
entire sympatric speciation continuum must be considered to fully
understand the role of insect microbiota variations in the
speciation process of phytophagous insects. For example, even
though changes in insect gut microbiome can happen over a
short time frame [106], multi-generational studies [107] are
essential to determine whether even transient insect-microbial
associations enable the insect to explore a new host plant range,
thereby providing future ecological and evolutionary potential.
Many hypotheses have been proposed regarding the impact of
transient and horizontally acquired microbes during host range
expansion and host shift events [12], however, experimental
testing of these hypotheses is scarce. Similarly, while an extensive
descriptive literature is available on the microbial composition of
different populations or host races [108,109], evolutionary studies
focusing on the mechanisms underlying the acquisition and
maintenance of microbial communities are still limited [110–112].
Sexual communication, mate choice, and reproduction
Success of an insect population or species relies crucially on its
ability to reproduce. Reproductive success depends on a range of
phenotypic traits, including fecundity (gamete production and
survival), rate of reproduction, and successful mating. Mating
success is affected by mediation of intraspecific communication
[113], specificity of mating behaviours, and mate selection.
Evidence of microbial influence on reproductive fitness and
associated traits and behaviours has been accumulating across a
range of insect hosts. In bed bugs (Cimex lectularis), sexually
transmitted commensal bacteria can increase sperm mortality and
negatively influence fecundity [114]. Egg production and larval
development in mosquitoes (Aedes aegypti) is reduced in the
absence of its symbiotic bacteria community, an effect that was
rescued following reintroduction of key bacterial species—Serratia
sp. and Elizabethkingia sp. [115,116]. A gene of the symbiotic
double-stranded RNA virus of Drosophila biauraria encodes a
male-killing protein. The gene may have been acquired by the
virus through shuffling of genomic segments, or reassortment,
and may provide opportunities for intraspecific variation in insect
microbiomes affecting reproduction [117]. On the other extreme,
females of the parasitic wasp Asobara tabida become incapable of
oogenesis when Wolbachia-free [118], with intraspecific variation
in Wolbachia strains committing variable impacts on oogenesis
and cytoplasmic incompatibility [119]. These examples highlight
the differential impacts of microbial partners on insect host
fecundity.
Mate signalling and choice assays in fruit flies have revealed
intricate interactions with microbes associated with gut and
reproductive organs. Bacillus spp. and other related bacteria
localised in the rectum of B. dorsalis males can produce its sex
pheromones, tri- and tetra-methylpyrazine [120]. Bacterial origin
of the female sex hormone has also been reported in grass grub
beetle (Costelytra zealandica), as a breakdown product of tyrosine
by colleterial gland resident bacteria Morganella morgani
[121,122]. Attraction and mate selection in a variety of tephritid
fruit fly species is also enhanced in the presence of specific
bacterial symbionts [123] (Fig. 2).
Finally, microbial mediation of insect communication via
pheromones and semiochemicals has received renewed interest
recently [113,124]. Broadly, bacteria can produce a remarkable
variety of compounds that interfere with insect-insect commu-
nications as well as affect insect behavioural outcomes [124].
CONSEQUENCES FOR ADAPTATION AND EVOLUTION
Any changes to insect host biology have the potential to affect the
population structure and dynamics. The right combination of
microbial partners may support a resilient population, while a shift
can contribute to population decline or invasion. Microbiome
variation may be an adaptive trait, subject to natural selection,
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that increases host fitness according to a model on imprecise
vertical transmission [48]. By extending phenotypic plasticity of
the host, microbes can enable populations to be more flexible in
changing environments, which ultimately affects adaptation and
evolution.
Implications for biodiversity, conservation, and biosecurity
Microbiomes change when arthropods encounter new or
restrictive environments. Population size bottlenecks, common
in conservation efforts, and biocontrol programmes, can result in
loss of microbiome diversity [125], and consequently in reduced
host fitness [126]. Captive rearing alone can result in loss or gain of
harmless and beneficial microbes that then cause reductions in
host fitness on the return of host populations to the wild [96,127].
By conferring resistance to parasitoids in aphids, defensive
symbionts can pose a serious challenge to the development of
biological control agents [128,129].
In the wild, could transfer of microbes between introduced host
species and related indigenous host species cause unwanted
changes, for example in host ranges of herbivorous arthropods?
Rare cases of unpredicted non-target attack by introduced weed
biocontrol agents all had explanations that were not microbiome-
associated [130]. However, host shifts have been associated with
microbiome change in a moth pest [107] and were created
experimentally across generations in an aphid [131]. The potential
contribution of microbes to insect invasions is exemplified by the
hypothesis that a swap between pest and non-pest symbiont
genotypes or a symbiont mix during host hybridisation led to the
plataspid stink bug Megacopta cribraria becoming invasive in the
USA [132]. Can microbiome manipulations also have conservation
benefits? For example, the threatened Australian butterfly,
Ornithoptera richmondia oviposits on an introduced invasive weed
Aristolochia littoralis on which its larvae die [133]. Can we transfer
microbiome components from butterfly species that naturally feed
on A. littoralis in the weeds’native range to the threatened
Australian butterfly, turning an “ecological trap”plant into a host
plant (and potentially assisting in native biocontrol of the weed)?
Ultimately, understanding and manipulating arthropod micro-
biomes may allow us to reduce biosecurity risks, improve
performance of beneficial arthropods, and enhance conservation
of rare indigenous arthropods.
OUTSTANDING QUESTIONS AND FUTURE RESEARCH
DIRECTIONS
Current challenges in microbiome research are to go beyond
descriptive studies towards functional analysis at lower taxonomic
levels (within phyla, families, genera, and species) of microbe-
microbe and microbe-host interactions, community effects and
the targeted manipulation of microbiomes [1,7,8]. This requires
measuring and modelling the resilience of host-associated
microbial communities in non-model species in the field to
generate quantifiable data [11,134,135]. Apart from better
understanding the drivers and consequences of insect micro-
biome changes, a major challenge for future research is to better
understand how to influence and manage microbiomes of insects
[30,136–141]. We can take advantage of methods that have been
developed for human, plant or soil microbiomes and adopt them
to improve the analyses of insect microbiomes [30,142–144]. We
selected several examples of novel methods that can further
elucidate the complex interactions within insect microbiomes and
how to manipulate them (Table 1). For example, altering the
microbiome composition of insect pests may reduce the severity
of the damage they inflict, or honeybees may become more
resilient to climate change through altering the gut biome. As
there are many drivers of insect microbiome composition,
tractable manipulation in the field remains a major challenge
[140]. We propose that the most immediate opportunities for
microbiome management are therefore with insect species that
are reared under controlled conditions and then released, like for
biological control of pests such as predators or parasitoids, or
herbivores used to control weeds. Improving their efficiency, for
Fig. 2 Vertically transmitted Klebsiella oxytoca influences Oriental fruit fly mate selection. The Oriental fruit fly(Bactrocera dorsalis)
harbours a diversity of gut symbionts, of which vertically transmitted Enterobacteriaceae bacteria Klebsiella oxytoca are reported to enhance
mate selection [151]. In the absence of this symbiont, significant reduction in mate attraction (via olfaction, p< 0.0001) and mating outcome
(via sperm deposition, p< 0.0001) for gnotobiotic virgin females has been reported [152]. Subsequently reinfected with this symbiont, virgin
female flies regain both mate attraction and sperm accumulation responses from male flies [152]. By increasing the likelihood of successful
mating, K. oxytoca along with other symbionts can facilitate the invasion success of the Oriental fruit fly.
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Table 1. Novel methods to study intraspecific variation in microbial communities of insects.
Method Description Applications Benefits Challenges Reference
Paratransgenesis of
obligate symbionts
Symbiont-mediated RNAi to
silence host genes or genes of
other symbionts
Studying symbiosis
Preventing insects from
vectoring pathogens
Controlling pest insects
Improved field safety because
obligate symbionts are less likely
to colonise other insects than
facultative symbionts
Technology to cultivate many
obligates in axenic culture
Engineering obligates in situ
Social and regulatory acceptance
[136,
138,139,141]
Host-mediated
symbiont selection
Guiding the host to acquire and
maintain microbial components
via exposure to specific
conditions, such as stress
Microbiome manipulation
Improving host resilience
Controlling pest insects
Replacing the method of
artificially adding or
removing taxa
Microbial communities that are
adapted to the selected
conditions and create the
desired host effect
Microbiome stability and
resilience
Potential for off-target or
undesirable side effects on host
fitness and microbial community
dynamics
Translation from plant to insect
microbiomes
[30,137,140]
Co-occurrence network
analysis
Assessing relationships across
diverse and complex microbial
communities
Studying organisation and
structure of communities
Identification of keystone
species
Identification of factors that
determine community
structure
Inferring taxa interactions
Observing the structure and
function of diverse communities
in situ
Interpretation of results
Over- or underestimation of the
true complexity
Need for complementary
experimental studies to confirm
functions and interactions
Translation from soil to insect
microbiomes
[135,145–147]
Microbiome association
studies
Linking microbiome analyses to
phenotypic descriptions
Studying symbiosis
Identification of mechanisms
that connect microbial
community features to
specific host traits
Observing the structure and
function of diverse communities
in situ
Translation from human or soil to
insect microbiomes and
ecological settings
Biological confirmation of
computational results
Careful experimental design and
robust analysis and data
interpretation
[142–144]
C. Lange et al.
6
The ISME Journal

example via the introduction of symbionts that enhance fecundity
or longevity during rearing, can have far reaching consequences,
and this is in urgent need of investigation.
We highlight the following four questions to be addressed to
improve the understanding of intraspecific changes in insect
microbiomes and the management of insect resilience or invasion:
1. What determines the functional resilience of host-associated
microbial communities? While the core microbiome repre-
sents the most common and most abundant taxa, species
with rare occurrence and low abundance can also be
important for the functional stability and adaptation of the
microbiome. These community members may be transient,
but can supply functional redundancy, which may be critical
under stressful conditions [12]. Learning more about the
functions of individual species or strains, and the interaction
networks within the microbiome will improve our under-
standing of resilience of microbiome functions [135,
145–147].
2. How can we achieve targeted manipulation of insect
microbiomes for specific purposes (“microbiome engineer-
ing”), such as pest and pathogen control and biocontrol?
The experimental design for host-mediated symbiont
selection needs to be optimised. We need to develop tools
to cultivate obligate symbionts in axenic culture to then
engineer them or to engineer obligate symbionts in situ
[136,138,139,141,148]. We also need to understand more
about the complex functional relationships between sym-
bionts and hosts and other host-associated organisms, such
as plants and parasitoids to manipulate microbiomes
successfully and safely.
3. How can we quantify microbiota switches that naturally
occur in insects? To study the microbiome shift within
populations, we need to assess the prevalence and
abundance of taxa through time in the field. This will give
us the rate of microbiome change in real-world scenarios,
rather than anecdotal descriptions.
4. How do microbiome-affected processes, such as host range
and mate selection influence each other? These are complex
interactions, leading to species-specific outcomes that need
to be studied with careful consideration for selected
conditions. To answer this question, we cannot rely on
results for model organisms but need to directly investigate
the species and systems in question.
CONCLUSION
The multiplicity of transmission routes and sources of variation in
insect microbiota have been well identified. However, the extent of
microbial shifts occurring through these different routes remains
poorly known. In addition to naturally occurring exchanges among
sympatric species, the continuous increase in invasive species
introductions may open the door to the transmission of new
microbial symbionts, horizontally, between introduced and native
species. As opposed to new abilities that might arise from “random”
genetic mutations, the horizontal acquisition of new microbial
species and the abilities they confer can be immediate. If adaptive,
these abilities could spread through the population very rapidly,
with significant consequences on the insect and its ecosystem.
Consequences may include the ability for phytophagous insects to
exploit new host plants (and cause greater plant damage), or the
ability to cope with environmental stress (such as climate change),
evade natural enemies or withstand diseases. A better fundamental
understanding of intraspecific microbiome dynamics will improve
the protection of insect biodiversity and the management of
invasive insects, which will benefit the environment as well as local
and regional economies.
DATA AVAILABILITY
No datasets were generated or analysed during the current study.
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ACKNOWLEDGEMENTS
We would like to thank Nicolette Faville and Cissy Pan for the graphic design of the
figures. This work was funded by the Strategic Science Investment Fund from the
New Zealand Ministry of Business, Innovation and Employment. MCL and SB were
also supported by the EntomoCentre research network (Région Centre Val de Loire).
AUTHOR CONTRIBUTIONS
All authors contributed equally to conceive and design the work, draft and revise the
manuscript, and approve the final version.
FUNDING
Open Access funding enabled and organized by CAUL and its Member Institutions.
COMPETING INTERESTS
The authors declare no competing interests.
ADDITIONAL INFORMATION
Correspondence and requests for materials should be addressed to Claudia Lange.
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