# Crosstalk between the microbiota and insect postembryonic development

Maxime Girard, Claire Valiente Moro, Patricia Luis, Guillaume Minard

► To cite this version:

Maxime Girard, Claire Valiente Moro, Patricia Luis, Guillaume Minard. Crosstalk between the microbiota and insect postembryonic development. Trends in Microbiology, 2023, 31 (2), pp.181-196. 10.1016/j.tim.2022.08.013. hal-03811799

HAL Id: hal-03811799

https://hal.inrae.fr/hal-03811799v1

Submitted on 31 Mar 2025

HAL is a multi-disciplinary open access
archive for the deposit and dissemination of sci-
entific research documents, whether they are pub-
lished or not. The documents may come from
teaching and research institutions in France or
abroad, or from public or private research centers.

L'archive ouverte pluridisciplinaire HAL, est
destinée au dépôt et à la diffusion de documents
scientifiques de niveau recherche, publiés ou non,
émanant des établissements d'enseignement et de
recherche français ou étrangers, des laboratoires
publics ou privés.

<figure><img src="image_1.png" /></figure>

Distributed under a Creative Commons Attribution 4.0 International License
1

Crosstalk between the microbiota and insect post-embryonic development

2

3

Authors:

4

Maxime Girard¹, Patricia Luis¹, Claire Valiente Moro¹ and Guillaume Minard¹

5

6

¹Univ Lyon, Université Claude Bernard Lyon 1, CNRS, INRAE, VetAgro Sup, UMR Ecologie

7

Microbienne, F-69622, Villeurbanne, France.

8

9

Corresponding Author:

10

Guillaume MINARD,

11

Laboratoire d'Ecologie Microbienne,

12

8 rue Raphael Dubois,

13

69622 Villeurbanne Cedex.

14

15

#The authors contributed equally

16

17

18

19

20

21

22

23

24

25

26

27

28

29

30

31

32

33

34
**Abstract:**

Insect sequential development evolves from a simple molt towards complete metamorphosis. Like any multicellular host, insects interact with a complex microbiota. In this review, factors driving the microbiota dynamics were pointed out along their development. Special focus was put on tissue renewal, shift in insect ecology and microbial interactions. Conversely, how the microbiota modulates its host development through nutrient acquisition, hormonal control and cellular or tissue differentiation were exemplified. Such modifications might have long-term carryover effects on insect physiology. Finally, remarkable microbe-driven control of insect behaviors along their life cycle was highlighted. Increasing knowledge on those interactions might offer new insights on how insects respond to their environment as well as perspectives on pest or vector control strategies.

**Keywords:** crosstalk, microbiota, post-embryonic, development
69

## Reciprocal interactions between insect development and microbiota

70

71

72

73

74

75

76

77

78

79

80

81

82

83

84

85

86

87

88

89

90

91

92

93

94

95

96

97

98

99

100

101

Insects are ubiquitous and represent more than 70% of the overall animal species [1]. They play a pivotal role for the functioning of ecosystem, such as seed dispersal, pollen transportation, organic matter recycling or soil fertilization [2]. At the same time, some species are also considered as major pests or pathogen vectors for plants and animals [3]. Insects are diverse in size, form, behavior and life cycle allowing them to successfully colonize a wide variety of ecosystems [4]. The insect post-embryonic development begins after the egg is hatched and lasts until adult (or *imago*) emergence. It is characterized by successive **ecdysis** (see Glossary) or molt that enables them to grow, develop new organs and renew their exoskeleton [5]. Transition from a juvenile stage to a differentiated adult generally involves major morphological, physiological and ecological changes. This entire process is termed as **metamorphosis** (see Glossary). Insects are thus classified within distinct developmental categories depending on their ability to accomplish complete metamorphosis (Box 1) [6]. It has become increasingly clear that complex microbial communities (microbiota) are influencing many functions of their host including development [7] and more specifically for insects [8]. Interactions with insect hosts might drastically vary depending on whether the output is positive, negative or neutral for each of the partners involved. They are commonly classified along a gradient ranging from mutualism (beneficial interaction for both partners) to competition (detrimental interaction for both partners). However, things are never black or white and the nature of such interactions vary drastically according to the ecological context. Microorganisms can efficiently colonize various tissues of their insect hosts such as the cuticle, hemolymph, and gut epithelium [9]. Intimate interactions lead to the evolution of insect cells (e.g. bacteriocytes, mycetocytes) and tissues (e.g. mycangium, crypts) dedicated to symbiosis [10]. On an average, the digestive tract carries a higher **microbial diversity** and density as compared with other organs because of its regular food provision which is seeded by the microbes [11]. The environment is essential for continuous supply of microorganisms and shaping insect-microbe as well as microbe-microbe interactions [12,13] but inter-individual transmissions could also occur, either between individuals from the same generation (horizontal transmission) or across generations (vertical transmission). Host-microbiota interactions might thus be considered as important drivers of insect development.

This review emphasized current knowledge on the host-microbiota interactions during post-embryonic development of insects. First, the factors shaping microbiota dynamics across insect development were focused. Then, the main cellular and physiological roles of microbiota on
insect development as well as their carryover effects on the adult stage, in addition to the modifications induced on the post-embryonic insect behavior were elaborated.

# 1. Factors influencing microbiota dynamics across insect development:

## 1.1. Tissue restructuring reshapes microbiota niches:

Insect development, including the size and shape of insect is gradually transformed to achieve the adult form. Most insect organs also display substantial modifications to follow the rhythm imposed by endocrinal regulation [14]. Therefore, niches where microorganisms live evolve with development. The amplitude of this restructuring is influenced by the developmental strategy which ranges from limited morphological changes in ametaboles to a spectacular remodeling of the individuals in holometaboles [5]. However, to date proper studies depicting the influence of ametaboles molting on their associated microbial communities are lacking. Some studies on microbial community changes across development in Hemimetabola and Holometabola are provided in the **Table 1**. In general, intracellular symbionts remain more stable along the development and experience, and have less turnover than extracellular symbionts that live at the surface of tissues, that are delaminated during ecdysis [8].

The gut has been the most studied tissue to investigate microbiota-insect interaction [11]. Its structure and micro-environmental conditions (pH, enzymes, immunity, etc.) change a lot during the development with a gradient ranged from juvenile molts to incomplete and then complete metamorphosis (see Box 1). Elimination of the gut epithelium (and eventually the peritrophic matrix) in combination with changes in physiochemical conditions, enzymes and a specific immune response drastically alter the microbial communities living in the gut lumen or within the epithelial cells [11,36]. In a few examples in holometabolous insects, e.g. in moth and mosquitoes, a partial sterilization of this organ during metamorphosis is evident [29,36,37]. However, studies on mosquitoes showed that specific microorganisms acquired at the larval stage could be maintained during the whole insect life [32], but this apparent stability might also be the result of extinction-recolonization events [38]. In ants of the genera *Camponotus* and *Colobopsis*, the gut microbiota has been shown to remain stable all along development [20]. Consequently, drastic gut restructuring that occur through insect development can directly shatter microbiota niches but does not necessarily get rid of the microbiota. This could partially be explained by the simple fact that many insects are interacting with mutualistic
135
136
137
138

microorganisms. Loosing those symbionts might expose the insect to fitness decrease. As an example, removal of the extracellular symbiont *Enterococcus mundtii* in the greater wax moth *Galleria melonella* during pupation favors the invasion of the host by opportunistic pathogens [39].

139
140
141
142
143
144
145
146
147
148
149
150
151
152
153
154
155
156
157
158
159
160
161
162
163
164
165
166
167

Endosymbiotic microorganisms live within cell hosts and are expected to be more dependent on host tissue maintenance. However, studies showed that cell/tissue tropism of those symbionts might vary according to the insect cellular and morphological restructuring. For instance, *Wolbachia* is a genus of intracellular parasitic bacteria that affects arthropods' reproduction [40]. These bacteria are associated with reproductive organs of many insect species and benefit from maternal inheritance [41]. *Wolbachia* is specifically addressed to ovary and have a specific tropism for stem cells that will then give rise to germlines [42]. They have also been detected in many different insect tissues such as brain, salivary glands, fat body and a few others [43]. The spread of *Wolbachia* from reproductive organs to the other tissues takes place all along development and their density amongst insect tissues has been shown to gradually increase in some insect species such as the Asian citrus psyllid *Diaphorina citri* [44]. The process enabling *Wolbachia* to spread is not yet fully explained, although the most promising hypothesis is that the hemocytes help them to invade the insects, particularly during the metamorphosis of holometabola [45]. Another instance of tropism modification during insect development could be observed with the endosymbiotic bacteria *Arsenophonus nasoniae* in the parasitoid wasp *Nasonia vitripennis*. The bacteria have been proved to move from mouth, legs and abdomen during its larval stage to the ovipositor organ at the pupal stage [46]. This translocation finally enables the bacteria to be vertically transmitted in higher density to the offspring. Mechanisms involved in this transmission process are still unknown but it has been suggested that such tropism might be facilitated by the bacteria preference for growing on developing chitinous structures that are predominant in the ovipositor due to many chitin binding protein identified in its genome [47]. **Insect-endosymbiont** interactions across development has even sometimes lead to the appearance of dedicated organs. In the tortoise beetles of the Cassidinae family, symbiotic organs are connected with gut and ovaries [48]. Interestingly, those dedicated structures were developed in adults independently from their infection status suggesting that evolution has driven the host developmental process towards the acceptance of the endosymbiotic bacteria. In the rice weevil *Sitophilus oryzae*, modifications occurring during metamorphosis involves drastic remodeling of the dedicated symbiotic organ (bacteriome) [49]. During metamorphosis from larvae to pupae, bacteriomes
168

migrate from the foregut to the midgut. This translocation is also associated with changes in
bacterial transcriptomes that become more virulent and invade host stem cells during pupation
[49]. Consequently, endosymbiont colonization patterns are closely related to the
metamorphosis process of their host. Studies on endosymbionts of ametabolous and
hemitabolous insects are still lacking and could help to decipher the contribution of
endosymbiosis in all insect developmental processes.

169

170

171

172

173

174

175

## 1.2. Microbe-microbe interactions during the insect development

176

Temporal and spatial dynamics of the insect microbiota during development can also be
dependent on complex microbe-microbe interactions in their host. This could be mediated
through beneficial interactions. *Lactobacillus plantarum* is one of the most studied bacterial
symbiont associated with *Drosophila melanogaster* (Diptera) due to its beneficial role on host
nutrition during development [50]. The bacteria *L. plantarum* promotes growth by sustaining
its host gut peptidase activity, which facilitates proteolysis of food and provides optimal amino
acid levels. However, this mutualistic interaction could be ruptured by co-infection with the
fruit fly pathogen *Pectobacterium carotovorum* [51]. Mechanisms associated with this
antagonistic activity have not yet been elucidated but a cellular crosstalk between nutritional
and immune pathways have been hypothesized. On the contrary, *L. platarum* has also been
shown to cooperate with *Acetobacter pomorum* in a tripartite mutualistic relationship that
provides essential amino acids and vitamins to their host in poor nutritional environment [52].
Therefore, microbe-microbe interactions could also be important drivers in the regulation of
host development.

177

178

179

180

181

182

183

184

185

186

187

188

189

190

On contrary, antagonistic interactions between microbes might also occur during the
insect development. In the cockroach *Shelfordella lateralis*, the microbiota colonizes larvae
through both vertical transmission and environmental acquisition [53]. Vertical transmission is
ensured by egg smearing (i.e. females cover the egg surface with microbial cells). This
transmission leads to a stable microbiota among the adults through priority effect. In other
terms, the first microbial cells that colonize the insect gut (here through vertical transmission)
limit the gut colonization by other communities later. It has also been shown that multiple stable
microbial communities could be reached depending on the extinction-colonization events
occurring in the microbiota during the insect development [53]. The lepidopteran symbiont
*Enterococcus munditii*, is also responsible for biocontrol of the insect microbiota [54]. The

191

192

193

194

195

196

197

198

199
200
201

symbiont produces a toxin named mundticin that forms a chemical barrier that limits the colonization probability and growth of opportunistic bacteria.

202
203
204
205
206
207
208
209

The examples on microbe-microbe interactions during the development have been evidenced in a limited number of systems, further high throughput interaction dynamic essays with sequential supplementation of microorganisms to axenic insects and/or interaction network modeling would probably help the researchers to better disentangle those complex relationships. Indeed, interaction networks are highly variable and further knowledge is needed to predict the factors influencing the community patterns in adult stage insects, as it was recently highlighted in the black soldier fly *Hermetia illucens* [55].

210
211

**1.3. Influence of insect ecology on the assembly and stability of insect associated microbiota across development**

212
213
214
215
216
217
218
219
220
221
222
223
224
225
226
227
228
229
230
231

Many Hemimetabola *sensus stricto* and Holometabola exploit different diets and habitats between juvenile and adult life stages. Such ecological shifts could trigger consequences for insect microbiota structure and/or density [56]. For instance, the transition from aquatic larval stage to terrestrial adult in dragonflies has been associated with a reduction in bacterial **richness** over their development since dragonflies are insectivorous and might encounter different preys depending on their habitat [57,58]. Indeed, changes in diet can lead to differential selection and colonization events of microorganisms in the gut. Similar observations were performed in the firebugs *Pyrrhocoris apterus* for which community composition was correlated with changes in trophic behavior along the development [18]. However, despite the diet, many other abiotic and biotic components of the insect habitat might lead to differences in the probability of acquisition and selection of microbes. For instance, the green stinkbug *Nezara viridula* (Hemiptera) has been shown to lose its main bacterial symbionts in response to air temperature elevation (2.5°C above normal rearing conditions) [59]. Another example is the microbiota composition of the arbovirus vector *Culicoides imicola* that is correlated with soil moisture during larval stages, and host preferences during the adult blood meals [60]. Finally, human-induced selection through rearing has been associated with an increase in bacterial diversity in domesticated skilkworm *Bombyx mori* (Lepidoptera) as compared to the environmental populations of *Acronita major* and *Diaphania pyloalis* [61]. These examples revealed a major role of environmental conditions, diets and human manipulations on insect microbiota.
232

Some insects help microorganisms' horizontal transmission across different life stages.

233

In such cases, social interactions are an important driver of the microbial community acquisition and assembly during the insect life. Adults of the burying beetle *Nicrophorus vespilloides* use oral and anal secretions to support larval development by inoculating nutritive enzymes, microbial mutualists and antimicrobial compounds against microbial competitors in larval foods [62]. Therefore, the insect microbiota could be first transmitted through parental care before varying through different developmental stages due to individual ecological preference.

234

235

236

237

238

239

Many examples have shown that insect microbiota composition varies during the development due to multiple factors (Figure 1), some members of the microbiota could in turn influence their hosts' life cycle.

240

241

242

243

## 2. Microbiota-based strategies for the control of insect development:

244

### 2.1. Microbiota interaction with host's physiology and immunity:

245

A hormonal cascade governs the insect development. In some **instars** (e.g. larval instars in holometabola), this cascade responds to nutrition and tissue development of the insect (Box 2). In various systems, the molecular crosstalk between these signals and the microbiota contributes to modulate the insect growth. The bean bug, *Riptortus pedestris* is a hemimetabolous *sensus lato* insect in which the midgut forms invaginations called crypts, harboring symbiotic bacteria of the genus *Burkholderia* [63]. Those symbionts are acquired during the second larval instars and stimulate the host to produce both Juvenile Hormone-III (JH-III) and hexamerin, a protein used to store nitrogen when nutrient deficiency occurs [64].

246

247

248

249

250

251

252

253

Consequently, the time spent to reach the adult stage is ~37% shorter in presence of symbionts. It was also recently shown that *Burkholderia* symbionts are involved in waste recycling and compensate for certain nutritional deficiencies in essential amino acids and B vitamins [65].

254

255

256

Altogether, these nutritional interactions could be involved in the acceleration of insect development. Microbiota also induces the development of *Drosophila* through nutritional compensation and endocrine modulation [66]. A total of 22 essential nutrients are required for the development of **axenic** individuals from larvae to adults. Larvae mono-associated with bacteria especially *A. pomorum* and *L. plantarum* (i.e. often colonize individuals under laboratory conditions) develop faster on deficient media as compared to axenic relatives. In particular, *A. pomorum* produces all the essential amino acids as well as certain vitamins such as thiamine and riboflavin. On top of that they compensate for deficiency of nutrients by substituting them with analogous compounds (e.g. Choline). Both *A. pomorum* and *L. plantarum* also interact with genes involved in the endocrine system. The first one produces an

257

258

259

260

261

262

263

264

265
oxidative enzyme (pyrroloquinoline quinone-dependent alcohol dehydrogenase) which regulates the insulin/insulin-like growth factors and triggers, in turn, the insect development [67]. The latter increases the expressions of a gene involved in metamorphosis induced by ecdysone (E74B) and an insulin receptor (lnR) [68]. However, such in-depth disentangling of molecular interactions should involve *A. thailandicus* in the near future, since recent studies pointed out strong relevance of this symbiont with natural populations of *D. melanogaster* [50]. Likewise, mosquitoes rely on non-specific interactions with living facultative aerobic-anaerobic microorganisms (bacteria, algae or yeast) acquired from water of the larval breeding site to start their larval development [32,38,69-72]. It was later demonstrated that riboflavin (B2 vitamin) is an essential micronutrient provided by those microorganisms which leads to the activation of enzymes involved in energetic metabolism and gut hypoxia [73]. When hypoxia occurs, a transcription factor (HIF-α), which is linked to the regulation of various key stages of molting (gut growth, ecdysone synthesis, neutral lipids storage) is stabilized [74].

Influence of microbiota on nutritional and hormonal response of insects during the development was also reported in bees. The microbiota of *Apis mellifera* plays important role in the development of young adults right after metamorphosis from pupa to adult [75]. The daily accumulation of biomass is 82% lower in young axenic adults obtained from axenic pupa as compared to conventional ones that recolonized at the time of emergence. Axenic individuals suffer from repression of insulin-like-peptide genes (i.e. *ilp1* and *ilp2*). These peptides are produced by neurosecretory cells in response to diet and promote the systemic development of individuals [76]. Other authors showed that JH-III hormone and its derivatives were enhanced by the bee microbiota and differed among mono-associated young bees, supporting, here again, a microbiota mediated regulation of the hormonal growth signal [77]. Another study highlighted the role of *Zygosaccharomyces* fungus in the development of the Brazilian bee *Scaptotrigona depilis* [78]. When it is absent, only 8% of the larvae reach the pupal stage, while 71% of them reach this stage when it is present. Supplementation of food with ergosterol compensates the absence of fungi. Bees are unable to synthesize sterols, which serve as precursors for ecdysteroids, hence, ergosterol produced by fungi provides the precursor for ecdysteroids.

Insects rely on innate immunity and lack memory cells but levels of immune gene expression could be up-regulated by microorganisms to respond faster through a memory-like mechanism called immune priming [79]. As an example, axenic larvae of the red pal weevil obtained from sterilized eggs have shown decreased expression of 35 immune genes as compared to relatives carrying an unaltered microbiota [80]. This was a burden for the ability of 4th instar larvae to clear the hemolymph from infections and survive to sepsis.
In some cases, the role of insect-associated microorganisms is intertwined among immune, nutritional and endocrine systems. Acetic acids produced by bacteria associated with *D. melanogaster* interact with the immune system of the fly (i.e. they trigger the Imd pathway) and this molecular cascade increases the transcription of genes encoding Tachykinin. This endocrine peptide positively regulates the insulin signaling and leads to optimal lipid metabolism and development [81]. This study showed that microbiota maintains both immunity and metabolism homeostasis across the insect development using strongly interconnected pathways. Such interactions might be more common than expected since acetic acid and other short-chain fatty acids are produced by many host-associated microorganisms when fermenting dietary fiber, proteins and peptides [82]. By contrast, microbiota could sometimes be considered as a burden for insect growth as evidenced for the leaf beetle *Plagiodera versicolora* [83]. The body size was significantly higher in axenic individuals than in conventional ones, and transcriptomic analysis revealed that genes involved in carbohydrate metabolism were overexpressed and immunity related genes were modulated. The authors speculated that the microbiota could compete with the energy requirement of the insect and triggers immune responses at a cost. For instance, parasitic wasps use microbe-associated adverse functions to control their insect-host development. Polydnavirus are a family of enveloped DNA viruses associated with parasitoid hymenoptera. Viral genes of the *ank* family alter endocytic trafficking pathway in prothoracic glands of parasitized insects [84]. In *D. melanogaster* (a host of parasitoids), *ank* genes induce a developmental arrest followed by premature death of third instar larvae, which could only be restored by an injection of ecdysone. In another host-polydnavirus-parasitoid system, Wang *et al.*, evidenced that virus-mediated down regulation of ecdysone uses the host RNA interference pathways [85]. Such pathways are normally used by the insect for gene regulation and as a immune response to viral infections [86]. After parasitoid egg hatching, specialized cells produce miRNA released by the parasitoid that leads to gene silencing of the host ecdysone receptor.

Overall, the multiple examples reported in this section showed that focus on the insect microbiota might help to understand the complex molecular process that leads to full development of individuals (Figure 2).

329

## 2.2. Microbiota contribution to cell and tissue development

Involvement of the microbiota in digestive tract development has also been reported in cockroach *P. americana* [87]. Morphometric analysis showed that axenic cockroaches suffered from gut atrophy, which was partially restored when their diet was supplemented with

300

301

302

303

304

305

306

307

308

309

310

311

312

313

314

315

316

317

318

319

320

321

322

323

324

325

326

327

328

329

330
microorganisms. Posterior midgut and anterior hindgut were intensively impacted. Microorganisms specifically colonizing those regions could be responsible for a healthy gut development in cockroaches. Similar gut atrophy was observed in bees when adults were obtained from germ free pupae [88]. During the development of the rice weevil *Sitophilus oryzae*, a synchronized molecular and cellular “choreography” occurs with its bacterial endosymbiont *Sodalis pierantonius* [89]. The symbiont colonizes stem cells at the edge of pupae midgut. Once bacteria are internalized, they induce cell differentiation into bacteriocytes, allowing the formation of the bacteriome, an organ dedicated to symbiosis. Similarly *Burkholderia insecticola* colonizes *Riptotrus pedestris* during the 3rd instar larvae and then induces midgut closure during the 4th larval instar [90]. In *D. melanogaster*, the bacteria *L. plantarum* stimulates the production of reactive oxygen species (ROS) within larval enterocytes and increases the number of larval stem cells that lead to the formation of the midgut in adults [91]. These stem cells are protected from ROS but their proliferation responds to a ROS-dependent signal transduced by enterocytes. Altogether, these results support a host cellular reprogramming by bacterial endosymbionts during insect development.

The gut is by far the most studied tissue, whereas other striking examples have also been reported. The turtle ant larvae suffer from nitrogen deprivation in their conventional food. However, their bacterial microbiota recycle nitrogen wastes and enable the correct formation of the cuticle including its macromolecular structure, impermeability, thickness and mechanical properties by participating to the production of Tyrosine and Phenylalanine amino acids, the formation of chitin, cuticular proteins and catecholamine cross-linkers [92]. Another study suggested that bacteria involved in the ant cuticle formation process are mostly environmentally acquired and poorly specific [93]. Symbionts could also contribute to the development of insect immune cells. *Wigglesworthia* a maternally acquired primary endosymbiont of the tsetse fly trigger odorant binding protein 6 [94,95], and has shown to control the hematopoiesis of its host through generation of a specific type of hemocyte. These cells are found in hemolymph (i.e. a fluid that is equivalent of blood for insects) and contribute in fighting infections as well as protecting insects from fluid loss at wound sites. The authors discovered an orthologous process in the fruit fly *D. melanogaster* except that it was triggered by environmentally acquired symbionts.

## 2.3. Carryover effects of the early microbiota on adult traits

Conditions experienced during the early life of insects might affect their traits along other life stages and across different habitats [96]. Implication of the carryover effects of the
368

369

370

371

372

373

374

375

376

377

378

379

380

381

382

383

384

385

386

387

388

389

390

391

392

393

394

395

396

397

398

Fluctuation in microbiota composition during the development of insects that experience limited changes in habitat and morphology (paurometabola) tend to be weaker than in insects experiencing more changes (hemimetabola s.s. and holometabola), although with

399

400

401

microbiota have previously been specifically studied for insect vectors due to their consequences of pathogen transmission. *Aedes* mosquito larvae mainly acquire their bacteria from water habitats. Larvae that were mono or co-associated with different bacteria during their larval stage develop into adults with contrasted lipid metabolism, starvation resistance, immunity and ability to efficiently replicate and transmit dengue [97,98]. Similarly, Tsetse fly larvae conduct an intrauterine development during which they acquire their primary bacterial endosymbiont *Wigglesworthia glossinida* [99]. Growing larvae that is deprived from the symbiont leads to the production of adults with a strong permissiveness towards trypanosome parasites. This was inferred to the dislocation of peritrophic membrane (that forms a physical gut barrier) and down regulation of insect immunity.

## 2.4. Microbiota influence on insect behaviors

Certain insects that depend on vertical transmission of mutualistic symbionts exhibit adaptation towards a behavior that maximizes the symbiont transmission during immature life stages. This was deeply investigated on stinkbugs (Pentatomidae). In the pataplastid stinkbug *Megacopta punctatissima* the bacterial symbiont *Ishikawella capsulata* is transmitted by mothers to their progeny when the mother lay eggs [100,101]. Right after hatching, nymphs were detected and the capsules were probed that contained symbiont cells to be seeded by their symbionts. Individuals that fail to find their symbiotic capsules stay as wandering nymphs. Interestingly, pentatomid stinkbugs *Sibaria englemani* experience a modification of nymph behavior linked to symbiont vertical transmission [102]. In this case, symbionts are transmitted by the mother at the egg surface, when newly emerging first instar nymphs hatch from eggs, they tend to probe the chorion to ensure a sufficient oral acquisition of the symbiotic cells.

Finally, the presence/absence of microbiota in insects might lead to nutritional behavior modifications that compensate the lack of nutrient provision from microbes. When the Queensland fruit fly *Bactrocera tryoni* was reared under axenic conditions, larvae spent quite amount of time foraging for food [103]. Foraging times were lower in presence of a vertically acquired microbiota and intermediated on the sole presence of an environmentally acquired microbiota. Composition of the diet also induced variations in the foraging time.

## Concluding Remarks and Future Perspectives:
some exception for insects that go through strong transgenerational exchange of microbiota (e.g. through social interactions and parental caring). A comparative study showed that the microbiota of adults and juveniles are more divergent in holometabola than in hemimetabola insects [104]. This suggests that insect metamorphosis enhances microbiota renewal. Acquisition of metamorphosis is often considered as an advantageous adaptive trait [8], but the role of metamorphosis in microbiota assembly and protection against pathogens should probably be better regarded. Although major changes occurring throughout the host life cycle do influence microbial communities, these could in turn control host development. Several studies tend to show that many nutritional symbioses also indirectly modulate hormones and insect development. The most striking characteristic of studies is the lack of investigations on Ametabola. More information is needed to better estimate the extent in which insect developmental strategy influences the microbial community transitions along the insect life and development control. As it was already pointed out in other reviews [105], fluxomic analysis (i.e. the rate of metabolic reactions in an organism) are currently very limited to emphasize the knowledge of insect-microbiota interactions. Although recent studies have begun to address insect metabolomics profiles [106,107], only a few have investigated insect-microbiota relationship [108]. It was believed that both transcriptomic and metabolomics analyses would help elucidating mechanisms underlying these interactions.

From an applied perspective, knowledge on the insect-microbiota interactions during post-embryonic development could improve pest management and vector control strategies. Use of microorganisms or their derivatives could be used for interfering with the insect hormonal signal, nutrition or immunity at different targeted life stages. Their carryover effects might also help to limit the adverse effects linked to adult life stages in some species. Finally, the identification of symbionts that efficiently colonize the relevant targeted life stages or insect tissues might help the application of paratransgenesis [109]. To that end, better knowledge on the molecular and cellular mechanisms governing host-microbiota relationships during insect development as well as their eco-evolutionary consequences are needed. Development of axenic insect models in each developmental strategy would probably help addressing those specific points.

431

432 **Authors' contribution:**

MG wrote the first version of the manuscript during his Master thesis under the supervision of CVM and GM. PL edited and improved the manuscript.
436 **Conflict of interest statement:**

437 The authors declare no conflict of interest.

439 **Acknowledgement:**

440 We sincerely thank Pr. Jens Rolff for his helpful comments that improved the manuscript
441 quality. Publication fees are funded by the ANSES program proof of concept (grant name:
442 ASCONTROL). This review was also performed within the framework of the CNRS-EC2CO
443 program (grant name: INTERASCO).

445 **References:**

446 1 Samways, M. (2018) Insect Conservation for the Twenty-First Century.

447 2 Schowalter, T.D. (2016) *Insect Ecology: An Ecosystem Approach*, Academic Press.

448 3 Shields, V.D.C. (2017) *Biological Control of Pest and Vector Insects*,

449 4 Mora, C. et al. (2011) How many species are there on Earth and in the ocean? *PLoS Biol* 9,
e1001127

451 5 Rolff, J. et al. (2019) Complete metamorphosis of insects. *Philos Trans R Soc Lond B Biol Sci* 374, 20190063

453 6 Truman, J.W. (2019) The Evolution of Insect Metamorphosis. *Current Biology* 29, R1252–R1268

454 7 Simon, J.-C. et al. (2019) Host-microbiota interactions: from holobiont theory to analysis. *Microbiome* 7, 5

456 8 Hammer, T.J. and Moran, N.A. (2019) Links between metamorphosis and symbiosis in holometabolous insects. *Philosophical Transactions of the Royal Society B* DOI: 10.1098/rstb.2019.0068

459 9 Blow, F. and Douglas, A.E. (2019) The hemolymph microbiome of insects. *J Insect Physiol* 115, 33–39

461 10 Stubbendieck, R.M. et al. (2019) Convergent evolution of signal-structure interfaces for maintaining symbioses. *Current Opinion in Microbiology* 50, 71–78

463 11 Engel, P. and Moran, N.A. (2013) The gut microbiota of insects – diversity in structure and function. *FEMS Microbiol Rev* 37, 699–735

465 12 Hannula, S.E. et al. (2019) Foliar-feeding insects acquire microbiomes from the soil rather than the host plant. *Nature Communications* 10, 1254

467 13 Guégan, M. et al. (2018) The mosquito holobiont: fresh insight into mosquito-microbiota interactions. *Microbiome* 6, 49

469 14 Lin, X. and Smagghe, G. (2019) Roles of the insulin signaling pathway in insect development and organ growth. *Peptides* 122, 169923

471 15 Brown, J.J. et al. (2020) Ontogeny, species identity, and environment dominate microbiome dynamics in wild populations of kissing bugs (Triatominae). *Microbiome* 8, 1–16

473 16 Chen, Z. et al. Differences in the Diversity and Structure of the Gut Microbiome in Different Life Stages of the American Cockroach (*Periplaneta americana*). 27-Jan-(2022)

475 17 Ren, Z. et al. (2022) Dynamics of Microbial Communities across the Life Stages of *Nilaparvata lugens* (Stål). *Microb Ecol* 83, 1049–1058

477 18 Sudakaran, S. et al. (2012) Geographical and ecological stability of the symbiotic mid-gut microbiota in European firebugs, *Pyrrhocoris apterus* (Hemiptera, Pyrrhocoridae). *Mol Ecol* 21, 6134–6151

480 19 Nobles, S. and Jackson, C.R. (2020) Effects of Life Stage, Site, and Species on the Dragonfly Gut Microbiome. *Microorganisms* 8, E183
482 20 Ramalho, M.O. et al. (2017) Species-specific signatures of the microbiome from *Camponotus* and *Colobopsis* ants across developmental stages. *PLOS ONE* 12, e0187461

483 21 Zhukova, M. et al. (2017) Diversity and Transmission of Gut Bacteria in *Atta* and *Acromyrmex* Leaf-Cutting Ants during Development. *Front Microbiol* 8, 1942

484 22 Kowallik, V. and Mikheyev, A.S. (2021) Honey Bee Larval and Adult Microbiome Life Stages Are Effectively Decoupled with Vertical Transmission Overcoming Early Life Perturbations. *mBio* 12, e0296621

489 23 Mejía-Alvarado, F.S. et al. (2021) Structure and Dynamics of the Gut Bacterial Community Across the Developmental Stages of the Coffee Berry Borer, *Hypothenemus hampei*. *Front Microbiol* 12, 639868

492 24 Zhang, Z. et al. (2018) Bacterial and fungal gut communities of *Agrilus mali* at different developmental stages and fed different diets. *Sci Rep* 8, 15634

494 25 Suárez-Moo, P. et al. (2020) Diversity and Composition of the Gut Microbiota in the Developmental Stages of the Dung Beetle *Copris incertus* Say (Coleoptera, Scarabaeidae). *Frontiers in Microbiology* 11,

497 26 Chouaia, B. et al. (2019) Developmental stages and gut microenvironments influence gut microbiota dynamics in the invasive beetle *Popillia japonica* Newman (Coleoptera: Scarabaeidae). *Environmental Microbiology* 21, 4343–4359

500 27 Chen, B. et al. (2016) Biodiversity and Activity of the Gut Microbiota across the Life History of the Insect Herbivore *Spodoptera littoralis*. *Scientific Reports* 6, 29505

502 28 Wang, X. et al. (2020) Variability of Gut Microbiota Across the Life Cycle of *Grapholita molesta* (Lepidoptera: Tortricidae). *Front Microbiol* 11, 1366

504 29 Alfano, N. et al. (2019) Changes in Microbiota Across Developmental Stages of *Aedes koreicus*, an Invasive Mosquito Vector in Europe: Indications for Microbiota-Based Control Strategies. *Front. Microbiol.* 10,

507 30 Duguma, D. et al. (2015) Developmental succession of the microbiome of *Culex* mosquitoes. *BMC Microbiol.* 15, 140

509 31 de Jonge, N. et al. (2020) Housefly (Musca domestica L.) associated microbiota across different life stages. *Sci Rep* 10, 7842

511 32 Coon, K.L. et al. (2014) Mosquitoes rely on their gut microbiota for development. *Mol. Ecol.* 23, 2727–2739

513 33 Scolari, F. et al. (2021) Exploring Changes in the Microbiota of *Aedes albopictus*: Comparison Among Breeding Site Water, Larvae, and Adults. *Frontiers in Microbiology* 12,

515 34 Gusmão, D.S. et al. (2010) Culture-dependent and culture-independent characterization of microorganisms associated with *Aedes aegypti* (Diptera: Culicidae) (L.) and dynamics of bacterial colonization in the midgut. *Acta Trop* 115, 275–281

518 35 Tawidian, P. et al. (2021) Host-Environment Interplay Shapes Fungal Diversity in Mosquitoes. *mSphere* DOI: 10.1128/mSphere.00646-21

520 36 Moll, R.M. et al. (2001) Meconial peritrophic membranes and the fate of midgut bacteria during mosquito (Diptera: Culicidae) metamorphosis. *J. Med. Entomol.* 38, 29–32

522 37 González-Serrano, F. et al. (2020) The Gut Microbiota Composition of the Moth *Brithys crini* Reflects Insect Metamorphosis. *Microb Ecol* 79, 960–970

524 38 Coon, K.L. et al. (2017) Bacteria-mediated hypoxia functions as a signal for mosquito development. *Proc. Natl. Acad. Sci. U.S.A.* 114, E5362–E5369

526 39 Johnston, P.R. and Rolff, J. (2015) Host and Symbiont Jointly Control Gut Microbiota during Complete Metamorphosis. *PLOS Pathog* 11, e1005246

528 40 Werren, J.H. et al. (2008) *Wolbachia*: master manipulators of invertebrate biology. *Nat. Rev. Microbiol.* 6, 741–751

530 41 Dobson, S.L. et al. (1999) *Wolbachia* infections are distributed throughout insect somatic and germ line tissues. *Insect Biochem Mol Biol* 29, 153–160

532 42 Toomey, M.E. et al. (2013) Evolutionarily conserved *Wolbachia*-encoded factors control pattern of stem-cell niche tropism in *Drosophila* ovaries and favor infection. *Proceedings of the National Academy of Sciences* 110, 10788–10793

535 43 Pietri, J.E. et al. (2016) The rich somatic life of *Wolbachia*. *MicrobiologyOpen* 5, 923–936
<table>
  <tr>
    <td>536</td>
    <td>44</td>
    <td>Ren, S. et al. (2018) Localization and dynamics of Wolbachia infection in Asian citrus psyllid Diaphorina citri, the insect vector of the causal pathogens of Huanglongbing. Microbiologyopen</td>
  </tr>
  <tr>
    <td>537</td>
    <td>538</td>
    <td>7, e00561</td>
  </tr>
  <tr>
    <td>539</td>
    <td>45</td>
    <td>Braquart-Varnier, C. et al. (2015) The Hematopoietic Organ: A Cornerstone for Wolbachia</td>
  </tr>
  <tr>
    <td>540</td>
    <td>541</td>
    <td>Propagation Between and Within Hosts. Frontiers in Microbiology, 6, 46 Nadal-Jimenez, P. et al. (2019) Genetic manipulation allows in vivo tracking of the life cycle of the son-killer symbiont, Arsenophonus nasoniae, and reveals patterns of host invasion, tropism and pathology. Environ Microbiol 21, 3172–3182</td>
  </tr>
  <tr>
    <td>542</td>
    <td>543</td>
    <td></td>
  </tr>
  <tr>
    <td>544</td>
    <td>47</td>
    <td>Darby, A.C. et al. (2010) Characteristics of the genome of Arsenophonus nasoniae, son-killer bacterium of the wasp Nasonia. Insect Mol Biol 19 Suppl 1, 75–89</td>
  </tr>
  <tr>
    <td>545</td>
    <td>48</td>
    <td>Fukumori, K. et al. Evolutionary Dynamics of Host Organs for Microbial Symbiosis in Tortoise Leaf Beetles (Coleoptera: Chrysomelidae: Cassidinae). mBio 13, e03691-21</td>
  </tr>
  <tr>
    <td>546</td>
    <td>49</td>
    <td>Maire, J. et al. Endosymbiosis morphological reorganization during metamorphosis diverges in weevils. Commun Integr Biol 13, 184–188</td>
  </tr>
  <tr>
    <td>547</td>
    <td>50</td>
    <td>Pais, I.S. et al. (2018) Drosophila melanogaster establishes a species-specific mutualistic interaction with stable gut-colonizing bacteria. PLOS Biology 16, e2005710</td>
  </tr>
  <tr>
    <td>548</td>
    <td>51</td>
    <td>Erkosar, B. et al. (2015) Pathogen Virulence Impedes Mutualist-Mediated Enhancement of Host Juvenile Growth via Inhibition of Protein Digestion. Cell Host Microbe 18, 445–455</td>
  </tr>
  <tr>
    <td>549</td>
    <td>52</td>
    <td>Consuegra, J. et al. (2020) Metabolic Cooperation among Commensal Bacteria Supports Drosophila Juvenile Growth under Nutritional Stress. iScience 23, 101232</td>
  </tr>
  <tr>
    <td>550</td>
    <td>53</td>
    <td>Renelies-Hamilton, J. et al. (2021) Disentangling the Relative Roles of Vertical Transmission, Subsequent Colonizations, and Diet on Cockroach Microbiome Assembly. mSphere 6, e01023-20</td>
  </tr>
  <tr>
    <td>551</td>
    <td>54</td>
    <td>Shao, Y. et al. (2017) Symbiont-Derived Antimicrobials Contribute to the Control of the Lepidopteran Gut Microbiota. Cell Chemical Biology 24, 66–75</td>
  </tr>
  <tr>
    <td>552</td>
    <td>55</td>
    <td>Marasco, R. et al. (2022) Destabilization of the Bacterial Interactome Identifies Nutrient Restriction-Induced Dysbiosis in Insect Guts. Microbiology Spectrum 10, e01580-21</td>
  </tr>
  <tr>
    <td>553</td>
    <td>56</td>
    <td>McDonald, J.E. et al. (2020) Application of ecological and evolutionary theory to microbiome community dynamics across systems. Proceedings of the Royal Society B: Biological Sciences</td>
  </tr>
  <tr>
    <td>554</td>
    <td>57</td>
    <td>287, 20202886</td>
  </tr>
  <tr>
    <td>555</td>
    <td>58</td>
    <td>Nobles, S. and Jackson, C.R. (2020) Effects of Life Stage, Site, and Species on the Dragonfly Gut Microbiome. Microorganisms 8, 183</td>
  </tr>
  <tr>
    <td>556</td>
    <td>59</td>
    <td>Deb, R. et al. (2019) Host dietary specialization and neutral assembly shape gut bacterial communities of wild dragonflies. PeerJ 7, e8058</td>
  </tr>
  <tr>
    <td>557</td>
    <td>60</td>
    <td>Kikuchi, Y. et al. Collapse of Insect Gut Symbiosis under Simulated Climate Change. mBio 7, e01578-16</td>
  </tr>
  <tr>
    <td>558</td>
    <td>61</td>
    <td>Díaz-Sánchez, S. et al. (2018) Biotic and abiotic factors shape the microbiota of wild-caught populations of the arbovirus vector Culicoides imicola. Insect Molecular Biology 27, 847–861</td>
  </tr>
  <tr>
    <td>559</td>
    <td>62</td>
    <td>Chen, B. et al. (2018) Gut bacterial and fungal communities of the domesticated silkworm (Bombyx mori) and wild mulberry-feeding relatives. ISME J 12, 2252–2262</td>
  </tr>
  <tr>
    <td>560</td>
    <td>63</td>
    <td>Shukla, S.P. et al. (2018) Microbiome-assisted carrion preservation aids larval development in a burying beetle. PNAS 115, 11274–11279</td>
  </tr>
  <tr>
    <td>561</td>
    <td>64</td>
    <td>Kikuchi, Y. and Yumoto, I. (2013) Efficient colonization of the bean bug Riptortus pedestris by an environmentally transmitted Burkholderia symbiont. Appl Environ Microbiol 79, 2088–2091</td>
  </tr>
  <tr>
    <td>562</td>
    <td>65</td>
    <td>Ohbayashi, T. et al. (2019) Comparative cytology, physiology and transcriptomics of Burkholderia insecticola in symbiosis with the bean bug Riptortus pedestris and in culture. The ISME Journal 13, 1469–1483</td>
  </tr>
  <tr>
    <td>563</td>
    <td>66</td>
    <td>Consuegra, J. et al. (2020) Drosophila-associated bacteria differentially shape the nutritional requirements of their host during juvenile growth. PLoS Biol 18, e3000681</td>
  </tr>
  <tr>
    <td>564</td>
    <td>67</td>
    <td>Shin, S.C. et al. (2011) Drosophila Microbiome Modulates Host Developmental and Metabolic Homeostasis via Insulin Signaling. Science 334, 670–674</td>
  </tr>
  <tr>
    <td>565</td>
    <td>68</td>
    <td>Storelli, G. et al. (2011) Lactobacillus plantarum promotes Drosophila systemic growth by modulating hormonal signals through TOR-dependent nutrient sensing. Cell Metab 14, 403–414</td>
  </tr>
</table>
590 69 Valzania, L. et al. (2018) Both living bacteria and eukaryotes in the mosquito gut promote growth of larvae. *PLOS Neglected Tropical Diseases* 12, e0006638

591 70 Coon, K.L. et al. (2020) Predaceous *Toxorhynchites* mosquitoes require a living gut microbiota to develop. *Proceedings of the Royal Society B: Biological Sciences* 287, 20192705

592 71 Zouache, K. et al. (2022) Larval habitat determines the bacterial and fungal microbiota of the mosquito vector *Aedes aegypti*. *FEMS Microbiol Ecol* 98, fiac016

593 72 Minard, G. et al. (2018) Shared larval rearing environment, sex, female size and genetic diversity shape Ae. albopictus bacterial microbiota. *PLOS ONE* 13, e0194521

594 73 Wang, Y. et al. (2021) Riboflavin instability is a key factor underlying the requirement of a gut microbiota for mosquito development. *Proc Natl Acad Sci U S A* 118,

595 74 Valzania, L. et al. (2018) Hypoxia-induced transcription factor signaling is essential for larval growth of the mosquito *Aedes aegypti*. *Proc Natl Acad Sci U S A* 115, 457–465

600 75 Zheng, H. et al. (2017) Honeybee gut microbiota promotes host weight gain via bacterial metabolism and hormonal signaling. *Proceedings of the National Academy of Sciences of the United States of America* 114, 4775

601 76 Okamoto, N. and Yamanaka, N. (2015) Nutrition-dependent control of insect development by insulin-like peptides. *Curr Opin Insect Sci* 11, 21–30

602 77 Kešnerová, L. et al. (2017) Disentangling metabolic functions of bacteria in the honey bee gut. *PLoS Biol.* 15, e2003467

603 78 Paludo, C.R. et al. (2018) Stingless Bee Larvae Require Fungal Steroid to Pupate. *Sci Rep* 8, 1122

604 79 Prakash, A. and Khan, I. (2022) Why do insects evolve immune priming? A search for crossroads. *Developmental & Comparative Immunology* 126, 104246

605 80 Muhammad, A. et al. (2019) Intestinal Microbiota Confer Protection by Priming the Immune System of Red Palm Weevil *Rhynchophorus ferrugineus* Olivier (Coleoptera: Dryophthoridae).

606 81 Kamareddine, L. et al. (2018) The Drosophila Immune Deficiency Pathway Modulates Enteroendocrine Function and Host Metabolism. *Cell Metabolism* 28, 449–462.e5

607 82 Koh, A. et al. (2016) From Dietary Fiber to Host Physiology: Short-Chain Fatty Acids as Key Bacterial Metabolites. *Cell* 165, 1332–1345

608 83 Ma, M. et al. (2021) Metabolic and immunological effects of gut microbiota in leaf beetles at the local and systemic levels. *Integrative Zoology* 16, 313–323

609 84 Ignesti, M. et al. (2018) A polydnavirus-encoded ANK protein has a negative impact on steroidogenesis and development. *Insect Biochem Mol Biol* 95, 26–32

610 85 Wang, Z. et al. (2018) Parasitic insect-derived miRNAs modulate host development. *Nat Commun* 9, 2205

611 86 Bonning, B.C. and Saleh, M.-C. (2021) The Interplay Between Viruses and RNAi Pathways in Insects. *Annu Rev Entomol* 66, 61–79

612 87 Jahnes, B.C. et al. (2021) Microbial colonization promotes model cockroach gut tissue growth and development. *J Insect Physiol* 133, 104274

613 88 Zheng, H. et al. (2017) Honeybee gut microbiota promotes host weight gain via bacterial metabolism and hormonal signaling. *Proc Natl Acad Sci U S A* 114, 4775–4780

614 89 Maire, J. et al. (2020) Spatial and morphological reorganization of endosymbiosis during metamorphosis accommodates adult metabolic requirements in a weevil. *PNAS* 117, 19347–19358

615 90 Kikuchi, Y. et al. (2020) *Burkholderia insecticola* triggers midgut closure in the bean bug *Riptortus pedestris* to prevent secondary bacterial infections of midgut crypts. *ISME J* 14, 1627–1638

616 91 Reedy, A.R. et al. (2019) Commensal microbiota-induced redox signaling activates proliferative signals in the intestinal stem cell microenvironment. *Development* 146, dev171520

617 92 Duplais, C. et al. (2021) Gut bacteria are essential for normal cuticle development in herbivorous turtle ants. *Nat Commun* 12, 676

618 93 Birer, C. et al. (2020) Disentangling the assembly mechanisms of ant cuticular bacterial communities of two Amazonian ant species sharing a common arboreal nest. *Molecular Ecology* 29, 1372

619 94 Weiss, B.L. et al. (2012) Obligate Symbionts Activate Immune System Development in the Tsetse Fly. *The Journal of Immunology* 188, 3395–3403

620 95

621 96 Weiss, B.L. et al. (2012) Obligate Symbionts Activate Immune System Development in the Tsetse Fly. *The Journal of Immunology* 188, 3395–3403
645 95 Benoit, J.B. et al. (2017) Symbiont-induced odorant binding proteins mediate insect host hematopoiesis. *eLife* 6, e19535

647 96 O'Connor, C.M. et al. (2014) Biological carryover effects: linking common concepts and mechanisms in ecology and evolution. *Ecosphere* 5, art28

648 97 Dickson, L.B. et al. (2017) Carryover effects of larval exposure to different environmental bacteria drive adult trait variation in a mosquito vector. *Science Advances* 3, e1700585

651 98 Giraud, É. et al. (2021) Mosquito-bacteria interactions during larval development trigger metabolic changes with carry-over effects on adult fitness. *Mol Ecol* DOI: 10.1111/mec.16327

652 99 Weiss, B.L. et al. (2013) Trypanosome infection establishment in the tsetse fly gut is influenced by microbiome-regulated host immune barriers. *PLoS Pathog* 9, e1003318

653 100 Hosokawa, T. et al. (2008) Symbiont acquisition alters behaviour of stinkbug nymphs. *Biol Lett* 4, 45-48

654 101 Hosokawa, T. et al. (2006) Strict Host-Symbiont Cospeciation and Reductive Genome Evolution in Insect Gut Bacteria. *PLOS Biology* 4, e337

655 102 Bistolas, K.S.I. et al. (2014) Symbiont polyphyly, co-evolution, and necessity in pentatomid stinkbugs from Costa Rica. *Frontiers in Microbiology* 5,

656 103 Morimoto, J. et al. (2019) Commensal microbiota modulates larval foraging behaviour, development rate and pupal production in *Bactrocera tryoni*. *BMC Microbiol* 19, 1-8

657 104 Manthey, C. et al. (2021) *Complete metamorphosis and microbiota turnover in insects*, Evolutionary Biology.

658 105 Muñoz-Benavent, M. et al. (2021) Insects' potential: Understanding the functional role of their gut microbiome. *Journal of Pharmaceutical and Biomedical Analysis* 194, 113787

659 106 Beale, D.J. et al. (2022) Is there any biological insight (or respite) for insects exposed to plastics? Measuring the impact on an insects central carbon metabolism when exposed to a plastic feed substrate. *Science of The Total Environment* 831, 154840

660 107 Oliveira, N.C. et al. (2022) Non-targeted metabolomics reveals differences in the gut metabolic profile of the fall armyworm strains when feeding different food sources. *Journal of Insect Physiology* 139, 104400

661 108 Yang, F.-Y. et al. (2020) Differential Profiles of Gut Microbiota and Metabolites Associated with Host Shift of *Plutella xylostella*. *International Journal of Molecular Sciences* 21, 6283

662 109 Ogaugwu, C.E. and Durvasula, R.V. (2017) *Developing the Arsenal Against Pest and Vector Dipterans: Inputs of Transgenic and Paratransgenic Biotechnologies*, IntechOpen.

663 110 Grimaldi, D. et al. (2005) *Evolution of the Insects*, Cambridge University Press.

664 111 Zhang, J. et al. (2014) Functional Analysis of Insect Molting Fluid Proteins on the Protection and Regulation of Ecdysis*. *Journal of Biological Chemistry* 289, 35891-35906

665 112 Tettamanti, G. and Casartelli, M. (2019) Cell death during complete metamorphosis. *Philosophical Transactions of the Royal Society B: Biological Sciences* 374, 20190065

666 113 Franzetti, E. et al. (2015) The midgut of the silkmoth *Bombyx mori* is able to recycle molecules derived from degeneration of the larval midgut epithelium. *Cell Tissue Res* 361, 509-528

667 114 Lehane, M.J. (1997) Peritrophic matrix structure and function. *Annu Rev Entomol* 42, 525-550

668 115 Johnston, P.R. et al. (2019) Immune gene regulation in the gut during metamorphosis in a holo- versus a hemimetabolous insect. *Philosophical Transactions of the Royal Society B: Biological Sciences* 374, 20190073

669 116 Lemke, T. et al. (2003) Physicochemical Conditions and Microbial Activities in the Highly Alkaline Gut of the Humus-Feeding Larva of *Pachnoda ephippiata* (Coleoptera: Scarabaeidae). *Applied and Environmental Microbiology* 69, 6650-6658

670 117 Šustr, V. et al. (2014) Microprofiles of oxygen, redox potential, and pH, and microbial fermentation products in the highly alkaline gut of the saprophagous larva of *Penthetria holosericea* (Diptera: Bibionidae). *Journal of Insect Physiology* 67, 64-69

671 118 Terra, W.R. and Ferreira, C. (2012) 11 - Biochemistry and Molecular Biology of Digestion. In *Insect Molecular Biology and Biochemistry* (Gilbert, L. I., ed), pp. 365-418, Academic Press

672 119 Truman, J.W. and Riddiford, L.M. (2002) Endocrine insights into the evolution of metamorphosis in insects. *Annu Rev Entomol* 47, 467-500
699 120 Morgan, E.D. (2010) 4.03 - Insect Hormones. In *Comprehensive Natural Products II* (Liu, H.-W. (Ben) and Mander, L., eds), pp. 127–151, Elsevier

701 121 Cheong, S.P.S. *et al.* (2015) Evolution of Ecdysis and Metamorphosis in Arthropods: The Rise of Regulation of Juvenile Hormone. *Integr Comp Biol* 55, 878–890

703 122 Feyereisen, R. (2012) 8 - Insect CYP Genes and P450 Enzymes. In *Insect Molecular Biology and Biochemistry* (Gilbert, L. I., ed), pp. 236–316, Academic Press

705 123 Subramanian, S. and Shankarganesh, K. (2016) Chapter 20 - Insect Hormones (as Pesticides). In *Ecofriendly Pest Management for Food Security* (Omkar, ed), pp. 613–650, Academic Press

707 124 Gilbert, L.I. *et al.* (2000) The juvenile hormones: historical facts and speculations on future research directions. *Insect Biochem Mol Biol* 30, 617–644

709 125 Truman, J.W. and Riddiford, L.M. (2019) The evolution of insect metamorphosis: a developmental and endocrine view. *Philos Trans R Soc Lond B Biol Sci* 374, 20190070

711 126 Riddiford, L.M. (1994) Cellular and Molecular Actions of Juvenile Hormone I. General Considerations and Premetamorphic Actions. In *Advances in Insect Physiology* 24 (Evans, P. D., ed), pp. 213–274, Academic Press

714 127 Garelli, A. *et al.* (2012) Imaginal discs secrete insulin-like peptide 8 to mediate plasticity of growth and maturation. *Science* 336, 579–582

716 128 Pedigo, L.P. and Rice, M.E. (2014) *Entomology and Pest Management: Sixth Edition*, Waveland Press.

718 129 Taylor, F. (1981) Ecology and Evolution of Physiological Time in Insects. *The American Naturalist* 117, 1–23

720

721

722

723

724

725

726

727

728

729

730

731

732

733

734

735

736

737

738

739

740
741

742

743

744

745

746

Table 1: Dynamics of the bacterial microbiota across the development of insects or closely related taxa.

747
753

**Figure 1: Factors shaping the composition of the gut microbiota during the development of insects.** (A) Different path of microorganisms transmission do exist during insects life.

754

755

756

757

758

759

760

761

762

763

764

765

766

767

768

769

770

771

772

773

774

775

776

777

778

779

780

781

782

783

784

785

786

Microorganisms can be acquired from the environment or through transmission between individuals of the same generation through horizontal transmission or from parent to their offspring through vertical transmission. Variation in insect ecology can affect those transmission paths through different ways. As an example, parental care may increase the vertical transmission probability between parent and their offspring and either social interactions or parental cares tend to increase horizontal transmission between individuals of the same generation. Examples of insects harboring such behavior show a more homogenous microbial community throughout their development [20,62]. Alternatively, variations in insect habitat type or trophic interactions throughout the development increases the opportunities of microbes' environmental acquisition. Example of studies on insect with such biological cycle showed they tend to harbor a heterogenous microbial community [18,29]. (B) Host physiology and morphology changes during development may also affect the stability of their microbial community. For example, microbes may be eliminated when the tissue composition of the insect changes or when immunity and lytic enzymes (e.g. lysozyme) are enhanced during specific stages [39]. (C) Finally, some microorganisms may be more adapted to persist during the insect development. As an example, *Enterococcus munditii* increases their competitiveness and persistence throughout lepidopteran development through the release of bacteriocins [54].

**Figure 2: Functioning of the endocrine system during the insect development.** The cellular basis for tissue growth is induced by hormones named insuline-like peptides (ILP) secreted by the brain. When the nutrition is optimal, soft tissues of the insect develop. The critical size is reached when the cuticle is filled by soft tissues. In response, the brain activates its neurosectretory cells, which produce the prothoracicotropic hormones (PTTH). Recognition of PTTH enhances the production of ecdysone (20E) by the prothoracic glands, which induces molt (between early and late larval instars) or incomplete metamorphosis (between nymph and imago for the heterometabola) or complete metamorphosis (between pupae and imago for the holometabola). The juvenile hormones (JH) produced by endocrine cells in the *corpus allatum* can be synthesized during different stages in the heterometabola and holometabola but their decrease induce the metamorphosis. Many examples listed in this review highlighted the influence of the microbiota at different levels of this regulatory system as highlighted here with numbers [64–66,68,73,74,76,78,81,84,85].
787 **Box 1: The development of insects**

788 Insect developmental strategies are schematized in the **Figure IA**. Ametabolous insects are
789 characterized by the absence of metamorphosis during their development. They can molt but
790 larval stages are very similar to the *imago* even though their size can differ. This group contains
791 apterygote insects (i.e. wingless insects that do not belong to a phylogenetic group including
792 winged ancestors) that belong to two different orders: Archaeognatha and Zygentoma.
793 Hemimetabolous *s.l.* insects (sometimes referred as heterometabolous) proceed to an
794 incomplete metamorphosis during their development. The immature stages named pre-nymph
795 or nymph are followed by the *imago* stage but they are not similar to each other. Some
796 hemimetabolous *s.l.* insects are shifting from one habitat to another along their lifecycle and
797 are referred as hemimetabolous insects *sensus stricto*. On the other hand, some are living within
798 the same habitat throughout their lifecycle and are referred to as paurometabolous insects.
799 Hemimetabolous *s.s.* and paurometabolous insects are paraphyletic and such subgrouping of
800 hemimetabolous *s.l.* is only relevant from an ecological perspective. Finally, some insects
801 accomplish a complete metamorphosis and are qualified as holometabolous. They develop
802 though one or more larval instars, followed by the pupal stage and finally the *imago*.
803 Holometabolous insects account for a large part of the insect richness constituting more than
804 80% of the described species [110]. During ecdysis periods, insects tissues experience physical
805 and chemical restructuration that may drastically impact the colonization and maintenance
806 probability of microorganisms colonizing them [111]. Many organs (e.g. the midgut, the fat
807 body, head glands and neurons) reshape or sometimes degenerate during those phases with
808 more or less amplitude [112]. Renewal of the insect cuticle is accompanied with the production
809 of molting fluids that contains proteins involved in the ecdysis. If the function of some of them
810 is well known (e.g. chitinases, proteases) many unknown but essential proteins still need to be
811 characterized. Proteins from the immune system are also secreted through those fluids for
812 protection against pathogens. The gut is often very prompt to microbe colonization in many
813 metazoan including insects [11]. Modification in the gut structure occur during the insect
814 development do have strong consequences for the microbiota community. Gut delamination
815 occur when insects are molting or during metamorphosis may lead to the differentiation of stem
816 cells covering the basal pole of the epithelium into new cells types ([113], **Figure IB**). During
817 life stages in which insects are feeding, the gut is protected from chemical and biological
818 epithelium aggression by a polymer of chitin and proteins forming a peritrophic matrix. Two
819 types of peritrophic matrices (PM) can be retrieved in the insects [114]. Type I PM are broadly
820 distributed and cover completely the insect midgut. Type II PM is only retrieved in a restricted
panel of hemi- and holometabolan species (Diptera, Dermaptera, Embiodea and some Lepidoptera) and cover the anterior midgut. Both types of PM can be produced by the same species but at different stages and they are eliminated with the epithelium when insects are molting. In addition, the gut is highly immunogenic. Its immune response has been shown to vary according to developmental strategies and life stages with higher immune levels reported during the metamorphosis of holometabonlans [115]. Finally, digestive enzymes and physicochemical conditions (e.g. pH, redox potential, O₂) in the insects gut are shifting depending on the considered species and development stage [38,55,116,117,118].

<figure><img src="image_2.png" /><figcaption>**Figure I: Developmental strategies of insects.** (A) The different stages of each developmental strategies retrieved in insects are represented. Ametabola do not proceed to metamorphosis, Hemimetabola sensu lato (including Hemimetabola sensus stricto or Paurometabola) do experience an incomplete metamorphosis between Nymphs and Imago and Holometabola experience a complete metamorphosis between pupae and imago. (B) Example of gut remodeling during complete metamorphosis. Insects proceed to delamination of their gut epithelium and produce a new cell layer from differenciated stem cells. The cell types may drastically differ between life stages as well as the type of peritrophic matrix (either type I or II) secreted by the epithelium. Metamorphosis is often accompanied with changes in the gut physicochemical conditions, enzymes and immunity.</figcaption></figure>
843 **Box 2: Major regulatory mechanisms of growth**

844 Nervous and endocrine systems regulate the post-embryonic development of insects. Together,
845 they involve interdependent hormones [119] transmitting the signal that triggers or modulates
846 exuviation. Initiation of development is induced when sensory neurons detect the filling of
847 cuticle by insect tissues that reached a critical size [120]. Responses to this signal will then
848 involve three major groups of hormones [121]. First, neurosecretory cells in the brain release
849 neuropeptides such as prothoracicotropic hormone (PITH). PITH activates the prothoracic
850 glands located in the posterior part of the insect thorax and trigger the synthesis of ecdysteroids
851 such as ecdysone (also called molting hormone), which is then converted into its active form:
852 20-hydroxyecdysone (20E). Conversion of ecdysone to 20E is catalyzed by enzymes produced
853 in many peripheral tissues (e.g. fat body, midgut, Malpighian tubes, etc) [122]. Release of 20E
854 triggers physiological processes related to molting and metamorphosis including synthesis of
855 exoskeleton and removal of the old cuticle [123]. It also inactivates other endocrine signals via
856 the ecdysone pathway involved in the expression of genes during the development. In
857 hemimetabolous s.l. and holometabolous insects, a third group of hormones is involved, called
858 sesquiterpenoid juvenile hormones (JHs) [124]. JHs are produced and released by the corpora
859 allata (i.e. a pair of endocrine glands in the posterior part of insects' brain). JHs modulate
860 systemic growth of individuals between molting periods [125]. An increase in JHs levels,
861 upstream of exuviation, induces the molt of the individual from one larval stage to another.
862 However, a strong decrease in JHs levels during this same period triggers complete or
863 incomplete metamorphosis [126].

864 In addition to nervous and endocrine systems, other factors influence the development of
865 insects. Diet is particularly important accumulation of energy reserves, directly or indirectly
866 impacting the endocrine system [76,125]. During larval development, many peptide hormones
867 control the consumption of energy reserves as well as the cell and tissue growth in response to
868 the insect diet. These hormones belong to insulin-like peptides (ILP) group. They are mainly
869 secreted by specialized endocrine cells in the brain and induce feedback on exuviation [127].
870 Finally, temperature seems to be the climatic factor that most strongly impacts the development
871 of insects [128,129].

872

873

874 **Glossary:**

875 **Axenic:** condition in which the individuals are germ free.
876

**Diversity:** this term refers to the number of species and their relative abundance in one or
several specific environments.

877

878

**Endosymbiont:** an organism which habitat is the body or (in this context) the cells of its host.

879

**Ecdysis:** process by which hexapods replace their old cuticle (i.e their outer layer) by a new one.

880

881

**Instar:** the period between two stages during insect development.

882

**Metamorphosis:** it etymologically refers to “the after form”, a process by which organism go through radically different forms along development.

883

884

**Richness:** this term refers to the number of species in a specific environment.