Boosting Bug Farms: A Meta-Analysis on Probiotic Effects in Insect Rearing

15 Interest in insects and food as feed is rapidly growing. With this, however, comes a 16 move towards mass rearing and industrial scale production. This upscaling and 17 industrialisation of the rearing process is likely to result in high density rearing, which 18 in itself facilitates disease spread (Tarwater and Martin, 2001). Furthermore, the fact 19 that these insects are likely to be genetically closely related further increase the risk 20 of infectious disease outbreak (Ekroth et al., 2019; Gibson and Nguyen, 2021). In 21 tackling this risk, it is important that we do not resort to the mass use of antibiotics 22 that has been seen in the livestock industry. Instead, alternative rearing practices 23 should be developed. A practice that has received a huge increase in attention in the 24 .CC-BY 4.0 International licenseavailable under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (whichthis version posted April 4, 2025. ; https://doi.org/10.1101/2025.04.04.647199doi: bioRxiv preprint 2 last years is the idea that supplementation of insect feed with probiotics could 25 improve insect health and prevent pathogen spread. We carried out a meta-analysis 26 to systematically analyse the data on probiotic supplementation of insects reared for 27 food and feed available to date. The most commonly measured response to probiotic 28 supplementation was body weight gain followed by microbiome diversity. For body 29 weight gain, we collected 71 effect sizes from 28 studies and for microbiome 30 diversity ten effect sizes from six studies. We found that overall, probiotics tended to 31 increase insect growth rate but did not significantly impact microbiome diversity. Our 32 analysis also highlighted two key literature gaps. To date, data are only available on 33 one of the four insect species able to be sold as food in the EU, Tenebrio molitor. 34 Furthermore, there are currently only very few studies that have looked at protection 35 against pathogens by probiotic bacteria in insects reared for food and feed. So far, 36 the data look promising, but data on more insect species and looking at inhibitory 37 effects against pathogens are urgently needed. 38

probiotic supplementation; edible insects; insect growth; microbiota 39 40

41 Insect protein is rapidly gaining attention as a future food source (Hazarika and 42 Kalita, 2023; van Huis and Gasco, 2023). This is largely because insects as a protein 43 source represent a climate friendly and sustainable alternative to traditional meat. 44 Around 34% of global greenhouse gas production comes from the food industry 45 (Crippa et al., 2021), 72-78% of which come from meat production (Springmann et 46 al., 2018). Insect rearing results in the emission of a fraction of the greenhouse 47 gasses produced during traditional meat production. For example, the production of 48 .CC-BY 4.0 International licenseavailable under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (whichthis version posted April 4, 2025. ; https://doi.org/10.1101/2025.04.04.647199doi: bioRxiv preprint 3 1kg cricket protein produces 99% less greenhouse gas than 1kg of beef protein and 49 overall insect rearing results in just 6% of the greenhouse gas emissions per kg body 50 weight released during cattle rearing for beef (Jafir et al., 2024). Under the EU novel 51 food legislation (EU) 2015/2283, four insect species, Locusta migratoria, Acheta 52 domesticus, Alphitobius diaperinus and Tenebrio molitor, can be reared for and sold 53 as human food in the EU (Precup et al., 2022). This rapidly growing market presents 54 huge benefits for the equitable and sustainable production of food for a global human 55 population that is expected to exceed 10 million by 2025 (United Nations Department 56 of Economic and Social Affairs, Population Division, 2022). It also brings with it 57 potential challenges (Maciel-Vergara et al., 2021), notably, an increased risk of 58 disease spread. 59 Rearing of insects on the scales necessary to feed the growing human population 60 requires mass rearing at high densities. High density cultures of genetically similar 61 animals are known to present a particularly high risk for disease spread within 62 populations (Ekroth et al., 2019; Gibson and Nguyen, 2021). Furthermore, insects 63 can be reared on a range of organic side streams (Broeckx et al., 2021; van Huis, 64 2013; Van Peer et al., 2021; Vrontaki et al., 2024), which while reducing the 65 environmental impact further, introduces a diversity of bacteria into the digestive tract 66 of the insects which are likely to range from the potentially beneficial to the 67 potentially harmful (Marzoli et al., 2024; Savio et al., 2024a; Wynants et al., 2019). 68 The threat of potential pathogens in cultures of insects being reared for food and 69 feed is twofold. There are pathogens that threaten the insects themselves, including 70 entomopathogenic fungi (Dahal et al., 2022), bacterial entomopathogens, such as 71 Bacillus thuringiensis (Savio et al., 2024b) and viruses (Duffield et al., 2021). Human 72 food pathogens, in particular Salmonella enterica and Bacillus cereus have also 73 .CC-BY 4.0 International licenseavailable under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (whichthis version posted April 4, 2025. ; https://doi.org/10.1101/2025.04.04.647199doi: bioRxiv preprint 4 been found in the guts of edible insect species (Fasolato et al., 2018; Marzoli et al., 74 2024; Wynants et al., 2019). As insects, unlike traditional livestock are generally 75 prepared for human consumption with their guts intact, the presence of these 76 bacteria in the insect digestive tract poses a potential threat of transmission to the 77 human consumer. 78 Despite the threat posed by potential bacterial and fungal pathogens to insect 79 cultures, it is imperative that mass prophylactic administration of antimicrobials does 80 not become standard practice. The widespread use of antibiotics in the meat farming 81 industry (Ghimpe/i1 eanu et al., 2022) has significantly contributed to the global risk 82 presented by antimicrobial resistance (AMR) (Djordjevic et al., 2024). The insect 83 microbiota is known to be a potential source of AMR genes (Raka et al., 2024; Rawat 84 et al., 2023). Such genes are currently found in lower abundance in insects than 85 livestock (Raka et al., 2024) but would likely rapidly increase in frequency if selection 86 pressure through the prophylactic use of antibiotics was applied. It is critical that the 87 mistakes made in livestock farming are not repeated in mass insect rearing and that 88 alternative methodologies are developed to protect insect cultures and reduce the 89 risk of spreading antibiotic resistant bacteria into the food and feed system. 90 Recently, probiotics have been receiving increasing attention as a potential strategy 91 to increase the overall health of insect cultures (Dahal et al., 2022; Grau et al., 2017; 92 Savio et al., 2022). We know from research in the field of evolutionary ecology that 93 bacteria with potentially protective effects, also known as “defensive microbes”, have 94 the potential to provide stable, long-term defence against pathogens (Armitage et al., 95 2022; King and Bonsall, 2017; Vorburger and Perlman, 2018). For example, the gut 96 microbe Enterococcus mundtii works together with its host Galleria mellonella to 97 control the host microbiome during meta-morphosis, protecting against the 98 .CC-BY 4.0 International licenseavailable under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (whichthis version posted April 4, 2025. ; https://doi.org/10.1101/2025.04.04.647199doi: bioRxiv preprint 5 proliferation of pathogenic bacteria (Johnston and Rolff, 2015). Similarly, the 99 bacterial symbiont, Hamiltonella defensa, protects its aphid host against parasitoid 100 wasp attack (Kaech et al., 2022; Kwiatkowski et al., 2012; Wu et al., 2022), with 101 implications for pest control (Donner et al., 2023). 102 Many probiotics fall into the category of “defensive microbe”, in that they provide 103 protection against infection (Corr et al., 2007; Deriu et al., 2013; Do et al., 2024; 104 Fukuda et al., 2011; Piewngam et al., 2021, 2018). These probiotics can be 105 harnessed not just to support insect growth and nutritional health, but to protect 106 against infection and disease spread (Ford and King, 2016). In contrast to antibiotics, 107 probiotics or defensive bacteria present a dynamic and (co)evolving defensive agent, 108 that can change to counter adapt to a pathogen evolving resistance (Ford et al., 109 2017, 2016; Kwiatkowski et al., 2012). Furthermore, many human probiotics have 110 been shown to have beneficial effects on edible insects (e.g. (Lecocq et al., 2021; 111 Milanović et al., 2021)), meaning that supplementation may not only benefit the 112 health of the insect cultures but has the potential to provide benefits to the human 113 consumer as well. 114 Over the last three to four years, there has been a burst of publications on probiotic 115 supplementation in edible insect species. Here we use a meta-analytic approach to 116 summarise and systematically review these data, draw conclusions on what we 117 know so far and highlight literature gaps where we find that more research is 118 needed. 119 120

121 Literature search 122 .CC-BY 4.0 International licenseavailable under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (whichthis version posted April 4, 2025. ; https://doi.org/10.1101/2025.04.04.647199doi: bioRxiv preprint 6 Our initial aim was to find all papers reporting the results of studies where edible 123 insects had been supplemented with probiotics. We used the search terms including 124 “Tenebrio molitor probiotics”, “Bombyx mori probiotics”, “Hermetia illucens 125 probiotics”, “Apis mellifera probiotics”, “Acheta domesticus probiotics”, “Alphitobius 126 diaperinus probiotics”, “Locusta migratoria probiotics” and “edible insect probiotics” 127 to search the databases: PubMed, Google Scholar and Web of science. We then 128 looked at the papers citing the papers we had found and checked the references lists 129 of all discovered papers for more potential studies. 130 We noted that the most commonly measured parameters in the studies found were 131 insect growth rate and microbiome diversity (often measured as the Shannon index) 132 and therefore decided to carry out two meta-analysis the first asking whether 133 probiotic supplementation influenced insect growth rate and the second asking 134 whether probiotic supplementations influenced insect microbiome diversity. 135 Inclusion criteria 136 Papers meeting the following criteria were included in the meta-analyses: 137  The paper was published in a peer reviewed journal; 138  The paper was written in English; 139  The host species was insect species either already registered as a novel food 140 within the EU, which is under evaluation as a novel food in the EU or is regularly 141 consumed as protein source outside of the EU; 142  The paper presented data on, for meta-analysis one, insect growth rate or, for 143 meta-analysis two, microbiome diversity, for both a probiotic treated and a control 144 group; 145 .CC-BY 4.0 International licenseavailable under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (whichthis version posted April 4, 2025. ; https://doi.org/10.1101/2025.04.04.647199doi: bioRxiv preprint 7  Means and standard errors could be extracted, calculated or estimated from the 146 presented data either from graphs, raw data or other measures of average and 147 spread of the data. 148 Statistical analysis 149 All analyses were carried out in R (v4.4.1; R Core Team). For both the growth rate 150 and diversity data sets, we calculated Hedge’s g effect sizes and confidence 151 intervals using the “esc” package in R (Lüdecke, 2019). We then carried out a 152 multivariant meta-analyses using the rma.mv function in the “metafor” package 153 (Viechtbauer, 2025) with “study”, “insect” and “probiotic species” as random factors 154 except where insect and probiotic species were explicitly being tested as moderator 155 variables. For each of the two data sets we carried out a four moderator variable 156 analyses which each of the follow moderators as a fixed factor. We tested whether 1) 157 insect species, 2) probiotic species, 3) whether the probiotic was a lactobacillus or 158 not and 4) whether the probiotic was gram positive, or gram negative had an impact 159 on the magnitude or direction of the effect. We plotted funnel plots to visually assess 160 publication bias and calculated fail safe N values to estimate the number of datasets 161 that would need to be added to the analysis to change the outcome (Orwin, 1983). 162

163 Growth rate 164 Overall model for growth rate 165 Through our literature search we collected a total of 71 datasets from 24 publications 166 presenting data on growth rate under control and probiotic supplemented conditions 167 and that met our inclusion criteria (Table 1). These data showed that, overall, 168 supplementation with probiotics had a positive effect on growth (multi-variant meta-169 .CC-BY 4.0 International licenseavailable under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (whichthis version posted April 4, 2025. ; https://doi.org/10.1101/2025.04.04.647199doi: bioRxiv preprint 8 analysis: estimate = 2.7036 (ci.lower = 1.2541/ ci.upper = 4.1532), z = 3.6556, p = 170 0.0003), suggesting that overall supplementation with probiotics leads to heavier 171 insects (Figure 1). We noticed during our data collection that some feeding material 172 was fermented, this was specifically the case for black soldier fly larvae. As 173 fermentation potentially results in probiotic enrichment, we noted these cases in 174 Table 1. 175 Moderator variable analyses for growth rate 176 Insect species did not have a significant influence on the overall magnitude or 177 direction of the effect (QM = 2.0767, d.f. = 3, p = 0.5566), however grouping studies 178 by insect species demonstrates the disparity in numbers of studies across species 179 (Figure 1). While we were able to include 40 datasets using Hermetia illucens as 180 host, a species not yet registered in the EU as a novel food, but one of the insects 181 bred on an industrial scale for animal feeding or agro-industrial field purposes, no 182 studies have yet been carried out on three out of the four species that are able to be 183 sold for food in the EU. In fact, of all four species registered as novel food in the EU, 184 Locusta migratoria, Acheta domesticus, Alphitobius diaperinus and Tenebrio molitor, 185 only T. molitor has been used a host in published studies and in just 10 of the 71 186 included data sets. 187 To test what subcategories of probiotics might be effective, we tested for differences 188 between lactobacilli and non-lactobacilli and gram-positive and gram-negative 189 bacteria. There was no significant difference between lactobacilli and non-lactobacilli 190 (QM = 2.7487, d.f. = 1, p = 0.0973) or between gram-positive and gram-negative 191 probiotics (QM =2.4363, d.f. = 2, p = 0.2958). 192 Publication bias for growth rate 193 .CC-BY 4.0 International licenseavailable under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (whichthis version posted April 4, 2025. ; https://doi.org/10.1101/2025.04.04.647199doi: bioRxiv preprint 9 To test for publication bias we first visualized the relationship between effect sizes 194 and standard errors using a funnel plot (Figure 2A). As expected in the absence of 195 publication bias (Field and Gillett, 2010), we saw that we tended to have larger 196 standard errors with larger effect sizes and that we had both positive and negative 197 effect sizes in our data set. This indicates that there was no evidence of publication 198 bias. Furthermore, fail safe N analysis suggested that we would need to add 2393 199 data sets to our study to change the outcome, giving us a high level of confidence in 200 our results. 201 202 Microbiome diversity 203 Overall model for microbiome diversity 204 Compared to growth rate, we found fewer studies reporting data on the effects of 205 probiotic supplementation on microbiome diversity. Nevertheless, we extracted 10 206 datasets from six published papers (Table 2). Overall, there was no significant effect 207 of probiotic supplementation on microbiome diversity (estimate = -0.9449(lower ci= -208 2.9918/ upper ci = 1.1020), z = -0.9048, p = 0.3656), with studies showing a range of 209 both positive and negative effect sizes (Figure 3). 210 Moderator variable analysis for microbiome diversity 211 Again here, insect species did not have an impact on the magnitude or direction of 212 the effect (QM = 1.0816, d.f. = 2, p =0.5823), although the breakdown also 213 highlighted that in diversity measures, just as in growth rate, there is a bias in the 214 insect species in which research has been carried out, with the majority of the 215 research having been done on the black soldier fly, H. illucens (Figure 3). For this 216 .CC-BY 4.0 International licenseavailable under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (whichthis version posted April 4, 2025. ; https://doi.org/10.1101/2025.04.04.647199doi: bioRxiv preprint 10 analysis there were an insufficient number of studies to meaningfully test further 217 moderator variables. 218 Publication bias for microbiome diversity 219 We plotted a funnel plot to visualize potential publication bias for microbiome 220 diversity, however, due to the small number of datasets meeting the criteria for 221 inclusion in the meta-analysis for microbiome diversity it is difficult to draw clear 222

(Figure 2B). 223 Inhibitory effects of probiotics against pathogen infection 224 For the benefits of probiotics to be maximised, we would ideally supplement farmed 225 insects with probiotics that not only stimulate growth and gut health but that also 226 provide protection against pathogens. Unfortunately, to date there are too few 227 studies to carry out a meaningful meta-analysis on this specific question. To our 228 knowledge published work on the defensive properties of probiotics against 229 pathogens has been mainly carried out in T. molitor against entomopathogens. The 230 few published studies present mixed, yet potentially promising results. Lecocq et al 231 (Lecocq et al., 2021) showed that Pediococcus pentoceus has inhibitory effects in 232 vitro against a range of potentially relevant entomopathogens. Building on this, Dahal 233 et al (Dahal et al., 2022) showed in vivo that supplementation of T. molitor with P. 234 pentoceus, not only enhances growth but also seems to provide some degree of 235 protection to T. molitor against mortality induced by the highly virulent 236 entomopathogenic fungus Metarhizium brunneum, although the potential probiotic B. 237 subtilus trends towards negatively impacting survival upon infection (Dahal et al., 238 2022). The positive results with P. pentoceus were, however, further supported by 239 Savio et al (Savio et al., 2024a) who showed that P. pentoceus seems to provide 240 .CC-BY 4.0 International licenseavailable under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (whichthis version posted April 4, 2025. ; https://doi.org/10.1101/2025.04.04.647199doi: bioRxiv preprint 11 protection to T. molitor against coinfection with Bacillus thuringiensis and 241 Metarhizium brunneum, although there are some differences in the outcomes of the 242 studies, which suggests a degree of context dependency. 243 There is also evidence of probiotic protection against pathogens in the silk worm 244 Bombyx mori. Here supplementation with Lactobacillus casei resulted in protection 245 against infection by the microsporidian Nosema bombycis (Suraporn and Terenius, 246 2021) and supplementation with Lactobacillus lactis protection against infection with 247 Pseudomonas aeruginosa (Nishida et al., 2016). The mechanism of Lactobacillus 248 induced protection is thought to be via bacterial induced activation of the immune 249 system (Nishida et al., 2016). 250

251 There is a rapidly growing literature base on the supplementation of insects with 252 probiotics. Despite this growing literature, however, we only were able to extract a 253 sufficient number of data sets for meta-analysis of two measures: insect growth and 254 microbiome diversity. While probiotic supplementation significantly enhances insect 255 growth across tested species, probiotics do not have a clear effect on microbial 256 diversity, with studies showing both positive and negative effects. Furthermore, our 257 data clearly highlight some substantial literature gaps where more data are urgently 258 needed. Most striking is the restricted number of insects in which these kinds of test 259 have been carried out and the lack of studies on three of the four insect species 260 registered as novel food in the EU. 261 Across the data that are available we saw a very strong and robust positive effect of 262 probiotic supplementation on insect growth. Rearing insects on probiotics resulted in 263 heavier insects. By decomposing indigestible fibres, producing essential nutrients, 264 .CC-BY 4.0 International licenseavailable under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (whichthis version posted April 4, 2025. ; https://doi.org/10.1101/2025.04.04.647199doi: bioRxiv preprint 12 and enabling metabolic and signalling processes (Chabanol and Gendrin, 2024), the 265 probiotics play a key role in the nutrition and development of the insect host. 266 Moreover, they may aid the insects' ability to cope with stress factors (temperature, 267 toxins, chemicals, pathogens, etc.). This is a phenomenon known from other 268 mutualisms between insects and microbes (Armitage et al., 2022; Ford and King, 269 2016; Vorburger and Perlman, 2018). 270 We did not see a significant effect of probiotic supplementation on microbiome 271 diversity albeit based on a very small sample size. Although microbiota diversity, 272 often in the form of a Shannon index, is a commonly reported measure in probiotic 273 supplementation studies, it is difficult to judge what exactly a reduced or increased 274 Shannon index means for insect health (Johnson and Burnet, 2016). It is often 275 assumed that high diversity means a healthy microbiome, likely due do associations 276 with low diversity and human disease (Huttenhower et al., 2012). However, when 277 supplementing with probiotics at high doses we may, to an extent, replace the 278 unknown microbiota, which may be healthy or unhealthy, with bacterial species 279 known to benefit health. Thus, reduced diversity may be due to replacement with the 280 probiotic and not necessarily indicate poor or even reduced health. 281 The increase in growth observed across studies when insects are supplemented with 282 probiotics supports the idea that probiotics enhance insect health. What is more this 283 benefit to growth results in increased yields, bringing additional economic 284 advantages. In spite of the indication of enhanced health coming from the growth 285 rate data, however, we found that very little data were available on the susceptibility 286 to pathogens of probiotic supplemented insects. This is in spite of the fact that 287 probiotics are known to have protective effects in aquaculture settings (Chauhan and 288 Singh, 2019; Kuebutornye et al., 2020; Sharifuzzaman and Austin, 2017), and are 289 .CC-BY 4.0 International licenseavailable under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (whichthis version posted April 4, 2025. ; https://doi.org/10.1101/2025.04.04.647199doi: bioRxiv preprint 13 even being considered as an alternative to antibiotics in livestock farming (Leistikow 290 et al., 2022). Furthermore, the use of probiotics or “defensive microbes” has been 291 advocated as a method of pathogen control in applied settings in general (Ford and 292 King, 2016) as well as for the specific case of edible insects (Grau et al., 2017; Savio 293 et al., 2022). The studies that have been carried out looking at potential protection of 294 insects from pathogen attack by probiotics do show promising results, with evidence 295 that some probiotics inhibit the growth of pathogens (Dahal et al., 2022; Lecocq et 296 al., 2021; Nishida et al., 2016; Savio et al., 2024a; Suraporn and Terenius, 2021). 297 Much more data are needed, however, to fully explore the potential protective effects 298 of candidate probiotics against pathogen infection across the full range of edible 299 insect species. 300 Harnessing probiotics as an alternative to antibiotic treatment to prevent the 301 establishment and spread of disease within insect cultures presents a strategy 302 through which we may avoid the overuse of antibiotics seen in livestock rearing. 303 However, this strategy is not without risks. We know that probiotics can also be a 304 source of AMR genes (Daniali et al., 2020; Radovanovic et al., 2023; Savio et al., 305 2022; Tóth et al., 2021), which could mean that if probiotic treatment is unsuccessful 306 and antibiotics do have to be used, efficacy is impacted and the presence of 307 probiotics harbouring such genes may even enhance the rate of AMR evolution. On 308 the other hand, we can use this knowledge to ensure that probiotics are bred 309 exclusively from AMR free strains. Also here, this is a question that urgently needs to 310 be explored both experimentally and theoretically as the scale of insect production 311 for food and feed increases. 312 The microbiome is critically associated with a range of behavioural and physiological 313 function and how the immune response might be linked to probiotic supplementation 314 .CC-BY 4.0 International licenseavailable under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (whichthis version posted April 4, 2025. ; https://doi.org/10.1101/2025.04.04.647199doi: bioRxiv preprint 14 remains unclear. There has been much discussion as to how the host immune 315 system balances the need to allow the colonisation of mutualistic micro-organisms 316 while simultaneously fighting off pathogens (Betts et al., 2016; Hanson, 2024), as 317 research in the field develops, it is important we consider how potential immune 318 effects of microbiome manipulation may impact mass rearing. 319 Finally, it is important to consider the microbial composition of the feeding material 320 given to insects grown as food and feed. Our literature research showed that 321 particularly black soldier fly larvae are being fed a range of microbe rich (Mazza et 322 al., 2020; Rehman et al., 2019) and fermented material (Somroo et al., 2019; 323 Witriana et al., 2023). Fermentation can result in enrichment of probiotics (Heller, 324 2001). Given that fermentation of vegetable and other food waste provides an 325 opportunity to utilise organic side streams and increase the climate friendliness of 326 insects as food and feed further, the impact of these foods on insect health and 327 growth warrants further investigation (Antunes et al, unpublished data). 328 Our systematic analysis shows that a strong body of research is developing on the 329 topic of probiotic supplementation in edible insects. As research continues, we 330 highlight six key questions that remain to be addressed. These are: 1) Do growth 331 enhancing probiotics also provide protection against pathogens? 2) Does probiotic 332 supplementation reduce or increase the risk of AMR? 3) Do the effects we see in the 333 insect species studied so far extent to other edible insect species, particularly those 334 being sold as novel food in the EU? 4) Does probiotic supplementation impact insect 335 behaviour under mass rearing conditions? 5) How does probiotic supplementation 336 affect the insect immune system? 6) What role do diet substrates, and their 337 processing techniques play in shaping probiotic diversity and stability within the 338 insect gut? Our hope is that continued research in this field, addressing the 339 .CC-BY 4.0 International licenseavailable under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (whichthis version posted April 4, 2025. ; https://doi.org/10.1101/2025.04.04.647199doi: bioRxiv preprint 15 highlighted questions has the potential to greatly improve the sustainability and 340 efficiency of insect rearing for food and feed. 341 342

343 We have provided the first systematic review of the quantitative impacts of probiotic 344 supplementation on edible insects. Overall probiotics tend to boost insect growth, 345 which suggests that insect health is enhanced through supplementation. However, 346 there is no clear effect on microbiome diversity. There are also two clear literature 347 gaps highlighted by our study. First of all, the taxonomic restriction on the insect 348 species that have been studied to date. If probiotic supplementation is to be 349 considered as an implementation strategy to support insect health in commercial 350 rearing, it is important that those insects with the highest commercial potential are 351 testing. Secondly, our study highlights a lack of studies investigating the protective 352 effects of probiotics against pathogenic infection. As the focus on probiotics in the 353 edible insect industry grows, we hope these knowledge gaps will be filled. 354 Conflict of interest: 355 We have no conflicts of interest. 356 Funding statement: 357 This work was funded by an Investionsbank Berlin ProValid grant to CR 358 (VAL128/2023). 359 360

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It is made The copyright holder for this preprint (whichthis version posted April 4, 2025. ; https://doi.org/10.1101/2025.04.04.647199doi: bioRxiv preprint 31 Yu, G., Cheng, P ., Chen, Yanhong, Li, Y., Yang, Z., Chen, Yuanfeng, Tomberlin, J.K., 725 2011. Inoculating Poultry Manure With Companion Bacteria Influences Growth 726 and Development of Black Soldier Fly (Diptera: Stratiomyidae) Larvae. 727 Environmental Entomology 40, 30–35. https://doi.org/10.1603/EN10126 728 Yuan, S., Sun, Y., Chang, W., Zhang, J., Sang, J., Zhao, J., Song, M., Qiao, Y., 729 Zhang, C., Zhu, M., Tang, Y ., Lou, H., 2023. The silkworm (Bombyx mori) gut 730 microbiota is involved in metabolic detoxification by glucosylation of plant 731 toxins. 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Fems Microbiol. 743 Rev. 32, 723–735. https://doi.org/10.1111/j.1574-6976.2008.00123.x 744 745 746 747 Figure legends: 748 .CC-BY 4.0 International licenseavailable under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (whichthis version posted April 4, 2025. ; https://doi.org/10.1101/2025.04.04.647199doi: bioRxiv preprint 32 Figure 1. Forest plots showing effect sizes and confidence intervals for differences in 749 insect growth with and without probiotic supplementation with effect sizes and 95% 750 confidence intervals for each data set included in the study, negative effect sizes 751 show a lower body weight in insects reared with probiotics than reared without and 752 positive effect sizes show increased growth in insects reared with probiotic 753 supplementation. Colour depicts the insect species and size the sample size. 754 Figure 2. Funnel plots showing observed outcomes (effect sizes) and standard errors 755 for: (A) the first meta-analysis comparing growth; (B 756 757 ) the second meta-analysis comparing microbiome diversity. The white triangle 758 shows the expected distribution in the absence of publication bias. 759 Figure 3. Forest plots showing effect sizes and confidence intervals for differences in 760 insect microbiome diversity with and without probiotic supplementation with effect 761 sizes and 95% confidence intervals for each data set included in the study, negative 762 effect sizes show a lower microbial diversity in insects reared with probiotics than 763 reared without and positive effect sizes show increased microbial diversity in insects 764 reared with probiotic supplementation. Colour depicts the insect species and size the 765 sample size. 766 767 768 769 770 771 .CC-BY 4.0 International licenseavailable under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (whichthis version posted April 4, 2025. ; https://doi.org/10.1101/2025.04.04.647199doi: bioRxiv preprint 33 Table 1. Included studies and relevant information on the insect studies included in the first meta-analysis comparing body weights.

No. Insect species Bacterial species Lactobacillus? Gram positive or negative? Weight control SE control N control Weight probiotic SE probiotic N probioti c Ferm ented diet Lecocq et al. 2021 (Lecocq et al., 2021) 1 Tenebrio molitor Pediococcus pentosaceus yes positive 0,117 g ± 0,002 g 8 0,149 g ± 0,005 g 8 no Rizou et al. 2022 (Rizou et al., 2022) 2 Tenebrio molitor Bacillus subtilis no positive 160,3 mg ± 5,7 mg 4 205,5 mg ± 3,8 mg 4 no Rizou et al. 2022 (Rizou et al., 2022) 3 Tenebrio molitor Bacillus toyonensis no positive 157,4 mg ± 4,7 mg 4 179,1 mg ± 0 mg 4 no Rizou et al. 2022 (Rizou et al., 2022) 4 Tenebrio molitor Enterococcus faecalis no positive 146,1 mg ± 5,7 mg 4 197,9 mg ± 6,6 mg 4 no Dahal et al. 2022 (Dahal et al., 2022) 5 Tenebrio molitor Pediococcus pentosaceus yes positive 0,041 g ± 0,002 g 10 0,133 g ± 0,003 g 10 no Dahal et al. 2022 Dahal et al., 2022) 6 Tenebrio molitor Bacillus subtilis no positive 0,131 g ± 0,004 g 10 0,072 g ± 0 g 10 no Dahal et al. 2022 Dahal et al., 2022) 7 Tenebrio molitor Enterococcus faecium no positive 0,104 g ± 0,004 g 10 0,131 g ± 0,004 g 10 no Suraporn et al. 2015 (Suraporn 8 Bombyx Lactobacillus yes positive 1,18 g ± 0,05 g 3 1,26 g ± 0,05 g 3 no 98 .CC-BY 4.0 International licenseavailable under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (whichthis version posted April 4, 2025. ; https://doi.org/10.1101/2025.04.04.647199doi: bioRxiv preprint 34 et al., 2015) mori acidophilus Suraporn et al. 2021 (Suraporn and T erenius, 2021) 9 Bombyx mori Lactobacillus casei yes positive 2,55 g ± 0,06 g 3 2,84 g ± 0,06 g 3 no Taha et al. 2017 (T aha et al., 2017) 10 Bombyx mori Bifidobacterium bifidum no positive 2,66 g ± 0 g 4 3,02 g ± 0,02 g 4 no Saranya et al. 2019 (M et al., 2019) 11 Bombyx mori Staphylococcus gallinarum no positive 3,6 g ± 0,008 g 5 4,12 g ± 0,014 g 5 no Saranya et al. 2019 (M et al., 2019) 12 Bombyx mori Staphylococcus arlettae no positive 3,6 g ± 0,008 g 5 3,89 g ± 0,008 g 5 no Somroo et al. 2019 (Somroo et al., 2019) 13 Hermetia illucens Lactobacillus buchneri yes positive 126,4 g ± 1,1 g 3 146,4 g ± 1,8 g 3 yes Witriana et al. 2023 (Witriana et al., 2023) 14 Hermetia illucens Lactiplantibacillus plantarum yes positive 212,1 g ± 11,4 g 3 251,9 g ± 2,6 g 3 Ferm ented with the probi otics Witriana et al. 2023 (Witriana et al., 2023) 15 Hermetia illucens Limosilactobacillus fermentum yes positive 212,1 g ± 11,4 g 3 213,6 g ± 14,5 g 3 Ferm ented with the probi otics .CC-BY 4.0 International licenseavailable under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (whichthis version posted April 4, 2025. ; https://doi.org/10.1101/2025.04.04.647199doi: bioRxiv preprint 35 Witriana et al. 2023 (Witriana et al., 2023) 16 Hermetia illucens L. plantarum, L. fermentum yes positive 212,1 g ± 11,4 g 3 287,3 g ± 6,8 g 3 Ferm ented with the probi otics Callegari et al. 2020 (Callegari et al., 2020) 17 Hermetia illucens Bacillus licheniformis no positive 1,59 g ± 0,88 g 3 1,88 g ± 0,11 g 3 no Callegari et al. 2020 (Callegari et al., 2020) 18 Hermetia illucens Stenotrophomonas maltophilia no negative 1,59 g ± 0,88 g 3 1,59 g ± 0,14 g 3 no Callegari et al. 2020 (Callegari et al., 2020) 19 Hermetia illucens Escherichia coli no negative 1,59 g ± 0,88 g 3 1,92 g ± 0,08 g 3 no Callegari et al. 2020 (Callegari et al., 2020) 20 Hermetia illucens B. licheniformis, S. maltophilia no positive, negative 1,59 g ± 0,88 g 3 1,76 g ± 0,13 g 3 no Hasan et al. 2022 (Hasan et al., 2022) 21 Apis mellifera Lactobacillus rhamnosus yes positive 118,7 mg ± 9,6 mg 3 128,2 mg ± 7,4 mg 3 no Hasan et al. 2022 (Hasan et al., 2022) 22 Apis mellifera Lactobacillus brevis yes positive 119,9 mg ± 9,5 mg 3 124,1 mg ± 5,3 mg 3 no Hasan et al. 2022 (Hasan et al., 2022) 23 Apis mellifera Bacillus clausii no positive 119,9 mg ± 9,5 mg 3 138,9 mg ± 6,5 mg 3 no Savio et al. 2024 (Savio et 25 Tenebrio molitor Lactiplantibacillus plantarum yes positive 72,8 mg ± 9,7 mg 3 90,0 mg ± 8,9 mg 3 no .CC-BY 4.0 International licenseavailable under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (whichthis version posted April 4, 2025. ; https://doi.org/10.1101/2025.04.04.647199doi: bioRxiv preprint 36 al., 2024a) Savio et al. 2024 (Savio et al., 2024a) 26 Tenebrio molitor Pediococcus pentosaceus yes positive 72,8 mg ± 9,7 mg 3 89,7 mg ± 10,8 mg 3 no Zhong et al. 2017 (Zhong et al., 2017) 27 Tenebrio molitor Bifidobacterium bifidum, Clostridium butyricum, Bacillus subtilis, Bacillus licheniformis no positive 8,5 g ± 0,3 g 6 9,00 g ± 0,16g 6 no Yu et al. 2011 (Yu et al., 2011) 28 Hermetia illucens Bacillus subtilis S15 no positive 0,008 g ± 0,012 g 3 0,095 g ± 0,015 g 3 manu re Yu et al. 2011 (Yu et al., 2011) 29 Hermetia illucens Bacillus subtilis S16 no positive 0,008 g ± 0,012 g 3 0,092 g ± 0,013 g 3 manu re Yu et al. 2011 (Yu et al., 2011) 30 Hermetia illucens Bacillus subtilis S19 no positive 0,008 g ± 0,012 g 3 0,087 g ± 0,017 g 3 manu re Yu et al. 2011 (Yu et al., 2011) 31 Hermetia illucens Bacillus natto D1 no positive 0,008 g ± 0,012 g 3 0,0848 g ± 0,0014 g 3 manu re Kooienga et al. 2020 (Kooienga et al., 2020) 32 Hermetia illucens Arthrobacter no positive 10,2 g ± 2,12 g 4 9,6 g ± 0,4 g 4 no Kooienga et al. 2020 (Kooienga et al., 2020) 33 Hermetia illucens Bifidobacterium breve no positive 14,3 g ± 2,00 g 3 1,05 g ± 0,13 g 3 no Kooienga et al. 2020 (Kooienga et al., 2020) 34 Hermetia illucens Rhodococcus rhodochrous no positive 10,2 g ± 2,15 g 4 9,0 g ± 0,403 g 4 no .CC-BY 4.0 International licenseavailable under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (whichthis version posted April 4, 2025. ; https://doi.org/10.1101/2025.04.04.647199doi: bioRxiv preprint 37 Kooienga et al. 2020 (Kooienga et al., 2020) 35 Hermetia illucens Arthrobacter no positive 2,88 g ± 0,13 g 3 2,54 g ± 0,11 g 3 no Moustafa et al. 2019 (Moustafa and Soliman, 2019) 36 Bombyx mori Lactobacillus rhamnosus yes positive 2,325 g ± 0,114 g 3 3,51 g ± 0,31 g 3 no Moustafa et al. 2019 (Moustafa and Soliman, 2019) 37 Bombyx mori Bifidobacterium bifidum no positive 2,325 g ± 0,114 g 3 3,775 g ± 0,18 g 3 no Zeng et al. 2024 (Zeng et al., 2024) 38 Bombyx mori Bacillus spp. no positive 1,5 g ± 0,2 g 3 1,55 g ± 0,09 g 3 no Zeng et al. 2024 (Zeng et al., 2024) 39 Bombyx mori Bacillus cereus no positive 1,5 g ± 0,2 g 3 1,50 g ± 0,08 g 3 no Zeng et al. 2024 (Zeng et al., 2024) 40 Bombyx mori Bacillus huizhouensi no positive 1,5 g ± 0,2 g 3 1,19 g ± 0,09 g 3 no Zeng et al. 2024 (Zeng et al., 2024) 41 Bombyx mori Enterococcus casseliflavus no positive 1,5 g ± 0,2 g 3 1,68 g ± 0,06 g 3 no Zeng et al. 2024 (Zeng et al., 2024) 42 Bombyx mori Enterococcus mundtii 75-4 no positive 1,5 g ± 0,2 g 3 1,59 g ± 0,07 g 3 no Zeng et al. 2024 (Zeng et 43 Bombyx mori Enterococcus mundtii X-2 no positive 1,5 g ± 0,2 g 3 1,29 g ± 0,07 g 3 no .CC-BY 4.0 International licenseavailable under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (whichthis version posted April 4, 2025. ; https://doi.org/10.1101/2025.04.04.647199doi: bioRxiv preprint 38 al., 2024) Zeng et al. 2024 (Zeng et al., 2024) 44 Bombyx mori Pediococcus pentosaceus yes positive 2,325 g ± 0,114 g 3 3,78 g ± 0,18 g 3 no Zeng et al. 2024 (Zeng et al., 2024) 45 Bombyx mori Enterococcus faecium NL5-5 no positive 1,5 g ± 0,2 g 3 1,36 g ± 0,08 g 3 no Zeng et al. 2024 (Zeng et al., 2024) 46 Bombyx mori Enterococcus faecium ML21 no positive 1,5 g ± 0,2 g 3 1,41 g ± 0,03 g 3 no Zeng et al. 2024 (Zeng et al., 2024) 47 Bombyx mori Enterococcus faecium CA01 no positive 1,5 g ± 0,2 g 3 1,48 g ± 0,03 g 3 no Unban et al. 2022 (Unban et al., 2022) 48 Bombyx mori Enterococcus hirae no positive 4,2 g ± 0,04 g 3 4,53 g ± 0,07 g 3 no Rehman et al. 2019 (Rehman et al., 2019) 49 Hermetia illucens Paenibacillus polymyxa no positive 90,46 g ± 0,89 g 3 97,69 g ± 0,22 g 3 manu re Rehman et al. 2019 Rehman et al., 2019) 50 Hermetia illucens Bacillus spp SMO1 no positive 90,46 g ± 0,89 g 3 105,61 g ± 0,56 g 3 manu re Rehman et al. 2019 Rehman et al., 2019) 51 Hermetia illucens Bacillus spp. SMO 2 no positive 90,46 g ± 0,89 g 3 102,48 g ± 0,66 g 3 manu re Rehman et al. 2019 Rehman et al., 2019) 52 Hermetia illucens Bacillus spp. MRO2 no positive 90,46 g ± 0,89 g 3 112,49 g ± 0,69 g 3 manu re .CC-BY 4.0 International licenseavailable under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (whichthis version posted April 4, 2025. ; https://doi.org/10.1101/2025.04.04.647199doi: bioRxiv preprint 39 Rehman et al. 2019 Rehman et al., 2019) 53 Hermetia illucens Bacillus spp. SMO4 no positive 90,46 g ± 0,89 g 3 101,94 g ± 1,01 g 3 manu re Franks et al. 2021 (Franks et al., 2021) 54 Hermetia illucens Rhodococcus rhodochrous no positive 882.9 mg ± 29,59 mg 3 1498,1 mg ± 52,62 mg 3 no Mazza et al. 2020 (Mazza et al., 2020) 55 Hermetia illucens Bacillus subtilis no positive 0,089 g ± 0,004 g 3 0,118 g ± 0,004 g 3 manu re Mazza et al. 2020 (Mazza et al., 2020) 56 Hermetia illucens Kocuria marina no positive 0,089 g ± 0,004 g 3 0,113 g ± 0,005 g 3 manu re Mazza et al. 2020 (Mazza et al., 2020) 57 Hermetia illucens Micrococcus luteus no positive 0,089 g ± 0,004 g 3 0,114 g ± 0,006 g 3 manu re Mazza et al. 2020 (Mazza et al., 2020) 58 Hermetia illucens Enterococcus faecalis no positive 0,089 g ± 0,004 g 3 0,110 g ± 0,010 g 3 manu re Mazza et al. 2020 (Mazza et al., 2020) 59 Hermetia illucens Lysinibacillus boronitolerans no positive 0,089 g ± 0,004 g 3 0,118 g ± 0,012 g 3 manu re Mazza et al. 2020 (Mazza et al., 2020) 60 Hermetia illucens Sporosarcina koreensis no positive 0,089 g ± 0,004 g 3 0,103 g ± 0,008 g 3 manu re Mazza et al. 2020 (Mazza et al., 2020) 61 Hermetia illucens Gordonia sihwensis no positive 0,089 g ± 0,004 g 3 0,111 g ± 0,003 g 3 manu re Mazza et al. 2020 (Mazza 62 Hermetia illucens Enterobacter spp. no negative 0,089 g ± 0,004 g 3 0,104 g ± 0,003 g 3 manu re .CC-BY 4.0 International licenseavailable under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (whichthis version posted April 4, 2025. ; https://doi.org/10.1101/2025.04.04.647199doi: bioRxiv preprint 40 et al., 2020) Mazza et al. 2020 (Mazza et al., 2020) 63 Hermetia illucens Proteus mirabilis no negative 0,089 g ± 0,004 g 3 0,113 g ± 0,013 g 3 manu re Mazza et al. 2020 (Mazza et al., 2020) 64 Hermetia illucens Bacillus subtilis no positive 0,089 g ± 0,004 g 3 0,105 g ± 0,002 g 3 manu re Li et al. 2023 (Li et al., 2023) 65 Hermetia illucens Providencia spp. no negative 0,364 g ± 0,001 g 3 0,335 g ± 0,004 g 3 no Li et al. 2023 (Li et al., 2023) 66 Hermetia illucens Citrobacter spp. no negative 0,364 g ± 0,001 g 3 0,344 g ± 0,002 g 3 no Li et al. 2023 (Li et al., 2023) 67 Hermetia illucens Klebsiella spp. no negative 0,364 g ± 0,001 g 3 0,360 g ± 0,001 g 3 no Li et al. 2023 (Li et al., 2023) 68 Hermetia illucens Proteus spp. no negative 0,364 g ± 0,001 g 3 0,321 g ± 0,003 g 3 no Li et al. 2023 (Li et al., 2023) 69 Hermetia illucens Gordonia sihwensis spp. no positive 0,364 g ± 0,001 g 3 0,351 g ± 0,003 g 3 no Li et al. 2023 (Li et al., 2023) 70 Hermetia illucens Dysgonomonas spp. no negative 0,364 g ± 0,001 g 3 0,336 g ± 0,001 g 3 no Pei et al. 2022 (Pei et al., 2022) 71 Hermetia illucens Bacillus velezensis no postive 0,81 g ± 0,024 g 3 0,941 g ± 0,021 g 3 no Table 2. Included studies and relevant information on the insect studies included in the second meta-analysis comparing Shannon indices as an indication of bacterial community diversity. .CC-BY 4.0 International licenseavailable under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (whichthis version posted April 4, 2025. ; https://doi.org/10.1101/2025.04.04.647199doi: bioRxiv preprint 41

Insect species Bacterial species Shannon control SE control N control Shannon probiotic SE probiotic N probiotic Lecoq et al. 2021 (Lecocq et al., 2021) Tenebrio molitor Pediococcus pentoceus 0,73 ± 0,18 8 1,19 ± 0,07 8 Savio et al. 2024 (Savio et al., 2024a) Tenebrio molitor Lactiplantibacillus plantarum 1,31 ± 0,34 3 0,82 ± 0,10 3 Savio et al. 2024 (Savio et al., 2024a) Tenebrio molitor Pediococcus pentoceus 1,31 ± 0,34 3 1,68 ± 0,05 3 Meng et al. 2023 (Meng et al., 2023) Hermetia illucens Rhodopseudomonas palustris 1,71 ± 0,04 6 1,83 ± 0,03 6 Meng et al. 2023 (Meng et al., 2023) Hermetia illucens Rhodopseudomonas palustris 1,71 ± 0,04 6 2,49 ± 0,09 6 Kooienga et al. 2020 (Kooienga et al., 2020) Hermetia illucens Arthrobacter 2,79 ± 0,01 4 2,62 ± 0,01 4 Kooienga et al. 2020 (Kooienga et al., 2020) Hermetia illucens Rhodococcus rhodochrous 2,79 ± 0,01 4 2,75 ± 0,01 4 Yuan et al. 2023 (Yuan et al., 2023) Bombyx mori Bacillus subtilis 1,04 ± 0,20 5 1,33 ± 0,15 5 Yuan et al. 2023 (Yuan et al., 2023) Bombyx mori Bacillus subtilis 0,47 ± 0,13 5 0,81 ± 0,19 5 Pei et al. 2022 (Pei et al., 2022) Hermetia illucens Bacillus velezensis 2,61 ± 0,18 3 1,93 ± 0,06 3 .CC-BY 4.0 International licenseavailable under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (whichthis version posted April 4, 2025. ; https://doi.org/10.1101/2025.04.04.647199doi: bioRxiv preprint 42 .CC-BY 4.0 International licenseavailable under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (whichthis version posted April 4, 2025. ; https://doi.org/10.1101/2025.04.04.647199doi: bioRxiv preprint Data set Effect size Insect species Apis mellifera Bombyx mori Hermetia illucens Tenebrio molitor .CC-BY 4.0 International licenseavailable under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (whichthis version posted April 4, 2025. ; https://doi.org/10.1101/2025.04.04.647199doi: bioRxiv preprint A B .CC-BY 4.0 International licenseavailable under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (whichthis version posted April 4, 2025. ; https://doi.org/10.1101/2025.04.04.647199doi: bioRxiv preprint Insect species Bombyx mori Hermetia illucens Tenebrio molitor Data set Effect size .CC-BY 4.0 International licenseavailable under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (whichthis version posted April 4, 2025. ; https://doi.org/10.1101/2025.04.04.647199doi: bioRxiv preprint