# Reciprocal interactions between anthropogenic stressors and insect microbiota

Pierre Antonelli, Pénélope Duval, Patricia Luis, Guillaume Minard, Claire

Valiente Moro

► To cite this version:

Pierre Antonelli, Pénélope Duval, Patricia Luis, Guillaume Minard, Claire Valiente Moro. Reciprocal interactions between anthropogenic stressors and insect microbiota. Environmental Science and Pollution Research, 2022, 29 (43), pp.64469-64488. 10.1007/s11356-022-21857-9. hal-03788170

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# Reciprocal interactions between anthropogenic stressors and insect microbiota

Pierre Antonelli<fnref n="1" /> · Pénélope Duval<fnref n="1" /> · Patricia Luis<fnref n="1" /> · Guillaume Minard<fnref n="1" /> · Claire Valiente Moro<fnref n="1" />

Received: 29 April 2022 / Accepted: 1 July 2022 / Published online: 21 July 2022
© The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature 2022

## Abstract

Insects play many important roles in nature due to their diversity, ecological role, and impact on agriculture or human health. They are directly influenced by environmental changes and in particular anthropic activities that constitute an important driver of change in the environmental characteristics. Insects face numerous anthropogenic stressors and have evolved various detoxication mechanisms to survive and/or resist to these compounds. Recent studies highligted the pressure exerted by xenobiotics on insect life-cycle and the important role of insect-associated bacterial microbiota in the insect responses to environmental changes. Stressor exposure can have various impacts on the composition and structure of insect microbiota that in turn may influence insect biology. Moreover, bacterial communities associated with insects can be directly or indirectly involved in detoxification processes with the selection of certain microorganisms capable of degrading xenobiotics. Further studies are needed to assess the role of insect-associated microbiota as key contributor to the xenobiotic metabolism and thus as a driver for insect adaptation to polluted habitats.

**Keywords** Xenobiotics · Insects · Bacteria · Detoxification · Insect-microbiota interactions

## Introduction

Over the past 50 years, humans have significantly impacted their environment (Jacobson et al. 2019) at such magnitude that ecological and biogeochemical shifts are comparable to those described for major geological transitions (Corlett 2015). To illustrate those profound changes, the Anthropocene (derived from the Greek *anthropos* “a human being” and *kainos* “new”), a new geological era has been proposed to succeed the ongoing Holocene (Corlett 2015). Overproduction of greenhouse gases, natural habitat degradation, and massive release of human-made xenobiotics in the

environment are among the most important factors contributing to a sustained and rapid disruption of ecosystems. Xenobiotics (from xenos “foreign” and bios “life”) refer to a foreign substance within an organism.

Among these living organisms, insects represent 85% of animal biodiversity with about 1 million described species and strongly contribute to biogeochemical cycles (Stork 2018; Goulson 2019). For instance, they contribute to the reproduction of flowering plants (pollination by bees, bumblebees, butterflies), they are a source of food for other species (e.g., locusts, flies), or contribute to soil fertility by bioconversion of agricultural wastes (e.g., dung beetles, black soldier fly larvae, houseflies). Some species are important for human agricultural activities as they act as pest control (e.g., ladybugs, parasitoid wasps) whereas some others have adverse effects on economy crop pests and public health (pathogen vectors) (Bradshaw et al. 2016). Based on these important ecological roles, insect response to xenobiotics can be a proxy to estimate wider alterations of ecosystem functions as well as economy losses and epidemic risks.

Increasing exposure of insects to many xenobiotics has contributed to the development of resistance mechanisms (i) at the metabolic level by increasing the activity of detoxification and excretion enzymes, (ii) at the cellular level by

Responsible Editor: Giovanni Benelli

Guillaume Minard and Claire Valiente Moro contributed equally to this work.

<figure><img src="image_2.png" /></figure>

Claire Valiente Moro
claire.valiente-moro@univ-lyon1.fr

<footnote n="1">¹ Univ Lyon, Université Claude Bernard Lyon 1, CNRS, INRAE, VetAgro Sup, UMR Ecologie Microbienne, 69622 Villeurbanne, France</footnote>
modifying the xenobiotics' targets, and (iii) at the behavioral
level by contact avoidance with the xenobiotic or limitation
of the contact duration (Palli 2020; Gao et al. 2022). At the
same time, biology faces a significant paradigm shift. Higher
eukaryotic organisms are no longer considering as isolated
individual but also integrate the influence of their associ-
ated microbial communities (i.e., their microbiota). This
refers to a super-organism called holobiont (from the Greek
holo “everything” and bios “life”) (Morris 2018). Thus, the
intrinsic capacities of insects heavily rely on functions car-
ried by their microbiota, composed of a rich and diverse
community of bacteria, viruses, fungi, algae, and protists
that mainly colonize the digestive tract. The microbiota
influences insect's biological processes such as development,
physiology, nutrition (Feldhaar 2011; Guégan et al. 2018), or
resistance to biotic (e.g., pathogens) and abiotic (e.g., tem-
perature, xenobiotics) stresses (Dunbar et al. 2007; Douglas
2015; Agamennone et al. 2019; Shukla and Beran 2020).
In this context, understanding the effects of xenobiotics on
insect holobionts becomes an important asset in the current
ecological transition in order to better estimate consequences
for ecosystem functioning.

In this review, we synthesize current knowledge on the
impact of anthropic xenobiotics on insect-microbiota inter-
actions highlighting their effects on the microbiota composi-
tion and structure and its role in detoxification processes. We
focus specifically our attention on the bacterial component
of the microbiota as it was more intensely studied compared
with the rest of the microbiota. Studying the intricate links
between insects, their associated microbiota, and the xeno-
biotics may advance our understanding of insect xenobiotic
responses and could have practical applications in the field
through the use of biological pest and vector control methods.

Anthropogenic stressors: classification, origin, and impact

Anthropogenic stressors include a wide variety of com-
pounds and particles with heterogenous properties. The
effects of xenobiotic compounds are complex since they
can affect many organisms and are modulated by multiple
factors (host, nature, and concentration of the xenobiotic,
intensity of exposure). Therefore, within individuals, effects
cannot be easily generalized. Xenobiotics are released in the
environment either in their original forms or as by-products
after being metabolized by living organisms or transformed
by abiotic factors such as chemical or physical agents. Long-
term exposure to these compounds is called chronic expo-
sure while acute exposure refers to short contact. Xenobiotic
activity, their concentration, their dissemination routes, and
frequency as well as the characteristic of the focal ecosystem they affect determine their environmental consequences

(Dinka 2018). Xenobiotics can affect the health of organ-
isms but may also indirectly influence the microorganims
they host (following the concept of holobiont) as well as the
organisms with which they interact within the ecosystem
(e.g., trophic transmission from prey to predators). Direct
effects of xenobiotics on the host are very heterogeneous
and can, for example, result in an alteration of the hormonal
system (endocrine disruptors) (Williams et al. 2016), a mod-
ification of gene expression (Wang et al. 2020) or in some
cases lead to the death (Daisley et al. 2018). Deleterious
effects are often observed during acute and chronic expo-
sure, the latter taking longer to be detected. Furthermore,
intermediate concentrations can also lead to high response
of the individuals. This is especially the case when biphasic
effects are observed. Hormesis is a model in which interme-
diate concentrations lead to the highest values of individual
responses. Low-dose exposure is therefore as important as
high dose, and probably more frequent. This is the case with
bisphenol A, which is widely used in plastic products and
has been shown to disrupt neural differentiation in human-
derived neural progenitor cells, potentially disrupting brain
development (Fujiwara et al. 2018).

The pharmaceutical products are a wide range of anthro-
pogenic stressors. The first synthetic drug is acetylsalicylic
acid, which was discovered in 1897. This medicine is widely
used for more than 120 years because of its remarkable pain
relief as well as cardiovascular event prevention properties.
The global pharmaceutical markets currently represent 479
products considered essential (WHO 2021). Human and ani-
mal medicines used to prevent and/or treat diseases often
contaminate the environments through wastewater and ani-
mal agriculture manure. It was estimated that between 2010
and 2030, the global consumption of antimicrobials will
increase by 67%, from 63,151 ± 1560 t to 105,596 ± 3605
t (Van Boeckel et al. 2015). Among these pharmaceutical
products, there are very diverse molecules such as anti-
inflammatory compounds, painkillers, antidepressants, con-
traceptives, antiparasitics, or antibiotics. Their impact on
non-target species is a major environmental concern (Fent
et al. 2006). It has been reported that the presence in water
of drugs containing promethazine affects the reproduction
of planktonic crustaceans of the genus *Daphnia* (Furuhagen
et al. 2014). Another study showed that exposure to ibu-
profen, diclofenac, and propranolol decreases the metabolic
activities of the common mussel *Mytilus edulis* and its
abitlity to adhere to a substrate (Ericson et al. 2010).

Another important class of anthropogenic stressors com-
prise pesticides. They include all substances eliminating
or preventing damages caused by adverse organisms (i.e.,
pests). Among them, phytosanitary products are used to
increase agricultural production by avoiding pest devel-
opment. They include many classes of products, such as
insecticides (prevent insect damages), fungicides (which

<figure><img src="image_3.png" /></figure>
eliminate fungi), herbicides (weeding), nematicides (which kill nematodes and earthworms), rodenticides (used to get rid of various rodents such as rats, field mice...), and are responsible of significant environmental impacts. Sharma et al. (2019) reported that global pesticide usage in 2020 was approximately 3.5 million t, with herbicides accounting for 47.5% of the total amount, followed by insecticides (29.5%), fungicides (17.5%), and others (5.5%). As agricultural intensification is needed to respond to the global population increase (i.e., from 2.3 billion in 1950 to 7.5 billion in 2019 according to the United Nations), the utilization of phytosanitary products increased. The risk assessment constitutes therefore an essential tool for the evaluation of the potential effects of these compounds on organisms (classes of toxicity to human health from 1 to 8) and ecosystems (environmental impact) (Sauer et al. 2015). However, despite the development of technical guidance documents, many side effects due to their massive use disrupted the functioning of ecosystems. A significant example of harmfull impacts is the non-selective systemic herbicide glyphosate, the most used (by volume and land area treated) herbicide in the world (Benbrook 2016). It has been shown to induce cardiotoxicity in developing zebrafish *Danio rerio* resulting in heart rate and circulation decrease/absence (Gaur and Bhargava 2019) as well as adverse pregnancy outcomes in many organisms (de Araujo et al. 2016). In addition, the negative impact of pesticides on human, animal, and environmental health or One Health initiative is exemplified through their persistence use in the environment leading to increased human exposure in food that can cause important metabolic disorder (Duarte-Hospital et al. 2019). One of the most striking examples is honey in which more than 19 pesticide residues from organophosphate insecticides have already been detected following their incorporation by bees after pollination of treated fields (El-Nahal 2020). This can be explained by the fact that many of them, particularly organophosphates, are actually directly applied in the hives against *Varroa* mites.

Compounds of industrial origin are the final class of
anthropogenic stressors. The industry has strongly expanded
since the early nineteenth century. Although environmental
protection is of global concerns of many industries (improve-
ment of treatment and waste recycling), manufactured prod-
ucts and the waste they generate still remain a major issue.
This is particularly the case for plastic derivatives as the
production of plastics have reached 368 million t worldwide
in 2019 (Plastics Europe 2020). Following growing exposure
to plastic materials in the environment, some insects like
the mealworm *Tenebrio molitor* showed strong ability to
degrade these complex polymers (Yang et al. 2021). Given
this potential, they are considered as promising candidates
in bioremediation in the context of contaminated soils. Other
residues such as nanoparticles (compounds <100 nm) are

also increasingly used in textile, cosmetic, food, electronic,
and medicine industries (Nel et al. 2006; Buzea et al. 2007;
Ahamed et al. 2010). These compounds are easily dissemi-
nated in the environment and can significantly impact the life
history traits of many insect species such as their develop-
ment, reproduction, or survival but also their immunity (Li
et al. 2020a).

Although it may be challenging to evaluate the effects
of anthropogenic stressors on insect holobionts, this can be
critical for understanding their adverse effects since insects
provide ecological services such as pollination, pest control,
and decomposition.

Xenobiotics on insect microbiota and host consequences

Insects harbor large and diverse microbial communities whose composition and structure vary during their development (Hammer and Moran 2019). While direct impacts of xenobiotics on insect are often studied (Li et al. 2007; Gao et al. 2022), it is increasingly apparent that the insect microbiota can also influence those interactions (Li et al. 2020a, b, c). Some insect models and life cycle stages are particularly studied because of their health, environmental, or agronomic relevance. As an example, in bees, most of studies have concerned adults because it is during this stage that they forage on flowers and enter in contact with the xenobiotic. Conversely, the crop pest butterfly *Helicoverpa* is more particularly studied at the larval stage due to potential application of those investigations for pest control.

Impact of pesticides

Insecticides

Insecticides are widely used to control insect pests and vec-
tors of diseases. According to their source of origin, they
can be synthetic and natural (either organic or inorganic).
They can impact the insect microbiota through various
ways. Among chemical insecticides, methoprene (a growth
regulator blocking the development of insects at the larval
stage) causes a strong remodeling of bacterial composition
in *Ae. stimulans*, resulting in an increase in relative abun-
dance of Enterobacteriaceae and *Staphylococcus* (Li et al.
2011; Receveur et al. 2018; Zhao et al. 2020). Conversely,
three chemical insecticides (i.e., permethrin, malathion, and
imidacloprid) were tested against the Asian tiger mosquito
*Aedes albopictus*, the honeybee *Apis mellifera*, and the
fruit fly *Drosophila melanogaster* with poor consequences
on their bacterial microbiota composition (Daisley et al.
2017; Raymann et al. 2018; Juma et al. 2020). However, the
authors reported that imidacloprid can modify the bacterial
microbiota of *D. melanogaster* at the larval stage, result-
ing in higher abundances of bacteria belonging to the gen-
era *Acetobacter* and *Lactobacillus* which were previously
reported for their probiotic activity (Consuegra et al. 2020).
In some cases, specific microorganisms confer higher resist-
ance of insects to insecticides. In *Anopheles albimanus*, the
microbiota was suggested to lead to phenotypic resistance
toward fenitrothion, a neurotoxic insecticide (Dada et al.
2018). In the fall, armyworm *Spodoptera frugiperda*, bacte-
rial genera *Enterococcus*, and *Leclercia* are able to metabo-
lize certain insecticides (e.g., chlorpyrifos, delthametrine,
spinosad) into assimilable carbon sources (Gomes et al.
2020). Conversely, additive toxic effects of certain mem-
bers of the microbiota were also reported. For instance, the
reintroduction of bacterial species such as *Enterobacter
ludwigii*, *Citrobacter freundii*, and *Serratia marcescens* in
axenic individuals restored the sensitivity of the beetle to
insecticides (Polenogova et al. 2021). Similarly, some bac-
teria associated with *D. melanogaster* are able to metabolize
chlorpyrifos (a chemical insecticide of the family chlorinated
organophosphates) into a metabolite that is 10 times more
reactive and toxic for the insect (Daisley et al. 2018). Among
bioinsectides, *Bacillus thuringiensis* (commonly called Bt) is
a bacterial species used for its insecticidal properties (Bravo
et al. 2011). It is notably the most widely used bioinsecticide
in organic farming. Its activity relies on the production of
Cry and Cyt toxins in the insect gut after bacterial inges-
tion (Bel et al. 2020). The Bt effects on the microbiota vary
according to the insect host and the conditions of Bt expo-
sure (toxin variants, Bt formulation, concentrations...). They
are ranging from no impact in the mosquito *Aedes stimu-
lans* or the ladybugs *Propylea japonica* to a strong alteration
of the bacterial community structure, in the mosquito *Ae.
aegypti* (Receveur et al. 2018; Tetreau et al. 2018; Zhao et al.
2020). Such community remodeling were also observed in
the Colorado potato beetle *Leptinotarsa decemlineata* after
being exposed to Bt or avermectin (another bioinsecticide)
of (Polenogova et al. 2021). These insecticides led to an
increase of Enterobacteriaceae, *Bacillus*, and *Serratia* within
the insect microbiota. The authors suggested that these bac-
teria could play a role in accelerating Bt infection and aver-
mectin-induced toxicosis and that the additive effect between
both bioinsecticides could be linked with alterations in the
bacterial community. In the corn earworm *Helicoverpa
zea* and the old-world bollworm *Helicoverpa armigera*, an
increase in bacterial diversity was also observed in the pres-
ence of Bt-producing transgenic plants. More specifically, a
proliferation of bacteria of the *Enterococcaceae* family and
more particularly of species *Enterococcus casseliflavus* and
*Klebsiella oxytoca* was reported (Gracy et al. 2016; Degue-
non et al. 2021). Similarly, exposure to Bt increased the bac-
terial density within the gut and hemolymph of *Spodoptera
littoralis* larvae and the cabbage moth *Plutella xylostella*

(Caccia et al. 2016; Li et al. 2021). The colonization of the
hemocoel by high densities of bacteria also reflects a sep-
ticemia leading to higher mortality of individuals exposed
to toxins (Caccia et al. 2016). Conversely, in the beet army-
worm *Spodoptera exigua* and the red flour beetle *Tribolium
castaneum*, a low bacterial density leads to a higher sensi-
tivity of the insect to Bt treatment, while a high bacterial
density improves its resistance (Hernández-Martínez et al.
2010; Futo et al. 2015). Depending on the insect, Bt can thus
induce either an increase in the bacterial load that can lead
to sepsis or increasing resistance to insecticide.

Herbicides

Plants provide food source for pollinator or pest-control
insects and can serve as nesting sites or shelter to many
species. Other insect species negatively impact plant fit-
ness either by consuming plant tissues or infecting them
with vector-borne pathogens. These interactions promote
close contact between insects and herbicides which used
to protect crops from weeds and therefore insects are fre-
quently exposed to herbicides. Most studies on the influ-
ence of herbicides on insect microbiota concerns glypho-
sate, the world's most widely used broad-spectrum and
non-selective herbicide for control of grass and broad-
leaf weeds. It targets the key enzyme of the shikimate
pathway, 5-enolpyruvylshikimate-3-phosphate synthase
(EPSPS), which synthesizes three essential aromatic
amino acids (phenylalanine, tyrosine, and tryptophan) in
plants but also in certain bacteria (Motta et al. 2018).
The effect of glyphosate on the honey bee *Apis mellifera*
at different stages has extensively been studied. The core
microbiota of bees is dominated by eight bacterial species
that play a role in insect growth and immunity (Daisley
et al. 2020). Glyphosate has been shown to halve relative
abundance of four of these species (i.e., *Snodgrassella
alvi*, *Bifidobacterium*, *Lactobacillus* Firm-4, and *Lac-
tobacillus* Firm-5) during the adult stage (Motta et al.
2018). This herbicide also induces an increase in some
bacterial-genera abundance including part of the keystone
species (i.e., *Bartonella*, *Lactobacillus*, and *Frischella*)
(Blot et al. 2019; Motta et al. 2020). Moreover, those
studies also highlighted that glyphosate affected the bee
physiology (e.g., decrease of brood survival and larval
weight, increase mortality of bees from hives) in a dose-
dependent way. These modifications of the microbiota
are most often dose-dependent (Dai et al. 2018; Motta
et al. 2020) and strongly alter bee survival and pollinator
activity at high concentrations. In newly emerged adults,
exposure to glyphosate affects the gut colonization by
beneficial bacteria. When treated with commercial formu-
lation of glyphosate (0.1% Roundup), weakened individu-
als are thus more vulnerable to opportunistic infections

<figure><img src="image_4.png" /></figure>
by entomopathogens belonging to the genus *Serratia*. However, glyphosate would not promote infections by the microsporidian *Nosema ceranae*, an emerging parasite described as the major cause of bee decline in some areas (Burnham 2019). In addition, the acid aminomethylphosphonic (AMPA), a derivative from the degradation of glyphosate often found in treated soils does not induce any significant change in the bee microbiota composition and structure. These results suggest that modifications observed on the microbiota of newly emerged adult bees are due to glyphosate (Blot et al. 2019). The effect of glyphosate on bacterial microbiota has also been studied in the greater wax moth *Galleria melonella* and in the mosquito *Anopheles gambiae*, the major malaria vector, which feed on honeycombs containing significant amount of beeswax and floral nectar, respectively. The results show that glyphosate exposure induces changes in the bacterial microbiota composition and structure in treated individuals in comparison of their untreated counterparts (Smith et al. 2021). This resulted in a significant increase in the relative abundance of the bacterial genus *Asaia* and a decrease of Enterobacteriaceae. Such a dysbiosis has been linked with reduced survival of Lepidoptera following infection with the entomopathogenic fungus *Cryptococcus neoformans* and increased colonization by the protozoan parasite *Plasmodium falciparum* in the mosquito. These observations suggest that Enterobacteriaceae such as *Serratia marcescens* may reduce the probability of infection of *G. melonella* with fungus and that of *A. gambiae* with the protozoan (Smith et al. 2021). A decrease in Enterobacteriaceae in the insect microbiota following glyphosate exposure was also observed in *Leptinotarsa decemlineata* with drastic microbiota modifications at the adult stage (Gómez-Gallego et al. 2020). Interestingly, it was shown that chronic exposure to atrazine, a synthetic herbicide of the triazine class, impacts the bacterial microbiota of the parasitoid wasp *Nasonia vitripennis* and confers an insect higher herbicide resistance. This effect is not only transgenerational but also generalizable to other herbicides such as glyphosate (Wang et al. 2020). In addition to its effect on microbiota, atrazine was also toxic to the insect host at very high doses (LC50 at 45.50 ± 15.73 parts per million). Moreover, transcription analysis showed that the most downregulated genes were involved in immunity and odorant binding. This suggests that atrazine exposure on *N. vitripennis* affects the insect behavior and immune system.

More globally, pesticides may indirectly alter the trajectory of host-microbiota coevolution as revealed in honeybees through modulation of social behaviors and the insect gut-brain axis (Daisley et al. 2022). On top of their direct impacts on insects per se, we should also critically

evaluate the pesticide influence on their microbiota and
the functions it ensures.

Impact of pharmaceutical products

Pharmaceuticals are commonly used in human health and
breeding. Antibiotics are more particularly used to cure
and prevent infections by pathogens but can also serve
as growth hormones. Due to the increasing resistance to
antibiotics, current research aims to find potential alterna-
tives (Gadde et al. 2017; Lillehoj et al. 2018). Antibiot-
ics are bacterial antagonists and can therefore disrupt the
insect bacterial microbiota. Insects enter in contact with
drugs either in aquatic and terrestrial environments con-
taminated by pollution. Occurrence of antibiotic residues
in the environment is highly increased by medical and vet-
erinary activities (between 40 and 90% of the administered
antibiotic is excreted in the feces and urine) or applica-
tion of contaminated manure on the agricultural lands as
fertilizer as well as irrigation of crops with wastewater
(Polianciuc et al. 2020). In some cas, as for honeybee,
insects can directly be treated with antibiotics to prevent
or treat bacterial infections (e.g., honeybee). The german
cockroach *Blatella germanica* remains one of the most dif-
ficult indoor insect species to control because of its ability
to develop resistance toward many insecticides. Recent
studies have assessed the efficiency of integrated manage-
ment methods based on antibiotic treatments that disrupt
insect-associated mutualistic bacteria (Zhang and Zhang
2018). A treatment combining levofloxacin and gen-
tamicin caused a significant decrease in bacterial density
and diversity (Li et al. 2020a, b, c). After 14 days, while
bacterial density reached similar values to that observed
in the initial state, shifts in the microbiota composition
were reported and consisted in an increase of Bacteroi-
detes and Fusobacteria and a decrease of Firmicutes. Such
disturbance of *B. germanica* microbiota did not alter the
host physiology. Gendrin et al. (2015) are the first to report
the influence of high antibiotic usage on the transmission
of vector-borne diseases. They showed that antibiotics in
ingested blood enhance the susceptibility of *A. gambiae*
mosquitoes to malaria infection by disturbing their gut
microbiota. The massive proliferation of bacteria seen at
24 h after the blood meal was reduced by 70% in the pres-
ence of antibiotic treatment and associated with a near
clearance of Enterobacteriaceae and an *Asaia* increase.
Antibiotic-induced dysbiosis not only increases mosquito
susceptibility to *Plasmodium* infection but also alters
their survival and fecundity. The antibiotic tetracycline is
widely used in honeybees to cure hives infections. It was
shown that antibiotic treatment of *A. mellifera* induced
higher mortality rates as well as gut dysbiosis that in turn
increased susceptibility to opportunistic pathogens (Ray-
mann et al. 2017). A recent study showed that the impact
of tetracycline on honeybee microbiota is dose-dependent
(Jia et al. 2022). While the residue of tetracycline treat-
ment does not exert lethal effects on gut bacterial com-
munities, high dose of antibiotics induced a microbiota
dysbiosis (Jia et al. 2022). Tetracyclines were found
to be the most abundant antibiotic groups found in pig
manure with about to 376,210 µg/kg (Frey et al. 2022).
Tetracycline is also used to treat infections in cattles and
can enter in contact with insects such as the dung beetle
*Aphodius fossor* which feeds on the animal's excrement.
Antibiotic-induced restructuring of dung beetle microbi-
ota was not associated with significant differences in the
insect development, survival, or reproduction (Hammer
et al. 2016). Nevertheless, long-lasting transgenerational
effects on insect microbiome after tetracycline treatment
at subinhibitory concentrations were suggested in the cab-
bage root fly *Delia radicum* with potentially tetracycline
resistant and heritable bacterial genera (Ourry et al. 2020).
Although water treatment is a health priority, many chemi-
cals may be carried out through water. The impact of dif-
ferent contaminants (hormones, antibiotics, paracetamol)
has been evaluated on the bacterial microbiota of *Culex
quiquefasciatus* mosquito larvae (Pennington et al. 2016).
Each treatment individually leads to an overall decrease
in the bacterial diversity while the mix of hormonal and
antibiotical treatments leads to the selection of bacteria
that were absent under control conditions or antibiotics
alone. These results suggest that the bacteria could use
hormones as substrates to grow.

Impact of nanoparticles

Silver nanoparticles (Np-Ag) have a drastic effect on the
bacterial microbiota of *D. melanogaster* larvae and act as
powerful antimicrobials by targeting in particular gram-
negative bacteria. After Np-Ag treatment of larvae, 91% of
the sequences obtained with metabarcoding were assigned
to the *Lactobacillus* (Gram-positive bacteria) genus and in
particular the species *Lactobacillus brevis*. Elimination of
Gram-negative bacteria was correlated with a lower larval
viability (83% vs. 4% in the absence and presence of nano-
particles, respectively) (Han et al. 2014). Their impact has
also been extensively studied on the silkworm *Bombyx mori*
since they are commonly used in sericulture in order to pre-
vent silkworm from getting infected with pathogens. A dose-
dependent effect was thus demonstrated on the microbiota.
A treatment at 20 mg/L of Np-Ag leads to an increase in
bacterial diversity and the abundance of certain bacterial
taxa (Firmicutes, Bacilli, and Enterococcus). On the other
hand, these taxa were completely replaced by others (e.g.,
*Blautia*, *Terrisporobacter...*) after the larvae were exposed

to 100 mg/L of Np-Ag. While high doses of titanium dioxide (TiO2) are toxic to the silkworm, intermediate ones (5 mg/L) enhance the immunity and insect growth rate. This treatment does not alter the dominant bacterial species but increases the abundance of subdominant species involved in nutrition and metabolism (Staphylococcus and Lachnospiraceae), or detoxification (Pseudomonas and Sphingomonas) and decreases that of Serratia marcescens, an opportunistic entomopathogen (Han et al. 2014). Similarly, copper and zinc oxide nanoparticles were shown to decrease abundance of probiotics (e.g., Acetobacter) and increase pathobionts (e.g., Pseudomonas, Bacillus, Escherichia, Enterococcus, and Ralstonia) in the silkworm gut bacterial microbiota following diet exposure (Muhammad et al. 2022). Another recent study demonstrates that nano-La2O3 can cause detrimental effects on honeybee health, potentially by causing dysbiosis of gut bacterial communities leading to the enrichment of bacterial pathogens Serratia and Frischella and the alteration of digestion-related taxa Bombella (Liu et al. 2021). Studies on the impact of nanoparticles on insects are still few. It is unknown how exposure to nanoparticles affect insect microbiota and its metabolic activity or which specific microbes are involved in their detoxification.

All these examples clearly demonstrate a direct impact of
anthropogenic stressors on the insect bacterial microbiota
and their deleterious consequences for the host (summarized
in Table 1). However, the microbiota can also modulate the
insect's response to xenobiotic compounds and contribute to
its resistance or sensitivity.

Stressor exposure favors insect performance  
through beneficial impact on microbiota

Despite deleterious effects, anthropogenic stressors may
also favor insect performance through positive impacts
on their microbiota. However, to date, these examples are
still scarce. For instance, in the silkworm *Bombyx mori*,
insecticide resistance develops faster, thanks to the selec-
tion of microbial symbionts. This beneficial impact results
from an increase in host fitness following essential amino
acids provisioning by bacteria allowing larvae to resist more
efficiently the deleterious effects of insecticides. Similarly,
exposure of *B. mori* to TiO₂ NPs alters the composition of
its gut microbiota, such an impact results in higher insect
growth and development rates, and immune response (Li
et al. 2020a, b, c). It also enhances the insect resistance to
insecticide. *D. melanogaster* flies treated with the carra-
geenan-oligosaccharide prebiotic showed higher gut micro-
biota diversity and increased abundance of the genus *Com-
mensalibacter* (Ma et al. 2021). This modification in their
microbiota was positively correlated with an extension of
the individuals' lifespan. Most of these examples highlight
indirect effects without demonstrating any clear causation.

<figure><img src="image_5.png" /></figure>
<table><caption>**Table 1** Anthropogenic xenobiotic impacts on bacterial microbiota and consequences for the insect host</caption><thead><tr><th rowspan="2">Xenobiotic<br>Group</th><th rowspan="2">Class</th><th rowspan="2">Name</th><th colspan="2">Insect</th><th colspan="2">Impact on bacterial<br>microbiota</th><th rowspan="2">Bacterial taxa<br>affected</th><th rowspan="2">Impact on insect<br>host</th><th rowspan="2">References</th></tr><tr><th>Common name</th><th>Species</th><th>General impact</th></tr></thead><tbody><tr><td><strong>Pesticides</strong></td><td><strong>Insecticides</strong></td><td><strong>Bioinsecticide</strong></td><td><strong>Bacillus thuri-<br>giensis (Bt)</strong></td><td><strong>Mosquito</strong></td><td><strong>Aedes aegypti</strong></td><td>Decreased diversity</td><td>UND</td><td>More insecticide<br>tolerance</td><td>Tetreau et al. 2018</td></tr><tr><td></td><td></td><td></td><td></td><td>Ladybug</td><td>Aedes simulans</td><td>No impact</td><td>-</td><td>No impact</td><td>Receveur et al. 2018</td></tr><tr><td></td><td></td><td></td><td></td><td>Beetle</td><td>Coleomegilla maculata</td><td>No impact</td><td>-</td><td>No impact</td><td>Li et al. 2011</td></tr><tr><td></td><td></td><td></td><td></td><td>Butterfly</td><td>Propylea japonica</td><td>No impact</td><td>-</td><td>No impact</td><td>Zhao et al. 2020</td></tr><tr><td></td><td></td><td></td><td></td><td></td><td>Plutella xylostella</td><td>Increased bacterial<br>load and decreased<br>diversity</td><td>UND</td><td>Lower insecticide<br>tolerance</td><td>Li et al. 2021</td></tr><tr><td></td><td></td><td></td><td></td><td></td><td>Spodoptera littoralis</td><td>Modifications in the<br>hemocoeel micro-<br>biota composition</td><td>Augmentation of<br><em>Clostridium</em> and<br><em>Serratia</em></td><td>Sepsis and death</td><td>Caccia et al. 2016</td></tr><tr><td></td><td></td><td></td><td></td><td></td><td>Helicoverpa zea</td><td>Increased bacterial<br>load and diversity</td><td>Augmentation of<br><em>Enterococcus</em></td><td>UND</td><td>Deguenon et al. 2021</td></tr><tr><td></td><td></td><td></td><td></td><td></td><td>Helicoverpa armigera</td><td>Increased diversity</td><td>UND</td><td>Multiple insecti-<br>icide resistance</td><td>R. Gandhi<br>et al. 2016</td></tr><tr><td></td><td></td><td></td><td></td><td></td><td>Spodoptera exigua</td><td>Increased bacterial<br>load</td><td>Augmentation of<br><em>Enterococcus</em></td><td>More insecticide<br>tolerance</td><td>Hernández-Martínez<br>et al. 2010</td></tr><tr><td></td><td></td><td></td><td></td><td>Colorado<br>potato beetle</td><td>Leptinotarsa decem-<br>lineata</td><td>Microbiota composi-<br>tion modification</td><td>Augmentation of<br>Enterobacteriaceae<br>Diminution of <em>Spi-<br>roplasma leptino-<br>tarsae</em></td><td>Lower insecticide<br>tolerance</td><td>Polenogova et al.<br>2021</td></tr><tr><td></td><td></td><td></td><td></td><td></td><td>Leptinotarsa decem-<br>lineata</td><td>Decreased diversity</td><td>Augmentation of<br>Enterobacteriaceae</td><td>UND</td><td>Kryukov et al. 2021</td></tr><tr><td></td><td></td><td></td><td></td><td>Mosquito</td><td>Aedes aegypti</td><td>Increased bacterial<br>load and diversity</td><td>Augmentation of<br>Flavobacteria and<br><em>Pseudomonas</em><br>/ Diminution of<br><em>Microbacterium</em></td><td>UND</td><td>Noskov et al. 2021</td></tr><tr><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>No impact</td><td>Juma et al. 2020</td></tr><tr><td></td><td>Organophos-<br>phate</td><td>Malathion</td><td>Fenitrothion</td><td></td><td>Aedes albopictus</td><td>No impact</td><td>Decreased diversity</td><td>More insecticide<br>tolerance</td><td>Dada et al. 2018</td></tr><tr><td></td><td></td><td></td><td></td><td></td><td>Anopheles albimanus</td><td>Decreased diversity</td><td>Diminution of<br>Actinomycetes<br>and Firmicutes<br>Augmentation of<br>Proteobacteria</td><td></td><td></td></tr></tbody></table>
<table><caption>**Table 1 (continued)**</caption><thead><tr><th rowspan="2">Xenobiotic Group</th><th rowspan="2">Class</th><th rowspan="2">Name</th><th colspan="2">Insect</th><th colspan="2">Impact on bacterial microbiota</th><th rowspan="2">Impact on insect host</th><th rowspan="2">References</th></tr><tr><th>Common name</th><th>Species</th><th>General impact</th><th>Bacterial taxa affected</th></tr></thead><tbody><tr><td>Pyrethroid</td><td>Permethrin</td><td>Aedes albopictus</td><td></td><td></td><td>No impact</td><td>-</td><td>No impact</td><td>Juma et al. 2020</td></tr><tr><td></td><td></td><td>Anopheles albimanus</td><td></td><td></td><td>Microbiota composition modification</td><td>Augmentation of Pantoea agglomerans and Pseudomonas fragi</td><td>Supposedly more insecticide tolerance</td><td>Dada et al. 2019</td></tr><tr><td>Flumethrin</td><td>Honeybee</td><td>Apis mellifera</td><td></td><td></td><td>Decreased abundance and diversity at low doses / Increased abundance and diversity at high doses</td><td>UND</td><td>More insecticide tolerance (at high doses)</td><td>Yu et al. 2021</td></tr><tr><td>Neonicotinoids</td><td>Imidaclopride</td><td></td><td></td><td></td><td>No impact</td><td>-</td><td>Increased mortality and susceptibility to bacterial infections</td><td>Raymann et al. 2018</td></tr><tr><td></td><td></td><td></td><td></td><td></td><td>Decreased diversity in the long term</td><td>Alteration of Frischella, lactobacilli and bifidobacteria</td><td>Reduced related nutritional and defense functions</td><td>Alberoni et al. 2021</td></tr><tr><td></td><td></td><td>Drosophila melanogaster</td><td></td><td></td><td>Microbiota composition modification</td><td>Augmentation of Acetobacter and Lactobacillus</td><td>Increased susceptibility to bacterial infections and tolerance to heat stress</td><td>Daisley et al. 2017</td></tr><tr><td>Thiaclopride</td><td>Honeybee</td><td>Apis mellifera</td><td></td><td></td><td>Decreased abundance and diversity</td><td>Diminution of Lactobacillus Firm-5 and Bombella apis</td><td>UND</td><td>Liu et al. 2020</td></tr><tr><td></td><td></td><td></td><td></td><td></td><td>Decreased diversity in the short term</td><td>Alteration of Frischella, lactobacilli and bifidobacteria</td><td>Reduced related nutritional and defense functions</td><td>Alberoni et al. 2021</td></tr><tr><td>Clothianidin</td><td></td><td></td><td></td><td></td><td>Increased diversity</td><td>UND</td><td>Decreased survival</td><td>El Khoury et al. 2021</td></tr></tbody></table>

<figure><img src="image_6.png" /></figure>
<table><caption>**Table 1 (continued)**</caption><thead><tr><th rowspan="2">Xenobiotic Group</th><th rowspan="2">Class</th><th rowspan="2">Name</th><th colspan="2">Insect</th><th rowspan="2">Impact on bacterial microbiota General impact</th><th rowspan="2">Bacterial taxa affected</th><th rowspan="2">Impact on insect host</th><th rowspan="2">References</th></tr><tr><th>Common name</th><th>Species</th></tr></thead><tbody><tr><td rowspan="10">Herbicides</td><td rowspan="5">Carbamate</td><td>Nitenpyrame</td><td></td><td></td><td>Microbiota composition modification</td><td>Augmentation of Bar-<br/>tonella, Bifidobacterium and Frischella.<br/>Diminution of Gilliamella</td><td>Higher mortality</td><td>Zhu et al. 2020</td></tr><tr><td>Guadipyr</td><td>Silkworm</td><td>Bombyx mori</td><td>Microbiota composition modification with increased diversity</td><td>Augmentation of Pseudomonas Diminution of Curvibacter</td><td>Higher mortality</td><td>Hou et al. 2021</td></tr><tr><td>Carbaryl</td><td>Honeybee</td><td>Apis mellifera</td><td>Microbiota composition modification with decreased bacterial load</td><td>Diminution of Alphaproteobacteria and augmentation of Snodgrasella alvi and Lkullaberegensis</td><td>Modification of bee's behaviour</td><td>Nogrado et al. 2019</td></tr><tr><td>Glyphosate</td><td>Butterfly</td><td>Galleria mellonella</td><td>Microbiota composition modification</td><td>Augmentation of Asaia and diminution of Enterobacteriaceae</td><td>Increased host sensibility to fungal infections</td><td>Smith et al. 2021</td></tr><tr><td>Honeybee</td><td>Mosquito</td><td>Anopheles gambiae</td><td>Microbiota composition modification</td><td>Augmentation of Asaia and diminution of Enterobacteriaceae</td><td>Enhance the susceptibility of mosquitoes to malaria infection</td><td>Smith et al. 2021</td></tr><tr><td rowspan="2">Carbamate</td><td rowspan="2">Glyphosate</td><td rowspan="2">Butterfly</td><td rowspan="2">Galleria mellonella</td><td rowspan="2">Microbiota composition modification</td><td rowspan="2">Diminution of core microbial species : Snodgrassella alvi, Lactobacillus Firm-4 et 5 and Bifidobacterium</td><td rowspan="2">Alteration of health and pol- lination activity, increased susceptibility to bacterial infections</td><td rowspan="2">Motta et al. 2018, 2020; Blot et al. 2019</td></tr><tr><td></td><td></td></tr><tr><td rowspan="2">Carbamate</td><td rowspan="2">Glyphosate</td><td rowspan="2">Butterfly</td><td rowspan="2">Galleria mellonella</td><td rowspan="2">Microbiota composition modification</td><td rowspan="2">Augmentation of Gammaproteobacteria, Lachnospiraceae</td><td rowspan="2">Reduced survival</td><td rowspan="2">Dai et al. 2018</td></tr><tr><td></td><td></td></tr></tbody></table>

<figure><img src="image_7.png" /></figure>
<table><caption>**Table 1 (continued)**</caption><thead><tr><th rowspan="2">Xenobiotic Group</th><th rowspan="2">Class</th><th rowspan="2">Name</th><th colspan="2">Insect</th><th colspan="2">Impact on bacterial microbiota</th><th rowspan="2">Bacterial taxa affected</th><th rowspan="2">Impact on insect host</th><th rowspan="2">References</th></tr><tr><th>Common name</th><th>Species</th><th>General impact</th><th>Augmentation of</th></tr></thead><tbody><tr><td rowspan="5">Triazine</td><td>AMPA</td><td>Honeybee</td><td>Colorado potato beetle</td><td>Leptinotarsa decem-lineata</td><td>Microbiota composition modification (higher in adults than in larvae)</td><td>Agrobacterium and diminution of Enterobacteriaceae</td><td>UND</td><td>Gómez-Gallego et al. 2020</td><td></td></tr><tr><td>Atrazine</td><td>Wasp</td><td>Apis mellifera (adults)</td><td>Nasonia vitripennis</td><td>No impact</td><td>-</td><td>No impact</td><td>Blot et al. 2019</td><td></td></tr><tr><td rowspan="2">Quaternary ammonium</td><td>Paraquat</td><td>Mosquito</td><td>Drosophila melanogaster</td><td>Microbiota composition modification with increased abundance and diversity</td><td>Augmentation of Serratia marcescens and Pseudomonas protegens</td><td>More insecticide tolerance to herbicides</td><td>Wang et al. 2020</td><td></td></tr><tr><td></td><td>Aedes albopictus</td><td>Drosophila melanogaster</td><td>Microbiota composition modification</td><td>Augmentation of Lactobacillales / diminution of Acetobacter and Rhodospirillales</td><td>UND</td><td>Brown et al. 2021</td><td></td></tr><tr><td></td><td></td><td></td><td></td><td>No impact</td><td>-</td><td>No impact</td><td>Juma et al. 2020</td><td></td></tr><tr><td rowspan="5">Pharmaceutical compounds</td><td>Carboxamide and Quinone outside inhibitors</td><td>Pristine (Boscalid and pyraclos-trobin)</td><td>Honeybee</td><td>Apis mellifera</td><td>Microbiota composition modification</td><td>Augmentation of Lactobacillales / diminution of Acetobacter and Rhodospirillales</td><td>UND</td><td>Brown et al. 2021</td><td></td></tr><tr><td>Organochlorine</td><td>Chlorothalonil</td><td>Fly</td><td>Musca domestica</td><td>Diminution of Gilliamella at low doses / Augmentation of Lactobacillus at high doses</td><td>Reduced nutritional function</td><td>DeGrandi-Hoffman et al. 2016</td><td></td><td></td></tr><tr><td>Antibiotic</td><td>Levofloxacin / Gentamicin</td><td></td><td></td><td>Microbiota composition modification</td><td>Diminution of Lactobacillales</td><td>UND</td><td>Kakumanu et al. 2016</td><td></td></tr><tr><td></td><td>Cockroach</td><td></td><td>Blatella germanica</td><td>Microbiota composition modification and increased diversity</td><td>Augmentation of Providencia, Halomonas and Morganella</td><td>Lack of development</td><td>Li et al. 2021</td><td></td></tr><tr><td></td><td></td><td></td><td></td><td>Decreased of bacterial load and diversity, Microbiota composition modification after 14 days</td><td>Augmentation of Bacteroidetes and Fusobacteria</td><td>UND</td><td>Li et al. 2020</td><td></td></tr></tbody></table>

<figure><img src="image_8.png" /></figure>
<table><caption>Table 1 (continued)</caption><thead><tr><th rowspan="2">Xenobiotic Group</th><th rowspan="2">Class</th><th rowspan="2">Name</th><th colspan="2">Insect</th><th rowspan="2">Impact on bacterial microbiota General impact</th><th rowspan="2">Bacterial taxa affected</th><th rowspan="2">Impact on insect host</th><th rowspan="2">References</th></tr><tr><th>Honeybee</th><th>Common name</th><th>Species</th></tr></thead><tbody><tr><td></td><td>Tetracyclin</td><td></td><td>Apis mellifera</td><td></td><td>Decreased bacterial abundance including core microbiota species</td><td>Diminution of Snodgrassella abvi, Lactobacillus Firm-4 and 5 et Bifidobacterium / Augmentation of Serratia and Halomonadaceae</td><td>Higher mortality</td><td>Raymann et al. 2017</td></tr><tr><td></td><td>Fly</td><td></td><td>Delia racidum</td><td></td><td>Microbiota composition modification and decreased diversity</td><td>Augmentation of Enterobacteriaceae and Pseudomonaceae / diminution of Acinetobacter, Providencia, Comamonas</td><td>UND</td><td>Ourry et al. 2020</td></tr><tr><td></td><td>Dung beetle</td><td></td><td>Aphodius fossor</td><td></td><td>Microbiota composition modification</td><td>UND</td><td>UND</td><td>Hammer et al. 2016</td></tr><tr><td></td><td>Doxycyclin</td><td></td><td>Blatella germanica</td><td></td><td>Decreased diversity</td><td>Augmentation of Gammaproteobacteria</td><td>Lower insecticide tolerance</td><td>Pietri et al. 2018</td></tr><tr><td></td><td>Lincomycin</td><td>Fly</td><td>Hermetia illucens</td><td></td><td>Decreased bacterial load and increased diversity</td><td>Augmentation of Firmicutes</td><td>No impact</td><td>Luo et al. 2022</td></tr><tr><td></td><td>Penicillin, Streptomycin</td><td>Mosquito</td><td>Anopheles gambiae</td><td></td><td>Decreased diversity and reduced bacterial proliferation after blood meal</td><td>Augmentation of Asaia and diminution of Enterobacteriaceae</td><td>Increased survival and fecondity rates</td><td>Gendrin et al. 2015</td></tr><tr><td></td><td>Hormones</td><td>Estrones, Norethisterone, Estradiol</td><td>Culex quinquefasciatus</td><td></td><td>Increased diversity</td><td>UND</td><td>UND</td><td>Pennington et al. 2016</td></tr><tr><td></td><td>Prebiotic</td><td>Carrageenan Oligosaccharide</td><td>Drosophila melanogaster</td><td></td><td>Increased diversity</td><td>Augmentation of Commensalibacter</td><td>Increased lifespan and fecondity</td><td>Ma et al. 2021</td></tr></tbody></table>
<table><caption>**Table 1 (continued)**</caption><thead><tr><th>Xenobiotic</th><th>Group</th><th>Class</th><th>Insect<br>Common name</th><th>Species</th><th>Impact on bacterial<br>microbiota<br>General impact</th><th>Bacterial taxa<br>affected</th><th>Impact on insect<br>host</th><th>References</th></tr></thead><tbody><tr><td rowspan="2">Industrial<br>com-<br>pounds</td><td>Nanoparti-<br>cules</td><td>Titanium<br>dioxyde</td><td></td><td></td><td>Modification of<br>the abundance of<br>certain taxa without<br>modifying the<br>dominant taxa</td><td>Augmentation of<br>Lachnospiraceae,<br>Pseudomonas and<br>Sphingomonas /<br>Diminution of<br>Serratia</td><td>Increased weight<br>and number of<br>cocoons</td><td>M. Li et al. 2020</td></tr><tr><td>Silver</td><td></td><td></td><td></td><td>Increased abundance<br>and diversity at low<br>doses / Decreased<br>abundance and<br>diversity at high<br>doses</td><td>Augmentation of Fir-<br>micutes, Bacilli and<br>Enterococcus at low<br>doses but diminu-<br>tion at high doses</td><td>Increased weight<br>at low doses</td><td>Lin et al. 2020</td></tr><tr><td></td><td>Drosophila</td><td>Drosophila mela-<br>nogaster</td><td></td><td></td><td>Decreased diversity<br>and dominance of<br>Lactobacilli</td><td>Augmentation of<br>Lactobacillus brevis<br>and Gram positive<br>bacteria / Diminu-<br>tion of Gram nega-<br>tive bacteria</td><td>Reduced survival<br>ans develop-<br>ment</td><td>Han et al. 2014</td></tr></tbody></table>

<figure><img src="image_9.png" /></figure>
It points out the importance for future research prospect dedicated to mechanistic interactions between xenobiotic exposure, microbiota, and insect health.

Anthropogenic stressors as human activities become more and more prevalent across the globe. However, due to different factors, such as life histories, previous exposition, and additive effects, the insect stress response is extremely context-dependent and difficult to predict or generalize. In order to cope with these stressors, insects evolved adaptive strategies that partly depend on their microbiota.

# Microbiota-mediated physiological insect response to xenobiotics

## Direct microbiota-mediated detoxification pathways

Insects evolved adaptive strategies to minimize the effects of xenobiotics that occur at different levels: behavioral, cellular, and metabolic (Haubruge and Amichot 1998). Cellular and metabolic adaptations can result in (i) modification of the xenobiotic target, (ii) stimulation of the excretory activity, and (iii) sequestration of the compound (Clark et al. 1995; Davies et al. 2008). Detoxification-based resistance mechanisms, such as enzyme overproduction and modification, were fully described in different insect models (Li et al. 2007; Misra et al. 2011; Gao et al. 2022). Among them are isoenzymes called cytochromes P450 monooxygenases can oxidize exogenous molecules (Feyereisen 1999, 2006, 2015). Other enzymes such as glutathione S-transferases, UDP glucuronosyltransferases, carboxylesterases, and ABC transporters (ATP-binding cassette) are able to modify some functional groups of xenobiotics (Pan et al. 2019; Yang et al. 2020; Mao et al. 2021). Such modifications reduce the compounds toxicity or increase their solubility so they can get excreted or degraded (Fig. 1). However, the insect microbiota can also be influenced by exposure to anthropogenic stressors and enhance enzyme activity through a wide range of metabolic pathways able to break down and/or modify xenobiotics (Itoh et al. 2018; Gangola et al. 2022). The role of bacterial microbiota in the detoxification of xenobiotics in insects was first described in 1967, in the apple maggot *Rhagoletis pomonella* (Boush and Matsumura 1967). It was shown that the bacterial symbiont *Pseudomonas melophthora* degrades up to six different insecticides.

The microbial enzymatic repertoire in the insect gut is very diverse and implies many enzymatic functions among which are microbial hydrolytic enzymes such as glycosidases, proteases, and sulfatases (Banerjee et al. 2022). Bacterial communities associated with insects can be directly involved in detoxification processes through (i) secretion of enzymes that

enable direct xenobiotic metabolization (acetylcholinesterase, carboxylesterase, glutathione S-transferase, and cytochrome P450) in the insect gut, (ii) xenobiotic reduction, (iii) through addition of acetyl and methyl groups, (iv) xenobiotic sequestration in bacterial cells, (v) bacterial efflux pumps capable of moving toxic compounds out of cells, or (vi) the formation of a protective biofilm to prevent xenobiotic to access host cells (Fig. 2). Comparative genomics analysis allowed identifying detoxifying genes in some bacterial symbiont isolates even though the degradation routes have not been yet fully identified (Siddiqui et al. 2022). For instance, nitro-reduction and oxidation are the main ways that some bacterial symbionts such as *Arsenophonus*, *Pseudomonas*, *Ensifer*, *Stenotrophomonas*, and *Variovorax* use to degrade the insecticide imidacloprid (Lu et al. 2016; Fusetto et al. 2017). *Pseudomonas* spp. and *Ensifer adhaerens* are able to metabolize the thiamethoxam pesticide by changing its N-nitroimino group to N-nitrosimine/nitrosoguanidine and urea (Hussain et al. 2016). *Citrobacter* and *Burkholderia* possess respectively phosphatase genes as well as methyl parathion-degrading enzyme, and an organophosphorus hydrolase able to degrade the trichlorphon and fenitrothion insecticides in the oriental fruit fly *B. dorsalis* and stinkbugs (Tago et al. 2005, 2006; Singh 2009; Cheng et al. 2017). An isolate of the genus *Burkholderia* was showed to be able to use fenitrothion as the sole carbon source for its growth (Kikuchi et al. 2012). Interestingly, *B. dorsalis* inoculated with this symbiont showed higher trichlorphon resistance compared to antibiotic-treated flies (Cheng et al. 2017). Most studies that describe the involvement of bacterial symbionts in detoxication activity were not examined in *insecta*. As biotic (presence of other microorganisms, multipartite interactions) and abiotic (pH, presence of nutrients, oxygen concentration, ...) factors strongly influence the within environment of insects, the outcome of detoxication success may be different in comparison to studies performed in vitro on bacterial isolates cultivated on artificial media. The resistance of the fruit fly *D. melanogaster* to chlorpyrifos insecticide depends on the bacteria present in the fly. While *Lactobacillus plantarum* degrades this compound into a more toxic metabolite (oxon chlorpyrifos), *Lactobacillus rhamnosus* is capable of sequestering the compound thus allowing enhanced insect survival (Daisley et al. 2018). Atrazine-degrading *atzA*, *atzB*, and *atzC* genes were identified in the *Acinetobacter tropicalis* symbionts associated with atrazine-resistant *D. melanogaster* (Brown et al. 2021). Those genes have been shown to be widespread and associated with plasmids from multiple atrazine-degrading isolates (Martinez et al. 2001). The gut microbiota of the mealworm *T. molitor* contribute to accelerate polystyrene biodegradation, and potential strategies for cultivation of plastic-degrading bacteria are currently investigated (Yang et al. 2021; Pivato et al. 2022). Finally, it was suggested that the gut bacterial microbiota of black soldier fly larvae *Hermetia illucens*
would be involved in tetracycline degradation. Those insects were often in contact with such antibiotic when feeding on organic animal waste (manure, corpses). Different *tet* genes involved in antibiotic tolerance and/or effective degradation were identified in the insect microbiota (97% in 12 days). A role of Bacteroidetes was suspected since their relative abundance strongly increases in the presence of tetracycline (46.2% when tetracycline is present vs. 1% when absent) (Cai et al. 2018). However, their formal intervention in this detoxification pathway has not yet been demonstrated. More recently, it was reported that gut bacterial microbiota of the black soldier fly larvae contributes to lincomycin degradation after isolating lincomycin-degrading strains from larval guts and composts (Luo et al. 2022).

Anthropogenic stressors can impact insect hosts and their microbiota independently but insect-microbiota interactions can, in turn, expand or constrain insect adaptation to stressor exposure. As previously mentioned, some insects have acquired bacterial symbionts to overcome the deleterious effects of stressor exposure allowing insects to adapt in polluted environments (Lemoine et al. 2020). This was the case for *Burkholderia* spp and *Pseudomonas* spp found in *Riptortus pedestris* and

*Spodoptera frugiperda* respectively that confer insecticide resistance to their hosts (Kikuchi et al. 2012; de Almeida et al. 2017). In most insect models for which bacterial detoxification were observed, the route of detoxifying bacteria acquisition still needs to be determined. Those can be seeded from the environment (mostly through nutrition) and/or transmitted among individuals through horizontal or vertical transfer.

Surprisingly, few results support the role of microorganisms in the shielding or efflux of anthropogenic stressors. Further studies are still needed to provide empirical evidence of microbial-assisted degradation of xenobiotic compounds and decipher the underlying mechanisms. Microbial enzymes sequence and activity differ from one species to another, and inter-individual variations occur in the composition and structure of insect-associated microbiota. Therefore, the diversity of chemical reactions catalyzed by the insect microbiota is difficult to predict. The high diversity of insect bacterial microbiota and intricate interactions among microbial communities suggest that this xenobiotic-degrading potential is currently underestimated and merit further investigation. Moreover, detoxication enzymes are often encoded in plasmids that can be horizontally transferred from species to species.

<figure><img src="image_10.png" /><figcaption>**Fig. 1** Main families of detoxication enzymes in insects. The xenobiotic metabolizing enzymes have a primary role in the metabolism and elimination of xenobiotics. They include (i) cytochromes P450 and carboxylesterase that facilitate the solubilization of the xenobiotic in water and its storage in less sensitive tissues or its excretion and (ii) glutathione S-transferases and UDP-glucuronosyltransferases that add hydrophilic groups to the xenobiotic allowing better mobilization and then its excretion. Once the xenobiotic has been modified by one of the enzymes, it can be degraded or excreted via specific transporters or ABC-type transporters. Created by BioRender.com</figcaption></figure>

<figure><img src="image_11.png" /></figure>
<figure><img src="image_12.png" /><figcaption>**Fig. 2** Direct and indirect insect microbiota-mediated detoxification pathways. The xenobiotic is acquired by ingestion of food. Once in the insect body, the xenobiotic interacts with the microbiota through different pathways. A Direct pathways would be involved: (i) biofilm growing on insect cells form a shield that blocks access of the xenobiotic to host cells and prevents contact with the host, (ii) the xenobiotic is sequestered in bacterial cells or in the biofilm matrix preventing its access to the host cells and then excreted via faeces, (iii) the xenobiotic enters the bacterial cells and then is released downstream into the gut via bacterial efflux systems (e.g., efflux pumps), (iv) bacteria in</figcaption></figure>

contact with the xenobiotic produce enzymes capable of modifying or degrading the xenobiotic, limiting its toxicity and facilitating its excretion. B Indirect pathways involve the activation of insect microbiota-mediated-host detoxification pathways like the enzymes P450s, Glutathion-S-Transferase (GST), or Esterase (EST). In addition, the microbiota can also induce the activation of the host immune system (IS). This stimulation could amplify detoxification process through pleiotropic effects of immunity genes or interactions between immunity and detoxification pathways. Created by BioRender.com

## Indirect microbiota-mediated detoxification pathways

Indirect microbiota-mediated xenobiotic detoxification relies on regulation of host defenses. This means that bacteria alone are not involved in xenobiotic resistance and detoxification but are rather able to promote the host response following stressor exposure. Honeybees treated with antibiotics are less able to degrade thiaclopride and tau-fluvalinate insecticides, which results in a lower survival rate, compared to untreated individuals (Wu et al. 2020). This phenotype is explained by increased expression of insect detoxification genes encoding in particular cytochromes P450 in the midgut colonized by bacteria. This interaction between the microbiota and the insect detoxification enzymes is supported by the fact that individuals harboring a microbiota and treated with a P450 enzyme inhibitor were unable to eliminate insecticides. Similarly, in the German cockroach,

*Blatella germanica*, resident bacterial microbiota increases resistance to indoxacarb, an insecticide from the carbamate family. It was suggested that bacteria would stimulate the Glutathione-S-Transferase (GST) detoxification enzyme of the host (Pietri et al. 2018). Similar microbiome-related enhancement of the GST activity was reported in multiple insect species such as in the mosquito *A. stephensis*, the leafhopper *Amplicephalus curtulus* and the honeybee *A. mellifera* (Arismendi et al. 2015; Soltani et al. 2017; Yu et al. 2021). Finally, other studies highlighted that bacterial microbiota triggers out the overexpression of esterases and catalases detoxification genes when *A. curtulus* and *A. stephensis* individuals were exposed to xenobiotics (Arismendi et al. 2015; Soltani et al. 2017).

Gut bacteria play an important role in insecticide resistance of the diamondback moth *Plutella xylostella* (Xia et al. 2018). Although *Enterococcus* sp. isolated from the insect gut was able to degrade chlorphyrifos in vitro, its
enhancement of insect resistance toward the insecticide was not associated to degradative enzymes but rather to vitamin C and acetylsalicylic acid produced by the bacteria. Indeed, other bacteria with similar in vitro ability to degrade the compounds did not increase the insect resistance. Vitamin C and acetylsalicylic acid produced by *Enterococcus* sp. were correlated with a specific modulation of insect antimicrobial peptides. It was therefore suggested that the influence of gut bacteria on insecticide resistance could depend on effects on the insect immune system in *P. xylostella* (Xia et al. 2018). These results suggest an indirect process mediated by the microbiota on the immune system for xenobiotic resistance. Interestingly, metatranscriptome sequencing in cockroaches revealed that a large part of downregulated differentially expressed genes were from microbial and viral origins, suggesting that selection for resistance could also be associated with elimination of microorganisms (Scharf et al. 2022). Similar results were observed regarding immune and detoxification of insect-associated microbiota in honeybee larvae (Yu et al. 2021). It was shown that bacterial microbiota served as a barrier in the insect gut and reduce the stress of flumethrin on honeybee larvae. Interestingly, as the concentration of flumethrin increases, larvae stimulate their immune system then detoxification system to protect against the potential detrimental effect of flumethrin. This could be explained either by (i) pleiotropic effects of insect immunity and insecticide resistance or (ii) the effects of some genes that participate in immunity that may be exploited in insecticide resistance (Xia et al. 2018). A crosstalk between microbiota and insect host genes can result from a shared stress response mechanisms that involve both xenobiotic resistance and immunity. Although the direct and indirect pathways involved in the microbiota-mediated xenobiotics degradation are described (Fig. 2), the molecular factors and pathways that lead to the expression and activity regulation of hosts detoxification genes are still unknown. Further studies are needed to investigate the insect-microbiome metabolism relationship and whether insect-associated microbiota metabolic interactions contribute to detoxication processes. Such advances will be essential and dependent upon studies using metabolomics to identify and differentiate metabolites from microbial and host origins.

## Conclusion

Numerous studies testify to a deleterious effect of xenobiotics in insects, and some of them testify the strong influence of the microbiota in insect response. Most of the studies are focused on bacteria while the microbiota is more complex and harbors a diversity of organisms (protists, viruses, archaea, eukaryotes) that should be included in future

investigations. Different types of responses were observed depending on the insect species, the bacterial microbiota composition, the xenobiotic, and the dose exposure that may lead to an alteration of key biological functions in insects. In addition, most of the studies are biased toward economic or health importance insect models (pollinators, vectors of pathogens, crop pests...) and most widely used xenobiotics (insecticides, herbicides, antibiotics...), which only partially reflects the complexity of these interactions. To date, other xenobiotics (e.g., anti-inflammatories such as ibuprofen) have not yet investigated. Given the limited literature on this topic and the complexity of those interactions, their effects on ecosystems properties and functioning remain difficult to predict.

**Author contribution** P.A. wrote the first version of the manuscript during his Master thesis under the supervision of C.V.M. and G.M. P.L. and P.D. edited and improved the manuscript.

**Funding** Author P.D. has received research support from Ecole Urbaine de Lyon. This review was performed within the framework of the CNRS MITI program (grant name: MosquiAdapt), the CNRS-EC2CO program (grant name: FAMOUS) and the Agence Nationale de la Recherche (grant name: SERIOUS).

**Data availability and materials** Not applicable.

## Declarations

**Ethics approval and consent to participate** Not applicable.

**Consent for publication** Not applicable.

**Competing interests** The authors declare no competing interests.

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