{"paper_id":"10cea836-dbdd-4f9e-93d5-293051b49140","body_text":"1\nBoosting Bug Farms: A Meta-Analysis on Probiotic Effects 1 \nin Insect Rearing  2 \nShort title: Probiotic effects in insect rearing  3 \nJulia Häbermann1, Bernardo Antunes2 (ORCID: 0000-0002-6810-5453), Hajo Haase1 (ORCID: 4 \n0000-0002-1622-8718), Jens Rolff2 (ORCID: 0000-0002-1529-5409), Claudia Keil1† (ORCID: 5 \n0000-0003-0317-0905) and Charlotte Rafaluk2*† (ORCID: 0000-0002-6245-7941) 6 \n1Department of Food Chemistry and T oxicology, Institute of Food Technology and Food 7 \nChemistry, Technische Universität Berlin, Straße des 17. Juni 135, 10623 Berlin, Germany; 8 \njulihaebi@yahoo.de (J.H.); c.keil@tu-berlin.de (C.K.); haase@tu-berlin.de (H.H.)  9 \n2Evolutionary Biology, Freie Universität Berlin, Institut für Biologie, Königin-Luise-Str. 1-3, 14195, 10 \nBerlin, Germany; bantunes@zedat.fu-berlin.de (B.A.); jens.rolff@fu-berlin.de (J.R.); 11 \ncharlotte.rafaluk@fu-berlin.de (C.R.) 12 \n†Equal contribution 13 \n*Correspondence: charlotte.rafaluk@fu-berlin.de 14 \nAbstract:  15 \nInterest in insects and food as feed is rapidly growing. With this, however, comes a 16 \nmove towards mass rearing and industrial scale production. This upscaling and 17 \nindustrialisation of the rearing process is likely to result in high density rearing, which 18 \nin itself facilitates disease spread (Tarwater and Martin, 2001). Furthermore, the fact 19 \nthat these insects are likely to be genetically closely related further increase the risk 20 \nof infectious disease outbreak (Ekroth et al., 2019; Gibson and Nguyen, 2021). In 21 \ntackling this risk, it is important that we do not resort to the mass use of antibiotics 22 \nthat has been seen in the livestock industry. Instead, alternative rearing practices 23 \nshould be developed. A practice that has received a huge increase in attention in the 24 \n.CC-BY 4.0 International licenseavailable under a \nwas not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprint (whichthis version posted April 4, 2025. ; https://doi.org/10.1101/2025.04.04.647199doi: bioRxiv preprint \n\n 2\nlast years is the idea that supplementation of insect feed with probiotics could 25 \nimprove insect health and prevent pathogen spread. We carried out a meta-analysis 26 \nto systematically analyse the data on probiotic supplementation of insects reared for 27 \nfood and feed available to date. The most commonly measured response to probiotic 28 \nsupplementation was body weight gain followed by microbiome diversity. For body 29 \nweight gain, we collected 71 effect sizes from 28 studies and for microbiome 30 \ndiversity ten effect sizes from six studies.  We found that overall, probiotics tended to 31 \nincrease insect growth rate but did not significantly impact microbiome diversity. Our 32 \nanalysis also highlighted two key literature gaps. To date, data are only available on 33 \none of the four insect species able to be sold as food in the EU, Tenebrio molitor. 34 \nFurthermore, there are currently only very few studies that have looked at protection 35 \nagainst pathogens by probiotic bacteria in insects reared for food and feed. So far, 36 \nthe data look promising, but data on more insect species and looking at inhibitory 37 \neffects against pathogens are urgently needed.  38 \n Keywords: probiotic supplementation; edible insects; insect growth; microbiota  39 \n 40 \nIntroduction 41 \nInsect protein is rapidly gaining attention as a future food source (Hazarika and 42 \nKalita, 2023; van Huis and Gasco, 2023). This is largely because insects as a protein 43 \nsource represent a climate friendly and sustainable alternative to traditional meat. 44 \nAround 34% of global greenhouse gas production comes from the food industry 45 \n(Crippa et al., 2021), 72-78% of which come from meat production (Springmann et 46 \nal., 2018). Insect rearing results in the emission of a fraction of the greenhouse 47 \ngasses produced during traditional meat production. For example,  the production of 48 \n.CC-BY 4.0 International licenseavailable under a \nwas not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprint (whichthis version posted April 4, 2025. ; https://doi.org/10.1101/2025.04.04.647199doi: bioRxiv preprint \n\n 3\n1kg cricket protein produces 99% less greenhouse gas than 1kg of beef protein and 49 \noverall insect rearing results in just 6% of the greenhouse gas emissions per kg body 50 \nweight released during cattle rearing for beef (Jafir et al., 2024). Under the EU novel 51 \nfood legislation (EU) 2015/2283, four insect species, Locusta migratoria, Acheta 52 \ndomesticus, Alphitobius diaperinus and Tenebrio molitor, can be reared for and sold 53 \nas human food in the EU (Precup et al., 2022). This rapidly growing market presents 54 \nhuge benefits for the equitable and sustainable production of food for a global human 55 \npopulation that is expected to exceed 10 million by 2025 (United Nations Department 56 \nof Economic and Social Affairs, Population Division, 2022).  It also brings with it 57 \npotential challenges (Maciel-Vergara et al., 2021), notably, an increased risk of 58 \ndisease spread.  59 \nRearing of insects on the scales necessary to feed the growing human population 60 \nrequires mass rearing at high densities. High density cultures of genetically similar 61 \nanimals are known to present a particularly high risk for disease spread within 62 \npopulations (Ekroth et al., 2019; Gibson and Nguyen, 2021). Furthermore, insects 63 \ncan be reared on a range of organic side streams (Broeckx et al., 2021; van Huis, 64 \n2013; Van Peer et al., 2021; Vrontaki et al., 2024), which while reducing the 65 \nenvironmental impact further, introduces a diversity of bacteria into the digestive tract 66 \nof the insects which are likely to range from the potentially beneficial to the 67 \npotentially harmful (Marzoli et al., 2024; Savio et al., 2024a; Wynants et al., 2019). 68 \nThe threat of potential pathogens in cultures of insects being reared for food and 69 \nfeed is twofold. There are pathogens that threaten the insects themselves, including 70 \nentomopathogenic fungi (Dahal et al., 2022), bacterial entomopathogens, such as 71 \nBacillus thuringiensis (Savio et al., 2024b) and viruses (Duffield et al., 2021). Human 72 \nfood pathogens, in particular Salmonella enterica and Bacillus cereus have also 73 \n.CC-BY 4.0 International licenseavailable under a \nwas not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprint (whichthis version posted April 4, 2025. ; https://doi.org/10.1101/2025.04.04.647199doi: bioRxiv preprint \n\n 4\nbeen found in the guts of edible insect species (Fasolato et al., 2018; Marzoli et al., 74 \n2024; Wynants et al., 2019). As insects, unlike traditional livestock are generally 75 \nprepared for human consumption with their guts intact, the presence of these 76 \nbacteria in the insect digestive tract poses a potential threat of transmission to the 77 \nhuman consumer.  78 \nDespite the threat posed by potential bacterial and fungal pathogens to insect 79 \ncultures, it is imperative that mass prophylactic administration of antimicrobials does 80 \nnot become standard practice. The widespread use of antibiotics in the meat farming 81 \nindustry (Ghimpe/i1 eanu et al., 2022) has significantly contributed to the global risk 82 \npresented by antimicrobial resistance (AMR) (Djordjevic et al., 2024). The insect 83 \nmicrobiota is known to be a potential source of AMR genes (Raka et al., 2024; Rawat 84 \net al., 2023). Such genes are currently found in lower abundance in insects than 85 \nlivestock (Raka et al., 2024) but would likely rapidly increase in frequency if selection 86 \npressure through the prophylactic use of antibiotics was applied. It is critical that the 87 \nmistakes made in livestock farming are not repeated in mass insect rearing and that 88 \nalternative methodologies are developed to protect insect cultures and reduce the 89 \nrisk of spreading antibiotic resistant bacteria into the food and feed system.  90 \nRecently, probiotics have been receiving increasing attention as a potential strategy 91 \nto increase the overall health of insect cultures (Dahal et al., 2022; Grau et al., 2017; 92 \nSavio et al., 2022). We know from research in the field of evolutionary ecology that 93 \nbacteria with potentially protective effects, also known as “defensive microbes”, have 94 \nthe potential to provide stable, long-term defence against pathogens (Armitage et al., 95 \n2022; King and Bonsall, 2017; Vorburger and Perlman, 2018). For example, the gut 96 \nmicrobe Enterococcus mundtii works together with its host Galleria mellonella to 97 \ncontrol the host microbiome during meta-morphosis, protecting against the 98 \n.CC-BY 4.0 International licenseavailable under a \nwas not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprint (whichthis version posted April 4, 2025. ; https://doi.org/10.1101/2025.04.04.647199doi: bioRxiv preprint \n\n 5\nproliferation of pathogenic bacteria (Johnston and Rolff, 2015). Similarly, the 99 \nbacterial symbiont, Hamiltonella defensa, protects its aphid host against parasitoid 100 \nwasp attack (Kaech et al., 2022; Kwiatkowski et al., 2012; Wu et al., 2022), with 101 \nimplications for pest control (Donner et al., 2023).  102 \nMany probiotics fall into the category of “defensive microbe”, in that they provide 103 \nprotection against infection (Corr et al., 2007; Deriu et al., 2013; Do et al., 2024; 104 \nFukuda et al., 2011; Piewngam et al., 2021, 2018). These probiotics can be 105 \nharnessed not just to support insect growth and nutritional health, but to protect 106 \nagainst infection and disease spread (Ford and King, 2016). In contrast to antibiotics, 107 \nprobiotics or defensive bacteria present a dynamic and (co)evolving defensive agent, 108 \nthat can change to counter adapt to a pathogen evolving resistance (Ford et al., 109 \n2017, 2016; Kwiatkowski et al., 2012). Furthermore, many human probiotics have 110 \nbeen shown to have beneficial effects on edible insects (e.g. (Lecocq et al., 2021; 111 \nMilanović  et al., 2021)), meaning that supplementation may not only benefit the 112 \nhealth of the insect cultures but has the potential to provide benefits to the human 113 \nconsumer as well.  114 \nOver the last three to four years, there has been a burst of publications on probiotic 115 \nsupplementation in edible insect species. Here we use a meta-analytic approach to 116 \nsummarise and systematically review these data, draw conclusions on what we 117 \nknow so far and highlight literature gaps where we find that more research is 118 \nneeded.  119 \n  120 \nMaterials and Methods 121 \nLiterature search  122 \n.CC-BY 4.0 International licenseavailable under a \nwas not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprint (whichthis version posted April 4, 2025. ; https://doi.org/10.1101/2025.04.04.647199doi: bioRxiv preprint \n\n 6\nOur initial aim was to find all papers reporting the results of studies where edible 123 \ninsects had been supplemented with probiotics. We used the search terms including 124 \n“Tenebrio molitor probiotics”, “Bombyx mori probiotics”, “Hermetia illucens 125 \nprobiotics”, “Apis mellifera probiotics”, “Acheta domesticus probiotics”, “Alphitobius 126 \ndiaperinus probiotics”, “Locusta migratoria probiotics” and “edible insect probiotics” 127 \nto search the databases: PubMed, Google Scholar and Web of science. We then 128 \nlooked at the papers citing the papers we had found and checked the references lists 129 \nof all discovered papers for more potential studies.  130 \nWe noted that the most commonly measured parameters in the studies found were 131 \ninsect growth rate and microbiome diversity (often measured as the Shannon index) 132 \nand therefore decided to carry out two meta-analysis the first asking whether 133 \nprobiotic supplementation influenced insect growth rate and the second asking 134 \nwhether probiotic supplementations influenced insect microbiome diversity. 135 \n Inclusion criteria 136 \nPapers meeting the following criteria were included in the meta-analyses: 137 \n The paper was published in a peer reviewed journal;  138 \n The paper was written in English;  139 \n The host species was insect species either already registered as a novel food 140 \nwithin the EU, which is under evaluation as a novel food in the EU or is regularly 141 \nconsumed as protein source outside of the EU; 142 \n The paper presented data on, for meta-analysis one, insect growth rate or, for 143 \nmeta-analysis two, microbiome diversity, for both a probiotic treated and a control 144 \ngroup; 145 \n.CC-BY 4.0 International licenseavailable under a \nwas not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprint (whichthis version posted April 4, 2025. ; https://doi.org/10.1101/2025.04.04.647199doi: bioRxiv preprint \n\n 7\n Means and standard errors could be extracted, calculated or estimated from the 146 \npresented data either from graphs, raw data or other measures of average and 147 \nspread of the data.  148 \nStatistical analysis  149 \nAll analyses were carried out in R (v4.4.1; R Core Team). For both the growth rate 150 \nand diversity data sets, we calculated Hedge’s g effect sizes and confidence 151 \nintervals using the “esc” package in R (Lüdecke, 2019). We then carried out a 152 \nmultivariant meta-analyses using the rma.mv function in the “metafor” package 153 \n(Viechtbauer, 2025) with “study”, “insect” and “probiotic species” as random factors 154 \nexcept where insect and probiotic species were explicitly being tested as moderator 155 \nvariables. For each of the two data sets we carried out a four moderator variable 156 \nanalyses which each of the follow moderators as a fixed factor. We tested whether 1) 157 \ninsect species, 2) probiotic species, 3) whether the probiotic was a lactobacillus or 158 \nnot and 4) whether the probiotic was gram positive, or gram negative had an impact 159 \non the magnitude or direction of the effect. We plotted funnel plots to visually assess 160 \npublication bias and calculated fail safe N values to estimate the number of datasets 161 \nthat would need to be added to the analysis to change the outcome (Orwin, 1983).  162 \nResults 163 \nGrowth rate  164 \nOverall model for growth rate 165 \nThrough our literature search we collected a total of 71 datasets from 24 publications 166 \npresenting data on growth rate under control and probiotic supplemented conditions 167 \nand that met our inclusion criteria (Table 1). These data showed that, overall, 168 \nsupplementation with probiotics had a positive effect on growth (multi-variant meta-169 \n.CC-BY 4.0 International licenseavailable under a \nwas not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprint (whichthis version posted April 4, 2025. ; https://doi.org/10.1101/2025.04.04.647199doi: bioRxiv preprint \n\n 8\nanalysis: estimate = 2.7036 (ci.lower = 1.2541/ ci.upper = 4.1532), z = 3.6556, p = 170 \n0.0003), suggesting that overall supplementation with probiotics leads to heavier 171 \ninsects (Figure 1). We noticed during our data collection that some feeding material 172 \nwas fermented, this was specifically the case for black soldier fly larvae. As 173 \nfermentation potentially results in probiotic enrichment, we noted these cases in 174 \nTable 1.  175 \nModerator variable analyses for growth rate 176 \nInsect species did not have a significant influence on the overall magnitude or 177 \ndirection of the effect (QM = 2.0767, d.f. = 3, p = 0.5566), however grouping studies 178 \nby insect species demonstrates the disparity in numbers of studies across species 179 \n(Figure 1). While we were able to include 40 datasets using Hermetia illucens as 180 \nhost, a species not yet registered in the EU as a novel food, but one of the insects 181 \nbred on an industrial scale for animal feeding or agro-industrial field purposes, no 182 \nstudies have yet been carried out on three out of the four species that are able to be 183 \nsold for food in the EU. In fact, of all four species registered as novel food in the EU, 184 \nLocusta migratoria, Acheta domesticus, Alphitobius diaperinus and Tenebrio molitor, 185 \nonly T. molitor has been used a host in published studies and in just 10 of the 71 186 \nincluded data sets.  187 \nTo test what subcategories of probiotics might be effective, we tested for differences 188 \nbetween lactobacilli and non-lactobacilli and gram-positive and gram-negative 189 \nbacteria. There was no significant difference between lactobacilli and non-lactobacilli 190 \n(QM = 2.7487, d.f. = 1, p = 0.0973) or between gram-positive and gram-negative 191 \nprobiotics (QM =2.4363, d.f. = 2, p = 0.2958).  192 \nPublication bias for growth rate 193 \n.CC-BY 4.0 International licenseavailable under a \nwas not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprint (whichthis version posted April 4, 2025. ; https://doi.org/10.1101/2025.04.04.647199doi: bioRxiv preprint \n\n 9\nTo test for publication bias we first visualized the relationship between effect sizes 194 \nand standard errors using a funnel plot (Figure 2A). As expected in the absence of 195 \npublication bias (Field and Gillett, 2010), we saw that we tended to have larger 196 \nstandard errors with larger effect sizes and that we had both positive and negative 197 \neffect sizes in our data set. This indicates that there was no evidence of publication 198 \nbias. Furthermore, fail safe N analysis suggested that we would need to add 2393 199 \ndata sets to our study to change the outcome, giving us a high level of confidence in 200 \nour results.  201 \n 202 \nMicrobiome diversity  203 \nOverall model for microbiome diversity  204 \nCompared to growth rate, we found fewer studies reporting data on the effects of 205 \nprobiotic supplementation on microbiome diversity. Nevertheless, we extracted 10 206 \ndatasets from six published papers (Table 2). Overall, there was no significant effect 207 \nof probiotic supplementation on microbiome diversity (estimate = -0.9449(lower ci= -208 \n2.9918/ upper ci = 1.1020), z = -0.9048, p = 0.3656), with studies showing a range of 209 \nboth positive and negative effect sizes (Figure 3). 210 \nModerator variable analysis for microbiome diversity  211 \nAgain here, insect species did not have an impact on the magnitude or direction of 212 \nthe effect (QM = 1.0816, d.f. = 2, p =0.5823), although the breakdown also 213 \nhighlighted that in diversity measures, just as in growth rate, there is a bias in the 214 \ninsect species in which research has been carried out, with the majority of the 215 \nresearch having been done on the black soldier fly, H. illucens (Figure 3). For this 216 \n.CC-BY 4.0 International licenseavailable under a \nwas not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprint (whichthis version posted April 4, 2025. ; https://doi.org/10.1101/2025.04.04.647199doi: bioRxiv preprint \n\n 10\nanalysis there were an insufficient number of studies to meaningfully test further 217 \nmoderator variables.  218 \nPublication bias for microbiome diversity 219 \nWe plotted a funnel plot to visualize potential publication bias for microbiome 220 \ndiversity, however, due to the small number of datasets meeting the criteria for 221 \ninclusion in the meta-analysis for microbiome diversity it is difficult to draw clear 222 \nconclusions (Figure 2B). 223 \nInhibitory effects of probiotics against pathogen infection 224 \nFor the benefits of probiotics to be maximised, we would ideally supplement farmed 225 \ninsects with probiotics that not only stimulate growth and gut health but that also 226 \nprovide protection against pathogens. Unfortunately, to date there are too few 227 \nstudies to carry out a meaningful meta-analysis on this specific question. To our 228 \nknowledge published work on the defensive properties of probiotics against 229 \npathogens has been mainly carried out in T. molitor against entomopathogens. The 230 \nfew published studies present mixed, yet potentially promising results. Lecocq et al 231 \n(Lecocq et al., 2021) showed that Pediococcus pentoceus has inhibitory effects in 232 \nvitro against a range of potentially relevant entomopathogens. Building on this, Dahal 233 \net al (Dahal et al., 2022) showed in vivo that supplementation of T. molitor with P.  234 \npentoceus, not only enhances growth but also seems to provide some degree of 235 \nprotection to T. molitor against mortality induced by the highly virulent 236 \nentomopathogenic fungus Metarhizium brunneum, although the potential probiotic B. 237 \nsubtilus trends towards negatively impacting survival upon infection (Dahal et al., 238 \n2022). The positive results with P. pentoceus were, however, further supported by 239 \nSavio et al (Savio et al., 2024a) who showed that P. pentoceus seems to provide 240 \n.CC-BY 4.0 International licenseavailable under a \nwas not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprint (whichthis version posted April 4, 2025. ; https://doi.org/10.1101/2025.04.04.647199doi: bioRxiv preprint \n\n 11\nprotection to T. molitor against coinfection with Bacillus thuringiensis and 241 \nMetarhizium brunneum, although there are some differences in the outcomes of the 242 \nstudies, which suggests a degree of context dependency. 243 \nThere is also evidence of probiotic protection against pathogens in the silk worm 244 \nBombyx mori. Here supplementation with Lactobacillus casei resulted in protection 245 \nagainst infection by the microsporidian Nosema bombycis (Suraporn and Terenius, 246 \n2021) and supplementation with Lactobacillus lactis protection against infection with 247 \nPseudomonas aeruginosa (Nishida et al., 2016). The mechanism of Lactobacillus 248 \ninduced protection is thought to be via bacterial induced activation of the immune 249 \nsystem (Nishida et al., 2016). 250 \nDiscussion 251 \nThere is a rapidly growing literature base on the supplementation of insects with 252 \nprobiotics. Despite this growing literature, however, we only were able to extract a 253 \nsufficient number of data sets for meta-analysis of two measures: insect growth and 254 \nmicrobiome diversity. While probiotic supplementation significantly enhances insect 255 \ngrowth across tested species, probiotics do not have a clear effect on microbial 256 \ndiversity, with studies showing both positive and negative effects. Furthermore, our 257 \ndata clearly highlight some substantial literature gaps where more data are urgently 258 \nneeded. Most striking is the restricted number of insects in which these kinds of test 259 \nhave been carried out and the lack of studies on three of the four insect species 260 \nregistered as novel food in the EU.  261 \nAcross the data that are available we saw a very strong and robust positive effect of 262 \nprobiotic supplementation on insect growth. Rearing insects on probiotics resulted in 263 \nheavier insects. By decomposing indigestible fibres, producing essential nutrients, 264 \n.CC-BY 4.0 International licenseavailable under a \nwas not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprint (whichthis version posted April 4, 2025. ; https://doi.org/10.1101/2025.04.04.647199doi: bioRxiv preprint \n\n 12\nand enabling metabolic and signalling processes (Chabanol and Gendrin, 2024), the 265 \nprobiotics play a key role in the nutrition and development of the insect host. 266 \nMoreover, they may aid the insects' ability to cope with stress factors (temperature, 267 \ntoxins, chemicals, pathogens, etc.). This is a phenomenon known from other 268 \nmutualisms between insects and microbes (Armitage et al., 2022; Ford and King, 269 \n2016; Vorburger and Perlman, 2018).   270 \nWe did not see a significant effect of probiotic supplementation on microbiome 271 \ndiversity albeit based on a very small sample size. Although microbiota diversity, 272 \noften in the form of a Shannon index, is a commonly reported measure in probiotic 273 \nsupplementation studies, it is difficult to judge what exactly a reduced or increased 274 \nShannon index means for insect health (Johnson and Burnet, 2016). It is often 275 \nassumed that high diversity means a healthy microbiome, likely due do associations 276 \nwith low diversity and human disease (Huttenhower et al., 2012). However, when 277 \nsupplementing with probiotics at high doses we may, to an extent, replace the 278 \nunknown microbiota, which may be healthy or unhealthy, with bacterial species 279 \nknown to benefit health. Thus, reduced diversity may be due to replacement with the 280 \nprobiotic and not necessarily indicate poor or even reduced health. 281 \nThe increase in growth observed across studies when insects are supplemented with 282 \nprobiotics supports the idea that probiotics enhance insect health. What is more this 283 \nbenefit to growth results in increased yields, bringing additional economic 284 \nadvantages. In spite of the indication of enhanced health coming from the growth 285 \nrate data, however, we found that very little data were available on the susceptibility 286 \nto pathogens of probiotic supplemented insects. This is in spite of the fact that 287 \nprobiotics are known to have protective effects in aquaculture settings (Chauhan and 288 \nSingh, 2019; Kuebutornye et al., 2020; Sharifuzzaman and Austin, 2017), and are 289 \n.CC-BY 4.0 International licenseavailable under a \nwas not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprint (whichthis version posted April 4, 2025. ; https://doi.org/10.1101/2025.04.04.647199doi: bioRxiv preprint \n\n 13\neven being considered as an alternative to antibiotics in livestock farming  (Leistikow 290 \net al., 2022). Furthermore, the use of probiotics or “defensive microbes” has been 291 \nadvocated as a method of pathogen control in applied settings in general (Ford and 292 \nKing, 2016) as well as for the specific case of edible insects (Grau et al., 2017; Savio 293 \net al., 2022). The studies that have been carried out looking at potential protection of 294 \ninsects from pathogen attack by probiotics do show promising results, with evidence 295 \nthat some probiotics inhibit the growth of pathogens (Dahal et al., 2022; Lecocq et 296 \nal., 2021; Nishida et al., 2016; Savio et al., 2024a; Suraporn and Terenius, 2021). 297 \nMuch more data are needed, however, to fully explore the potential protective effects 298 \nof candidate probiotics against pathogen infection across the full range of edible 299 \ninsect species. 300 \nHarnessing probiotics as an alternative to antibiotic treatment to prevent the 301 \nestablishment and spread of disease within insect cultures presents a strategy 302 \nthrough which we may avoid the overuse of antibiotics seen in livestock rearing. 303 \nHowever, this strategy is not without risks. We know that probiotics can also be a 304 \nsource of AMR genes (Daniali et al., 2020; Radovanovic et al., 2023; Savio et al., 305 \n2022; Tóth et al., 2021), which could mean that if probiotic treatment is unsuccessful 306 \nand antibiotics do have to be used, efficacy is impacted and the presence of 307 \nprobiotics harbouring such genes may even enhance the rate of AMR evolution. On 308 \nthe other hand, we can use this knowledge to ensure that probiotics are bred 309 \nexclusively from AMR free strains. Also here, this is a question that urgently needs to 310 \nbe explored both experimentally and theoretically as the scale of insect production 311 \nfor food and feed increases.  312 \nThe microbiome is critically associated with a range of behavioural and physiological 313 \nfunction and how the immune response might be linked to probiotic supplementation 314 \n.CC-BY 4.0 International licenseavailable under a \nwas not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprint (whichthis version posted April 4, 2025. ; https://doi.org/10.1101/2025.04.04.647199doi: bioRxiv preprint \n\n 14\nremains unclear. There has been much discussion as to how the host immune 315 \nsystem balances the need to allow the colonisation of mutualistic micro-organisms 316 \nwhile simultaneously fighting off pathogens (Betts et al., 2016; Hanson, 2024), as 317 \nresearch in the field develops, it is important we consider how potential immune 318 \neffects of microbiome manipulation may impact mass rearing.  319 \nFinally, it is important to consider the microbial composition of the feeding material 320 \ngiven to insects grown as food and feed. Our literature research showed that 321 \nparticularly black soldier fly larvae are being fed a range of microbe rich (Mazza et 322 \nal., 2020; Rehman et al., 2019) and fermented material (Somroo et al., 2019; 323 \nWitriana et al., 2023). Fermentation can result in enrichment of probiotics (Heller, 324 \n2001). Given that fermentation of vegetable and other food waste provides an 325 \nopportunity to utilise organic side streams and increase the climate friendliness of 326 \ninsects as food and feed further, the impact of these foods on insect health and 327 \ngrowth warrants further investigation (Antunes et al, unpublished data).  328 \nOur systematic analysis shows that a strong body of research is developing on the 329 \ntopic of probiotic supplementation in edible insects. As research continues, we 330 \nhighlight six key questions that remain to be addressed. These are: 1) Do growth 331 \nenhancing probiotics also provide protection against pathogens? 2) Does probiotic 332 \nsupplementation reduce or increase the risk of AMR? 3) Do the effects we see in the 333 \ninsect species studied so far extent to other edible insect species, particularly those 334 \nbeing sold as novel food in the EU? 4) Does probiotic supplementation impact insect 335 \nbehaviour under mass rearing conditions? 5) How does probiotic supplementation 336 \naffect the insect immune system? 6) What role do diet substrates, and their 337 \nprocessing techniques play in shaping probiotic diversity and stability within the 338 \ninsect gut? Our hope is that continued research in this field, addressing the 339 \n.CC-BY 4.0 International licenseavailable under a \nwas not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprint (whichthis version posted April 4, 2025. ; https://doi.org/10.1101/2025.04.04.647199doi: bioRxiv preprint \n\n 15\nhighlighted questions has the potential to greatly improve the sustainability and 340 \nefficiency of insect rearing for food and feed. 341 \n 342 \nConclusions 343 \nWe have provided the first systematic review of the quantitative impacts of probiotic 344 \nsupplementation on edible insects. Overall probiotics tend to boost insect growth, 345 \nwhich suggests that insect health is enhanced through supplementation. However, 346 \nthere is no clear effect on microbiome diversity. There are also two clear literature 347 \ngaps highlighted by our study. First of all, the taxonomic restriction on the insect 348 \nspecies that have been studied to date. If probiotic supplementation is to be 349 \nconsidered as an implementation strategy to support insect health in commercial 350 \nrearing, it is important that those insects with the highest commercial potential are 351 \ntesting. Secondly, our study highlights a lack of studies investigating the protective 352 \neffects of probiotics against pathogenic infection. As the focus on probiotics in the 353 \nedible insect industry grows, we hope these knowledge gaps will be filled.  354 \nConflict of interest: 355 \nWe have no conflicts of interest.  356 \nFunding statement: 357 \nThis work was funded by an Investionsbank Berlin ProValid grant to CR 358 \n(VAL128/2023). 359 \n 360 \nReferences: 361 \n.CC-BY 4.0 International licenseavailable under a \nwas not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprint (whichthis version posted April 4, 2025. ; https://doi.org/10.1101/2025.04.04.647199doi: bioRxiv preprint \n\n 16\nArmitage, S.A., Genersch, E., McMahon, D.P ., Rafaluk-Mohr, C., Rolff, J., 2022. 362 \nTripartite interactions: how immunity, microbiota and pathogens interact and 363 \naffect pathogen virulence evolution. Current Opinion in Insect Science 50, 364 \n100871. https://doi.org/10.1016/j.cois.2021.12.011 365 \nBetts, A., Rafaluk, C., King, K.C., 2016. Host and Parasite Evolution in a Tangled 366 \nBank. Trends in Parasitology 32, 863–873. 367 \nhttps://doi.org/10.1016/j.pt.2016.08.003 368 \nBroeckx, L., Frooninckx, L., Slegers, L., Berrens, S., Noyens, I., Goossens, S., 369 \nVerheyen, G., Wuyts, A., Van Miert, S., 2021. Growth of Black Soldier Fly 370 \nLarvae Reared on Organic Side-Streams. Sustainability 13, 12953. 371 \nhttps://doi.org/10.3390/su132312953 372 \nCallegari, M., Jucker, C., Fusi, M., Leonardi, M.G., Daffonchio, D., Borin, S., 373 \nSavoldelli, S., Crotti, E., 2020. Hydrolytic Profile of the Culturable Gut 374 \nBacterial Community Associated With Hermetia illucens. Front. Microbiol. 11. 375 \nhttps://doi.org/10.3389/fmicb.2020.01965 376 \nChabanol, E., Gendrin, M., 2024. Insects and microbes: best friends from the 377 \nnursery. Current Opinion in Insect Science 66, 101270. 378 \nhttps://doi.org/10.1016/j.cois.2024.101270 379 \nChauhan, A., Singh, R., 2019. Probiotics in aquaculture: a promising emerging 380 \nalternative approach. Symbiosis 77, 99–113. https://doi.org/10.1007/s13199-381 \n018-0580-1 382 \nCorr, S.C., Li, Y., Riedel, C.U., O’T oole, P .W., Hill, C., Gahan, C.G.M., 2007. 383 \nBacteriocin production as a mechanism for the antiinfective activity of 384 \nLactobacillus salivarius UCC118. Proceedings of the National Academy of 385 \nSciences 104, 7617–7621. https://doi.org/10.1073/pnas.0700440104 386 \n.CC-BY 4.0 International licenseavailable under a \nwas not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprint (whichthis version posted April 4, 2025. ; https://doi.org/10.1101/2025.04.04.647199doi: bioRxiv preprint \n\n 17\nCrippa, M., Solazzo, E., Guizzardi, D., Monforti-Ferrario, F., Tubiello, F.N., Leip, A., 387 \n2021. Food systems are responsible for a third of global anthropogenic GHG 388 \nemissions. Nat Food 2, 198–209. https://doi.org/10.1038/s43016-021-00225-9 389 \nDahal, S., Jensen, A.B., Lecocq, A., 2022. Effect of Probiotics on Tenebrio molitor 390 \nLarval Development and Resistance against the Fungal Pathogen 391 \nMetarhizium brunneum. Insects 13, 1114. 392 \nhttps://doi.org/10.3390/insects13121114 393 \nDaniali, M., Nikfar, S., Abdollahi, M., 2020. Antibiotic resistance propagation through 394 \nprobiotics. Expert Opinion on Drug Metabolism & Toxicology 16, 1207–1215. 395 \nhttps://doi.org/10.1080/17425255.2020.1825682 396 \nDeNieu, M., Mounts, K., Manier, M., 2019. Two gut microbes are necessary and 397 \nsufficient for normal cognition in Drosophila melanogaster. BioRxiv 593723. 398 \nDeriu, E., Liu, J.Z., Pezeshki, M., Edwards, R.A., Ochoa, R.J., Contreras, H., Libby, 399 \nS.J., Fang, F.C., Raffatellu, M., 2013. Probiotic Bacteria Reduce Salmonella 400 \nTyphimurium Intestinal Colonization by Competing for Iron. Cell Host & 401 \nMicrobe 14, 26–37. https://doi.org/10.1016/j.chom.2013.06.007 402 \nDjordjevic, S.P ., Jarocki, V.M., Seemann, T., Cummins, M.L., Watt, A.E., Drigo, B., 403 \nWyrsch, E.R., Reid, C.J., Donner, E., Howden, B.P., 2024. Genomic 404 \nsurveillance for antimicrobial resistance — a One Health perspective. Nat Rev 405 \nGenet 25, 142–157. https://doi.org/10.1038/s41576-023-00649-y 406 \nDo, H., Li, Z.-R., Tripathi, P .K., Mitra, S., Guerra, S., Dash, A., Weerasekera, D., 407 \nMakthal, N., Shams, S., Aggarwal, S., Singh, B.B., Gu, D., Du, Y ., Olsen, R.J., 408 \nLaRock, C., Zhang, W., Kumaraswami, M., 2024. Engineered probiotic 409 \novercomes pathogen defences using signal interference and antibiotic 410 \n.CC-BY 4.0 International licenseavailable under a \nwas not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprint (whichthis version posted April 4, 2025. ; https://doi.org/10.1101/2025.04.04.647199doi: bioRxiv preprint \n\n 18\nproduction to treat infection in mice. Nat Microbiol 9, 502–513. 411 \nhttps://doi.org/10.1038/s41564-023-01583-9 412 \nDonner, S.H., Beekman, M.M., Barth, K., Dicke, M., Zwaan, B.J., Verhulst, E.C., 413 \nPannebakker, B.A., 2023. Facultative endosymbionts of aphids on strawberry 414 \ncrops affect aphid-parasitoid interactions. Biol. Control 188, 105383. 415 \nhttps://doi.org/10.1016/j.biocontrol.2023.105383 416 \nDuffield, K.R., Hunt, J., Sadd, B.M., Sakaluk, S.K., Oppert, B., Rosario, K., Behle, 417 \nR.W., Ramirez, J.L., 2021. Active and Covert Infections of Cricket Iridovirus 418 \nand Acheta domesticus Densovirus in Reared Gryllodes sigillatus Crickets. 419 \nFront. Microbiol. 12. https://doi.org/10.3389/fmicb.2021.780796 420 \nEkroth, A.K.E., Rafaluk-Mohr, C., King, K.C., 2019. Host genetic diversity limits 421 \nparasite success beyond agricultural systems: a meta-analysis. Proceedings 422 \nof the Royal Society B: Biological Sciences 286, 20191811. 423 \nhttps://doi.org/10.1098/rspb.2019.1811 424 \nFasolato, L., Cardazzo, B., Carraro, L., Fontana, F., Novelli, E., Balzan, S., 2018. 425 \nEdible processed insects from e-commerce: Food safety with a focus on the 426 \nBacillus cereus group. Food Microbiology 76, 296–303. 427 \nhttps://doi.org/10.1016/j.fm.2018.06.008 428 \nField, A.P ., Gillett, R., 2010. How to do a meta-analysis. British Journal of 429 \nMathematical and Statistical Psychology 63, 665–694. 430 \nhttps://doi.org/10.1348/000711010X502733 431 \nFord, S.A., Kao, D., Williams, D., King, K.C., 2016. Microbe-mediated host defence 432 \ndrives the evolution of reduced pathogen virulence. Nature Communications 433 \n7, 13430. https://doi.org/10.1038/ncomms13430 434 \n.CC-BY 4.0 International licenseavailable under a \nwas not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprint (whichthis version posted April 4, 2025. ; https://doi.org/10.1101/2025.04.04.647199doi: bioRxiv preprint \n\n 19\nFord, S.A., King, K.C., 2016. Harnessing the Power of Defensive Microbes: 435 \nEvolutionary Implications in Nature and Disease Control. PLOS Pathogens 436 \n12, e1005465. https://doi.org/10.1371/journal.ppat.1005465 437 \nFord, S.A., Williams, D., Paterson, S., King, K.C., 2017. Co-evolutionary dynamics 438 \nbetween a defensive microbe and a pathogen driven by fluctuating selection. 439 \nMolecular Ecology 26, 1778–1789. https://doi.org/10.1111/mec.13906 440 \nFranks, K., Kooienga, E., Sanders, M., Pendarvis, K., Yang, F., Tomberlin, J.K., 441 \nJordan, H.R., 2021. The effect ofRhodococcus rhodochrous supplementation 442 \non black soldier fly (Diptera: Stratiomyidae) development, nutrition, and waste 443 \nconversion. Journal of Insects as Food and Feed 7, 397–408. 444 \nhttps://doi.org/10.3920/JIFF2020.0033 445 \nFukuda, S., Toh, H., Hase, K., Oshima, K., Nakanishi, Y ., Yoshimura, K., Tobe, T., 446 \nClarke, J.M., Topping, D.L., Suzuki, T., Taylor, T.D., Itoh, K., Kikuchi, J., Morita, 447 \nH., Hattori, M., Ohno, H., 2011. Bifidobacteria can protect from 448 \nenteropathogenic infection through production of acetate. Nature 469, 543–449 \n547. https://doi.org/10.1038/nature09646 450 \nGhimpe/i1 eanu, O.M., Pogurschi, E.N., Popa, D.C., Dragomir, N., Dră gotoiu, T ., 451 \nMihai, O.D., Petcu, C.D., 2022. Antibiotic Use in Livestock and Residues in 452 \nFood—A Public Health Threat: A Review. Foods 11, 1430. 453 \nhttps://doi.org/10.3390/foods11101430 454 \nGibson, A.K., Nguyen, A.E., 2021. Does genetic diversity protect host populations 455 \nfrom parasites? A meta‐ analysis across natural and agricultural systems. 456 \nEvolution Letters 5, 16–32. https://doi.org/10.1002/evl3.206 457 \n.CC-BY 4.0 International licenseavailable under a \nwas not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprint (whichthis version posted April 4, 2025. ; https://doi.org/10.1101/2025.04.04.647199doi: bioRxiv preprint \n\n 20\nGrau, T., Vilcinskas, A., Joop, G., 2017. Sustainable farming of the mealworm 458 \nTenebrio molitor for the production of food and feed. Zeitschrift für 459 \nNaturforschung C 72, 337–349. https://doi.org/10.1515/znc-2017-0033 460 \nHanson, M.A., 2024. When the microbiome shapes the host: immune evolution 461 \nimplications for infectious disease. Philosophical Transactions of the Royal 462 \nSociety B: Biological Sciences 379, 20230061. 463 \nhttps://doi.org/10.1098/rstb.2023.0061 464 \nHasan, A., Qazi, J.I., Muzaffer, N., Jabeen, S., Hussain, A., 2022. Effect of Organic 465 \nAcids and Probiotics on Growth of Apis mellifera Workers - ProQuest. 466 \nPakistan Journal of Zoology 54, 2501–3000. 467 \nHazarika, A.K., Kalita, U., 2023. Human consumption of insects. Science 379, 140–468 \n141. https://doi.org/10.1126/science.abp8819 469 \nHeller, K.J., 2001. Probiotic bacteria in fermented foods: product characteristics and 470 \nstarter organisms123. The American Journal of Clinical Nutrition 73, 374s–471 \n379s. https://doi.org/10.1093/ajcn/73.2.374s 472 \nHuttenhower, C., Gevers, D., Knight, R., Abubucker, S., Badger, J.H., Chinwalla, 473 \nA.T., Creasy, H.H., Earl, A.M., FitzGerald, M.G., Fulton, R.S., Giglio, M.G., 474 \nHallsworth-Pepin, K., Lobos, E.A., Madupu, R., Magrini, V., Martin, J.C., 475 \nMitreva, M., Muzny, D.M., Sodergren, E.J., Versalovic, J., Wollam, A.M., 476 \nWorley, K.C., Wortman, J.R., Young, S.K., Zeng, Q., Aagaard, K.M., Abolude, 477 \nO.O., Allen-Vercoe, E., Alm, E.J., Alvarado, L., Andersen, G.L., Anderson, S., 478 \nAppelbaum, E., Arachchi, H.M., Armitage, G., Arze, C.A., Ayvaz, T., Baker, 479 \nC.C., Begg, L., Belachew, T., Bhonagiri, V., Bihan, M., Blaser, M.J., Bloom, T., 480 \nBonazzi, V., Paul Brooks, J., Buck, G.A., Buhay, C.J., Busam, D.A., Campbell, 481 \nJ.L., Canon, S.R., Cantarel, B.L., Chain, P.S.G., Chen, I.-M.A., Chen, L., 482 \n.CC-BY 4.0 International licenseavailable under a \nwas not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprint (whichthis version posted April 4, 2025. ; https://doi.org/10.1101/2025.04.04.647199doi: bioRxiv preprint \n\n 21\nChhibba, S., Chu, K., Ciulla, D.M., Clemente, J.C., Clifton, S.W., Conlan, S., 483 \nCrabtree, J., Cutting, M.A., Davidovics, N.J., Davis, C.C., DeSantis, T .Z., Deal, 484 \nC., Delehaunty, K.D., Dewhirst, F.E., Deych, E., Ding, Y ., Dooling, D.J., 485 \nDugan, S.P., Michael Dunne, W., Scott Durkin, A., Edgar, R.C., Erlich, R.L., 486 \nFarmer, C.N., Farrell, R.M., Faust, K., Feldgarden, M., Felix, V.M., Fisher, S., 487 \nFodor, A.A., Forney, L.J., Foster, L., Di Francesco, V., Friedman, J., Friedrich, 488 \nD.C., Fronick, C.C., Fulton, L.L., Gao, H., Garcia, N., Giannoukos, G., Giblin, 489 \nC., Giovanni, M.Y ., Goldberg, J.M., Goll, J., Gonzalez, A., Griggs, A., Gujja, S., 490 \nKinder Haake, S., Haas, B.J., Hamilton, H.A., Harris, E.L., Hepburn, T.A., 491 \nHerter, B., Hoffmann, D.E., Holder, M.E., Howarth, C., Huang, K.H., Huse, 492 \nS.M., Izard, J., Jansson, J.K., Jiang, H., Jordan, C., Joshi, V., Katancik, J.A., 493 \nKeitel, W.A., Kelley, S.T., Kells, C., King, N.B., Knights, D., Kong, H.H., Koren, 494 \nO., Koren, S., Kota, K.C., Kovar, C.L., Kyrpides, N.C., La Rosa, P.S., Lee, 495 \nS.L., Lemon, K.P., Lennon, N., Lewis, C.M., Lewis, L., Ley, R.E., Li, K., Liolios, 496 \nK., Liu, B., Liu, Y ., Lo, C.-C., Lozupone, C.A., Dwayne Lunsford, R., Madden, 497 \nT., Mahurkar, A.A., Mannon, P .J., Mardis, E.R., Markowitz, V.M., Mavromatis, 498 \nK., McCorrison, J.M., McDonald, D., McEwen, J., McGuire, A.L., McInnes, P ., 499 \nMehta, T., Mihindukulasuriya, K.A., Miller, J.R., Minx, P .J., Newsham, I., 500 \nNusbaum, C., O’Laughlin, M., Orvis, J., Pagani, I., Palaniappan, K., Patel, 501 \nS.M., Pearson, M., Peterson, J., Podar, M., Pohl, C., Pollard, K.S., Pop, M., 502 \nPriest, M.E., Proctor, L.M., Qin, X., Raes, J., Ravel, J., Reid, J.G., Rho, M., 503 \nRhodes, R., Riehle, K.P ., Rivera, M.C., Rodriguez-Mueller, B., Rogers, Y .-H., 504 \nRoss, M.C., Russ, C., Sanka, R.K., Sankar, P., Fah Sathirapongsasuti, J., 505 \nSchloss, J.A., Schloss, P .D., Schmidt, T.M., Scholz, M., Schriml, L., Schubert, 506 \nA.M., Segata, N., Segre, J.A., Shannon, W.D., Sharp, R.R., Sharpton, T.J., 507 \n.CC-BY 4.0 International licenseavailable under a \nwas not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprint (whichthis version posted April 4, 2025. ; https://doi.org/10.1101/2025.04.04.647199doi: bioRxiv preprint \n\n 22\nShenoy, N., Sheth, N.U., Simone, G.A., Singh, I., Smillie, C.S., Sobel, J.D., 508 \nSommer, D.D., Spicer, P ., Sutton, G.G., Sykes, S.M., Tabbaa, D.G., 509 \nThiagarajan, M., Tomlinson, C.M., Torralba, M., Treangen, T.J., Truty, R.M., 510 \nVishnivetskaya, T.A., Walker, J., Wang, L., Wang, Z., Ward, D.V., Warren, W., 511 \nWatson, M.A., Wellington, C., Wetterstrand, K.A., White, J.R., Wilczek-Boney, 512 \nK., Wu, Y., Wylie, K.M., Wylie, T., Yandava, C., Ye, L., Ye, Y ., Yooseph, S., 513 \nYoumans, B.P., Zhang, L., Zhou, Y ., Zhu, Y., Zoloth, L., Zucker, J.D., Birren, 514 \nB.W., Gibbs, R.A., Highlander, S.K., Methé, B.A., Nelson, K.E., Petrosino, 515 \nJ.F., Weinstock, G.M., Wilson, R.K., White, O., The Human Microbiome 516 \nProject Consortium, 2012. Structure, function and diversity of the healthy 517 \nhuman microbiome. Nature 486, 207–214. 518 \nhttps://doi.org/10.1038/nature11234 519 \nJafir, M., Abbas, M., Irfan, M., Zia-Ur-Rehman, M., 2024. Chapter Ten - Greenhouse 520 \ngases emission from edible insect species, in: Rahimpour, M.R., Makarem, 521 \nM.A., Meshksar, M. (Eds.), Advances and Technology Development in 522 \nGreenhouse Gases: Emission, Capture and Conversion. Elsevier, pp. 205–523 \n225. https://doi.org/10.1016/B978-0-443-19231-9.00007-7 524 \nJohnson, K.V.-A., Burnet, P.W.J., 2016. Microbiome: Should we diversify from 525 \ndiversity? Gut Microbes 7, 455–458. 526 \nhttps://doi.org/10.1080/19490976.2016.1241933 527 \nJohnston, P .R., Rolff, J., 2015. Host and Symbiont Jointly Control Gut Microbiota 528 \nduring Complete Metamorphosis. PLOS Pathogens 11, e1005246. 529 \nhttps://doi.org/10.1371/journal.ppat.1005246 530 \n.CC-BY 4.0 International licenseavailable under a \nwas not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprint (whichthis version posted April 4, 2025. ; https://doi.org/10.1101/2025.04.04.647199doi: bioRxiv preprint \n\n 23\nKaech, H., Jud, S., Vorburger, C., 2022. Similar cost of Hamiltonella defensa in 531 \nexperimental and natural aphid-endosymbiont associations. Ecology and 532 \nEvolution 12, e8551. https://doi.org/10.1002/ece3.8551 533 \nKing, K.C., Bonsall, M.B., 2017. The evolutionary and coevolutionary consequences 534 \nof defensive microbes for host-parasite interactions. BMC Evolutionary 535 \nBiology 17, 190. https://doi.org/10.1186/s12862-017-1030-z 536 \nKooienga, E.M., Baugher, C., Currin, M., Tomberlin, J.K., Jordan, H.R., 2020. Effects 537 \nof Bacterial Supplementation on Black Soldier Fly Growth and Development 538 \nat Benchtop and Industrial Scale. Front. Microbiol. 11. 539 \nhttps://doi.org/10.3389/fmicb.2020.587979 540 \nKuebutornye, F.K.A., Abarike, E.D., Lu, Y ., Hlordzi, V., Sakyi, M.E., Afriyie, G., Wang, 541 \nZ., Li, Y ., Xie, C.X., 2020. Mechanisms and the role of probiotic Bacillus in 542 \nmitigating fish pathogens in aquaculture. Fish Physiol Biochem 46, 819–841. 543 \nhttps://doi.org/10.1007/s10695-019-00754-y 544 \nKwiatkowski, M., Engelstädter, J., Vorburger, C., 2012. On Genetic Specificity in 545 \nSymbiont-Mediated Host-Parasite Coevolution. PLOS Computational Biology 546 \n8, e1002633. https://doi.org/10.1371/journal.pcbi.1002633 547 \nLecocq, A., Natsopoulou, M.E., Berggreen, I.E., Eilenberg, J., Heckmann, L.-H.L., 548 \nNielsen, H.V., Stensvold, C.R., Jensen, A.B., 2021. Probiotic properties of an 549 \nindigenous Pediococcus pentosaceus strain on Tenebrio molitor larval growth 550 \nand survival. Journal of Insects as Food and Feed 7, 975–986. 551 \nhttps://doi.org/10.3920/JIFF2020.0156 552 \nLeistikow, K.R., Beattie, R.E., Hristova, K.R., 2022. Probiotics beyond the farm: 553 \nBenefits, costs, and considerations of using antibiotic alternatives in livestock. 554 \nFront. Antibiot. 1. https://doi.org/10.3389/frabi.2022.1003912 555 \n.CC-BY 4.0 International licenseavailable under a \nwas not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprint (whichthis version posted April 4, 2025. ; https://doi.org/10.1101/2025.04.04.647199doi: bioRxiv preprint \n\n 24\nLi, X.-Y ., Mei, C., Luo, X.-Y ., Wulamu, D., Zhan, S., Huang, Y.-P., Yang, H., 2023. 556 \nDynamics of the intestinal bacterial community in black soldier fly larval guts 557 \nand its influence on insect growth and development. Insect Science 30, 947–558 \n963. https://doi.org/10.1111/1744-7917.13095 559 \nLiberti, J., Engel, P ., 2020. The gut microbiota — brain axis of insects. Current 560 \nOpinion in Insect Science, Pests and resistance * Behavioural ecology 39, 6–561 \n13. https://doi.org/10.1016/j.cois.2020.01.004 562 \nLüdecke, D., 2019. esc: Effect Size Computation for Meta Analysis. 563 \nM, S., Krishnamoorthy, S.V., Murugesh, K.A., 2019. Fortification of mulberry leaves 564 \nwith indigenous probiotic bacteria on larval growth and economic traits of 565 \nsilkworm (Bombyx mori l.). J. Entomol. Zool. Stud. 7, 780–784. 566 \nMaciel-Vergara, G., Jensen, A. b., Lecocq, A., Eilenberg, J., 2021. Diseases in edible 567 \ninsect rearing systems. Journal of Insects as Food and Feed 7, 621–638. 568 \nhttps://doi.org/10.3920/JIFF2021.0024 569 \nMarzoli, F., Bertola, M., Pinarelli Fazion, J., Cento, G., Antonelli, P ., Dolzan, B., 570 \nBarco, L., Belluco, S., 2024. A systematic review on the occurrence of 571 \nSalmonella in farmed Tenebrio molitor and Acheta domesticus or their derived 572 \nproducts. International Journal of Food Microbiology 410, 110464. 573 \nhttps://doi.org/10.1016/j.ijfoodmicro.2023.110464 574 \nMazza, L., Xiao, X., ur Rehman, K., Cai, M., Zhang, D., Fasulo, S., Tomberlin, J.K., 575 \nZheng, L., Soomro, A.A., Yu, Z., Zhang, J., 2020. Management of chicken 576 \nmanure using black soldier fly (Diptera: Stratiomyidae) larvae assisted by 577 \ncompanion bacteria. Waste Management 102, 312–318. 578 \nhttps://doi.org/10.1016/j.wasman.2019.10.055 579 \n.CC-BY 4.0 International licenseavailable under a \nwas not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprint (whichthis version posted April 4, 2025. ; https://doi.org/10.1101/2025.04.04.647199doi: bioRxiv preprint \n\n 25\nMeng, L., Ma, L., Xu, J., Rong, K., Peng, N., Zhao, S., 2023. Effect of enzyme-580 \nassisted fermentation on quality, safety, and microbial community of black 581 \nsoldier fly larvae (Hermetia illucens L.) as a novel protein source. Food 582 \nResearch International 174, 113624. 583 \nhttps://doi.org/10.1016/j.foodres.2023.113624 584 \nMilanović , V., Cardinali, F., Belleggia, L., Garofalo, C., Pasquini, M., T avoletti, S., 585 \nRiolo, P ., Ruschioni, S., Isidoro, N., Osimani, A., Aquilanti, L., 2021. 586 \nExploitation of Tenebrio molitor larvae as biological factories for human 587 \nprobiotics, an exploratory study. Journal of Functional Foods 82, 104490. 588 \nhttps://doi.org/10.1016/j.jff.2021.104490 589 \nMoustafa, M.N., Soliman, S.A., 2019. Nutritional Efficiency and Economic Traits of 590 \nSilkworm Bombyx mori, L. Reared on Mulberry Leaves Fortified with 591 \nSynbiotics. Journal of Plant Protection and Pathology 10, 671–675. 592 \nhttps://doi.org/10.21608/jppp.2019.79460 593 \nNishida, S., Ono, Y., Sekimizu, K., 2016. Lactic acid bacteria activating innate 594 \nimmunity improve survival in bacterial infection model of silkworm. Drug 595 \nDiscoveries & Therapeutics 10, 49–56. 596 \nhttps://doi.org/10.5582/ddt.2016.01022 597 \nOrwin, R.G., 1983. A Fail-Safe N for Effect Size in Meta-Analysis. Journal of 598 \nEducational Statistics 8, 157–159. https://doi.org/10.2307/1164923 599 \nPei, Y., Zhao, S., Chen, X., Zhang, J., Ni, H., Sun, M., Lin, H., Liu, X., Chen, H., 600 \nYang, S., 2022. Bacillus velezensis EEAM 10B Strengthens Nutrient 601 \nMetabolic Process in Black Soldier Fly Larvae (Hermetia illucens) via 602 \nChanging Gut Microbiome and Metabolic Pathways. Front. Nutr. 9. 603 \nhttps://doi.org/10.3389/fnut.2022.880488 604 \n.CC-BY 4.0 International licenseavailable under a \nwas not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprint (whichthis version posted April 4, 2025. ; https://doi.org/10.1101/2025.04.04.647199doi: bioRxiv preprint \n\n 26\nPiewngam, P., Chiou, J., Ling, J., Liu, R., Pupa, P., Zheng, Y ., Otto, M., 2021. 605 \nEnterococcal bacteremia in mice is prevented by oral administration of 606 \nprobiotic Bacillus spores. Science Translational Medicine 13, eabf4692. 607 \nhttps://doi.org/10.1126/scitranslmed.abf4692 608 \nPiewngam, P., Zheng, Y., Nguyen, T.H., Dickey, S.W., Joo, H.-S., Villaruz, A.E., 609 \nGlose, K.A., Fisher, E.L., Hunt, R.L., Li, B., Chiou, J., Pharkjaksu, S., 610 \nKhongthong, S., Cheung, G.Y.C., Kiratisin, P ., Otto, M., 2018. Pathogen 611 \nelimination by probiotic Bacillus via signalling interference. Nature 562, 532–612 \n537. https://doi.org/10.1038/s41586-018-0616-y 613 \nPrecup, G., Ververis, E., Azzollini, D., Rivero-Pino, F., Zakidou, P ., Germini, A., 2022. 614 \nThe Safety Assessment of Insects and Products Thereof As Novel Foods in 615 \nthe European Union, in: Scaffardi, L., Formici, G. (Eds.), Novel Foods and 616 \nEdible Insects in the European Union: An Interdisciplinary Analysis. Springer 617 \nInternational Publishing, Cham, pp. 123–146. https://doi.org/10.1007/978-3-618 \n031-13494-4_7 619 \nRadovanovic, M., Kekic, D., Gajic, I., Kabic, J., Jovicevic, M., Kekic, N., Opavski, N., 620 \nRanin, L., 2023. Potential influence of antimicrobial resistance gene content in 621 \nprobiotic bacteria on the gut resistome ecosystems. Front. Nutr. 10. 622 \nhttps://doi.org/10.3389/fnut.2023.1054555 623 \nRaka, R.N., Zhang, L., Chen, R., Xue, X., 2024. Antibiotic Resistance Genes in 624 \nGlobal Food Transformation System: Edible Insects vs. Livestock. Foods 13, 625 \n3257. https://doi.org/10.3390/foods13203257 626 \nRawat, N., Anjali, Shreyata, Sabu, B., Jamwal, R., Devi, P .P ., Yadav, K., Raina, H.S., 627 \nRajagopal, R., 2023. Understanding the role of insects in the acquisition and 628 \n.CC-BY 4.0 International licenseavailable under a \nwas not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprint (whichthis version posted April 4, 2025. ; https://doi.org/10.1101/2025.04.04.647199doi: bioRxiv preprint \n\n 27\ntransmission of antibiotic resistance. Science of The Total Environment 858, 629 \n159805. https://doi.org/10.1016/j.scitotenv.2022.159805 630 \nRehman, K. ur, Ur Rehman, R., Somroo, A.A., Cai, M., Zheng, L., Xiao, X., Ur 631 \nRehman, A., Rehman, A., Tomberlin, J.K., Yu, Z., Zhang, J., 2019. Enhanced 632 \nbioconversion of dairy and chicken manure by the interaction of exogenous 633 \nbacteria and black soldier fly larvae. Journal of Environmental Management 634 \n237, 75–83. https://doi.org/10.1016/j.jenvman.2019.02.048 635 \nRizou, E., Kalogiouri, N., Bisba, M., Papadimitriou, A., Kyrila, G., Lazou, A., 636 \nAndreadis, S., Hatzikamari, M., Mourtzinos, I., Touraki, M., 2022. Amelioration 637 \nof growth, nutritional value, and microbial load of Tenebrio molitor 638 \n(Coleoptera: Tenebrionidae) through probiotic supplemented feed. Eur Food 639 \nRes Technol 248, 727–739. https://doi.org/10.1007/s00217-021-03925-5 640 \nSavio, C., Herren, P., Rejasse, A., Rios, A., Bourelle, W., Bruun-Jensen, A., Lecocq, 641 \nA., van Loon, J.J.A., Nielsen-LeRoux, C., 2024a. Minor impact of probiotic 642 \nbacteria and egg white on Tenebrio molitor growth, microbial composition, and 643 \npathogen infection. Front. Insect Sci. 4. 644 \nhttps://doi.org/10.3389/finsc.2024.1334526 645 \nSavio, C., Herren, P., Rejasse, A., van Loon, J.J.A., Nielsen-Leroux, C., 2024b. 646 \nBacillus thuringiensis serovar morrisoni biovar tenebrionis impact and 647 \npersistence in Tenebrio molitor larvae. Journal of Insects as Food and Feed 648 \n10, 1917–1931. https://doi.org/10.1163/23524588-00001028 649 \nSavio, C., Mugo-Kamiri, L., Upfold, J.K., 2022. Bugs in Bugs: The Role of Probiotics 650 \nand Prebiotics in Maintenance of Health in Mass-Reared Insects. Insects 13, 651 \n376. https://doi.org/10.3390/insects13040376 652 \n.CC-BY 4.0 International licenseavailable under a \nwas not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprint (whichthis version posted April 4, 2025. ; https://doi.org/10.1101/2025.04.04.647199doi: bioRxiv preprint \n\n 28\nSchretter, C.E., Vielmetter, J., Bartos, I., Marka, Z., Marka, S., Argade, S., 653 \nMazmanian, S.K., 2018. A gut microbial factor modulates locomotor behaviour 654 \nin Drosophila. Nature 563, 402–406. https://doi.org/10.1038/s41586-018-655 \n0634-9 656 \nSharifuzzaman, S. m., Austin, B., 2017. Probiotics for Disease Control in 657 \nAquaculture, in: Diagnosis and Control of Diseases of Fish and Shellfish. John 658 \nWiley & Sons, Ltd, pp. 189–222. https://doi.org/10.1002/9781119152125.ch8 659 \nSomroo, A.A., ur Rehman, K., Zheng, L., Cai, M., Xiao, X., Hu, S., Mathys, A., Gold, 660 \nM., Yu, Z., Zhang, J., 2019. Influence of Lactobacillus buchneri on soybean 661 \ncurd residue co-conversion by black soldier fly larvae (Hermetia illucens) for 662 \nfood and feedstock production. Waste Management 86, 114–122. 663 \nhttps://doi.org/10.1016/j.wasman.2019.01.022 664 \nSpringmann, M., Clark, M., Mason-D’Croz, D., Wiebe, K., Bodirsky, B.L., Lassaletta, 665 \nL., de Vries, W., Vermeulen, S.J., Herrero, M., Carlson, K.M., Jonell, M., 666 \nTroell, M., DeClerck, F., Gordon, L.J., Zurayk, R., Scarborough, P., Rayner, 667 \nM., Loken, B., Fanzo, J., Godfray, H.C.J., Tilman, D., Rockström, J., Willett, 668 \nW., 2018. Options for keeping the food system within environmental limits. 669 \nNature 562, 519–525. https://doi.org/10.1038/s41586-018-0594-0 670 \nSuraporn, S., Sangsuk, W., Chanhan, P., Promma, S., 2015. Effects of Probiotic 671 \nBacteria on the Growth Parameters of the Thai Silkworm, Bombyxmori. Thai 672 \nJournal of Agricultural Science 48, 29–33. 673 \nSuraporn, S., Terenius, O., 2021. Supplementation of Lactobacillus casei reduces 674 \nthe mortality of Bombyx mori larvae challenged by Nosema bombycis. BMC 675 \nRes Notes 14, 398. https://doi.org/10.1186/s13104-021-05807-1 676 \n.CC-BY 4.0 International licenseavailable under a \nwas not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprint (whichthis version posted April 4, 2025. ; https://doi.org/10.1101/2025.04.04.647199doi: bioRxiv preprint \n\n 29\nTaha, R.H., A., S.S., Kamel, H.M., 2017. Micro-Organisms Supplementation to 677 \nMulberry Silkworm, Bombyx mori L. Egyptian Academic Journal of Biological 678 \nSciences. A, Entomology 10, 57–64. 679 \nhttps://doi.org/10.21608/eajbsa.2017.12673 680 \nTarwater, P.M., Martin, C.F., 2001. Effects of population density on the spread of 681 \ndisease. Complexity 6, 29–36. https://doi.org/10.1002/cplx.10003 682 \nTóth, A.G., Csabai, I., Judge, M.F., Maróti, G., Becsei, Á., Spisák, S., Solymosi, N., 683 \n2021. Mobile Antimicrobial Resistance Genes in Probiotics. Antibiotics 10, 684 \n1287. https://doi.org/10.3390/antibiotics10111287 685 \nUnban, K., Klongklaew, A., Kodchasee, P ., Pamueangmun, P., Shetty, K., 686 \nKhanongnuch, C., 2022. Enterococci as Dominant Xylose Utilizing Lactic Acid 687 \nBacteria in Eri Silkworm Midgut and the Potential Use of Enterococcus hirae 688 \nas Probiotic for Eri Culture. Insects 13, 136. 689 \nhttps://doi.org/10.3390/insects13020136 690 \nUnited Nations Department of Economic and Social Affairs, Population Division, 691 \n2022. World Population Prospects 2022: Summary of Results. UN 692 \nDESA/POP/2022/TR/NO. 3. 693 \nvan Huis, A., 2013. Potential of Insects as Food and Feed in Assuring Food Security. 694 \nAnnual Review of Entomology 58, 563–583. https://doi.org/10.1146/annurev-695 \nento-120811-153704 696 \nvan Huis, A., Gasco, L., 2023. Insects as feed for livestock production. Science 379, 697 \n138–139. https://doi.org/10.1126/science.adc9165 698 \nVan Peer, M., Frooninckx, L., Coudron, C., Berrens, S., Álvarez, C., Deruytter, D., 699 \nVerheyen, G., Van Miert, S., 2021. Valorisation Potential of Using Organic 700 \n.CC-BY 4.0 International licenseavailable under a \nwas not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprint (whichthis version posted April 4, 2025. ; https://doi.org/10.1101/2025.04.04.647199doi: bioRxiv preprint \n\n 30\nSide Streams as Feed for Tenebrio molitor, Acheta domesticus and Locusta 701 \nmigratoria. Insects 12, 796. https://doi.org/10.3390/insects12090796 702 \nViechtbauer, W., 2025. metafor: Meta-Analysis Package for R. 703 \nVorburger, C., Perlman, S.J., 2018. The role of defensive symbionts in host–parasite 704 \ncoevolution. Biological Reviews 93, 1747–1764. 705 \nhttps://doi.org/10.1111/brv.12417 706 \nVrontaki, M., Adamaki-Sotiraki, C., Rumbos, C.I., Anastasiadis, A., Athanassiou, 707 \nC.G., 2024. Valorization of local agricultural by-products as nutritional 708 \nsubstrates for Tenebrio molitor larvae: A sustainable approach to alternative 709 \nprotein production. Environ Sci Pollut Res 31, 35760–35768. 710 \nhttps://doi.org/10.1007/s11356-024-33564-8 711 \nWitriana, N.I., Ardyati, T., Jatmiko, Y.D., 2023. The Use of probiotics in fermenting 712 \nfood wastes for production of black soldier fly larvae (Hermetia illucens L.; 713 \nDiptera: Stratiomyidae). Berkala Penelitian Hayati 29, 99–105. 714 \nhttps://doi.org/10.23869/bphjbr.29.3.20234 715 \nWu, T ., Monnin, D., Lee, R.A.R., Henry, L.M., 2022. Local adaptation to hosts and 716 \nparasitoids shape Hamiltonella defensa genotypes across aphid species. 717 \nProceedings of the Royal Society B: Biological Sciences 289, 20221269. 718 \nhttps://doi.org/10.1098/rspb.2022.1269 719 \nWynants, E., Frooninckx, L., Van Miert, S., Geeraerd, A., Claes, J., Van 720 \nCampenhout, L., 2019. Risks related to the presence of Salmonella sp. during 721 \nrearing of mealworms (Tenebrio molitor) for food or feed: Survival in the 722 \nsubstrate and transmission to the larvae. Food Control 100, 227–234. 723 \nhttps://doi.org/10.1016/j.foodcont.2019.01.026 724 \n.CC-BY 4.0 International licenseavailable under a \nwas not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprint (whichthis version posted April 4, 2025. ; https://doi.org/10.1101/2025.04.04.647199doi: bioRxiv preprint \n\n 31\nYu, G., Cheng, P ., Chen, Yanhong, Li, Y., Yang, Z., Chen, Yuanfeng, Tomberlin, J.K., 725 \n2011. Inoculating Poultry Manure With Companion Bacteria Influences Growth 726 \nand Development of Black Soldier Fly (Diptera: Stratiomyidae) Larvae. 727 \nEnvironmental Entomology 40, 30–35. https://doi.org/10.1603/EN10126 728 \nYuan, S., Sun, Y., Chang, W., Zhang, J., Sang, J., Zhao, J., Song, M., Qiao, Y., 729 \nZhang, C., Zhu, M., Tang, Y ., Lou, H., 2023. The silkworm (Bombyx mori) gut 730 \nmicrobiota is involved in metabolic detoxification by glucosylation of plant 731 \ntoxins. Commun Biol 6, 1–13. https://doi.org/10.1038/s42003-023-05150-0 732 \nZeng, Z., Tong, X., Yang, Y ., Zhang, Y ., Deng, S., Zhang, G., Dai, F., 2024. 733 \nPediococcus pentosaceus ZZ61 enhances growth performance and 734 \npathogenic resistance of silkworm Bombyx mori by regulating gut microbiota 735 \nand metabolites. Bioresource Technology 402, 130821. 736 \nhttps://doi.org/10.1016/j.biortech.2024.130821 737 \nZhong, J., Zhang, F., Peng, Y., Ji, Z., Li, H., Li, S., Zhang, X., Shi, Q., Zhang, J., 738 \n2017. Mixed culture of probiotics on a solid-state medium: An efficient method 739 \nto produce an affordable probiotic feed additive. Biotechnol Bioproc E 22, 740 \n758–766. https://doi.org/10.1007/s12257-017-0038-y 741 \nZilber-Rosenberg, I., Rosenberg, E., 2008. Role of microorganisms in the evolution 742 \nof animals and plants: the hologenome theory of evolution. Fems Microbiol. 743 \nRev. 32, 723–735. https://doi.org/10.1111/j.1574-6976.2008.00123.x 744 \n 745 \n 746 \n 747 \nFigure legends:  748 \n.CC-BY 4.0 International licenseavailable under a \nwas not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprint (whichthis version posted April 4, 2025. ; https://doi.org/10.1101/2025.04.04.647199doi: bioRxiv preprint \n\n 32\nFigure 1. Forest plots showing effect sizes and confidence intervals for differences in 749 \ninsect growth with and without probiotic supplementation with effect sizes and 95% 750 \nconfidence intervals for each data set included in the study, negative effect sizes 751 \nshow a lower body weight in insects reared with probiotics than reared without and 752 \npositive effect sizes show increased growth in insects reared with probiotic 753 \nsupplementation. Colour depicts the insect species and size the sample size. 754 \nFigure 2. Funnel plots showing observed outcomes (effect sizes) and standard errors 755 \nfor: (A) the first meta-analysis comparing growth; (B 756 \n 757 \n) the second meta-analysis comparing microbiome diversity. The white triangle 758 \nshows the expected distribution in the absence of publication bias. 759 \nFigure 3. Forest plots showing effect sizes and confidence intervals for differences in 760 \ninsect microbiome diversity with and without probiotic supplementation with effect 761 \nsizes and 95% confidence intervals for each data set included in the study, negative 762 \neffect sizes show a lower microbial diversity in insects reared with probiotics than 763 \nreared without and positive effect sizes show increased microbial diversity in insects 764 \nreared with probiotic supplementation. Colour depicts the insect species and size the 765 \nsample size. 766 \n 767 \n 768 \n 769 \n 770 \n 771 \n.CC-BY 4.0 International licenseavailable under a \nwas not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprint (whichthis version posted April 4, 2025. ; https://doi.org/10.1101/2025.04.04.647199doi: bioRxiv preprint \n\n 33\n \nTable 1. Included studies and relevant information on the insect studies included in the first meta-analysis comparing body weights. \nReference No. Insect \nspecies \nBacterial species  Lactobacillus? Gram positive \nor negative? \nWeight \ncontrol \nSE \ncontrol \nN \ncontrol \nWeight \nprobiotic \nSE \nprobiotic \nN \nprobioti\nc \nFerm\nented \ndiet \nLecocq et al. \n2021 (Lecocq \net al., 2021) \n1 \nTenebrio \nmolitor \nPediococcus \npentosaceus  \nyes positive 0,117 g ± 0,002 g  8 0,149 g ± 0,005 g 8 no \nRizou et al. \n2022 (Rizou et \nal., 2022) \n2 \nTenebrio \nmolitor \nBacillus subtilis  no positive 160,3 mg  ± 5,7 mg 4 205,5 mg ± 3,8 mg 4 no \nRizou et al. \n2022 (Rizou et \nal., 2022) \n3 Tenebrio \nmolitor \nBacillus toyonensis  no positive 157,4 mg\n ± 4,7 mg 4 179,1 mg ± 0 mg 4 no \nRizou et al. \n2022 (Rizou et \nal., 2022) \n4 Tenebrio \nmolitor \nEnterococcus faecalis  no positive 146,1 mg ± 5,7 mg 4 197,9 mg ± 6,6 mg 4 no \nDahal et al. \n2022 (Dahal et \nal., 2022) \n5 Tenebrio \nmolitor \nPediococcus \npentosaceus  \nyes positive 0,041 g ± 0,002 g  10 0,133 g ± 0,003 g 10 no \nDahal et al. \n2022 Dahal et \nal., 2022) \n6 Tenebrio \nmolitor \nBacillus subtilis  no positive 0,131 g ± 0,004 g  10 0,072 g ± 0 g 10 no \nDahal et al. \n2022 Dahal et \nal., 2022) \n7 Tenebrio \nmolitor \nEnterococcus faecium no positive 0,104 g ± 0,004 g  10 0,131 g ± 0,004 g 10 no \nSuraporn et al. \n2015 (Suraporn \n8 Bombyx Lactobacillus yes positive 1,18 g ± 0,05 g 3 1,26 g ± 0,05 g 3 no \n98 \n.CC-BY 4.0 International licenseavailable under a \nwas not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprint (whichthis version posted April 4, 2025. ; https://doi.org/10.1101/2025.04.04.647199doi: bioRxiv preprint \n\n 34\net al., 2015) mori acidophilus \nSuraporn et al. \n2021 (Suraporn \nand T erenius, \n2021) \n9 Bombyx \nmori \nLactobacillus casei  yes positive 2,55 g ± 0,06 g 3 2,84 g ± 0,06 g  3 no \nTaha et al. \n2017 (T aha et \nal., 2017) \n10 Bombyx \nmori \nBifidobacterium \nbifidum  \nno positive 2,66 g ± 0 g 4 3,02 g ± 0,02 g 4 no \nSaranya et al. \n2019 (M et al., \n2019) \n11 Bombyx \nmori \nStaphylococcus \ngallinarum  \nno positive 3,6 g ± 0,008 g  5 4,12 g ± 0,014 g 5 no \nSaranya et al. \n2019 (M et al., \n2019) \n12 \nBombyx \nmori \nStaphylococcus \narlettae  \nno positive 3,6 g ± 0,008 g  5 3,89 g ± 0,008 g 5 no \nSomroo et al. \n2019 (Somroo \net al., 2019) \n13 Hermetia \nillucens \nLactobacillus buchneri\n yes positive 126,4 g ± 1,1 g 3 146,4 g ± 1,8 g 3 yes \nWitriana et al. \n2023 (Witriana \net al., 2023) \n14 Hermetia \nillucens \nLactiplantibacillus \nplantarum \nyes positive 212,1 g ± 11,4 g 3 251,9 g ± 2,6 g 3 Ferm\nented \nwith \nthe \nprobi\notics \nWitriana et al. \n2023  (Witriana \net al., 2023) \n15 \nHermetia \nillucens \nLimosilactobacillus \nfermentum \nyes positive 212,1 g ± 11,4 g 3 213,6 g ± 14,5 g 3 Ferm\nented \nwith \nthe \nprobi\notics \n.CC-BY 4.0 International licenseavailable under a \nwas not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprint (whichthis version posted April 4, 2025. ; https://doi.org/10.1101/2025.04.04.647199doi: bioRxiv preprint \n\n 35\nWitriana et al. \n2023  (Witriana \net al., 2023) \n16 Hermetia \nillucens \nL. plantarum, L. \nfermentum \nyes positive 212,1 g ± 11,4 g 3 287,3 g ± 6,8 g 3 Ferm\nented \nwith \nthe \nprobi\notics \nCallegari et al. \n2020 (Callegari \net al., 2020) \n17 Hermetia \nillucens \nBacillus licheniformis no positive 1,59 g ± 0,88 g 3 1,88 g ± 0,11 g 3 no \nCallegari et al. \n2020 (Callegari \net al., 2020) \n18 \nHermetia \nillucens \nStenotrophomonas \nmaltophilia \nno negative 1,59 g ± 0,88 g 3 1,59 g ± 0,14 g 3 no \nCallegari et al. \n2020 (Callegari \net al., 2020) \n19 Hermetia \nillucens \nEscherichia coli no negative 1,59 g ± 0,88 g 3 1,92 g ± 0,08 g 3 no \nCallegari et al. \n2020 (Callegari \net al., 2020) \n20 Hermetia \nillucens \nB. licheniformis, S. \nmaltophilia \nno positive, negative  1,59 g ± 0,88 g 3 1,76 g  ± 0,13 g 3 no \nHasan et al. \n2022 (Hasan et \nal., 2022) \n21 Apis \nmellifera \nLactobacillus \nrhamnosus \nyes positive 118,7 mg  ± 9,6 mg 3 128,2 mg ± 7,4 mg 3 no \nHasan et al. \n2022  (Hasan \net al., 2022) \n22 Apis \nmellifera \nLactobacillus brevis yes positive 119,9 mg  ± 9,5 mg 3 124,1 mg ± 5,3 mg 3 no \nHasan et al. \n2022  (Hasan \net al., 2022) \n23 \nApis \nmellifera \nBacillus clausii no positive 119,9 mg  ± 9,5 mg 3 138,9 mg  ± 6,5 mg  3 no \nSavio et al. \n2024 (Savio et \n25 Tenebrio \nmolitor \nLactiplantibacillus \nplantarum \nyes positive 72,8 mg ± 9,7 mg 3 90,0 mg ± 8,9 mg 3 no \n.CC-BY 4.0 International licenseavailable under a \nwas not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprint (whichthis version posted April 4, 2025. ; https://doi.org/10.1101/2025.04.04.647199doi: bioRxiv preprint \n\n 36\nal., 2024a) \nSavio et al. \n2024 (Savio et \nal., 2024a) \n26 Tenebrio \nmolitor \nPediococcus \npentosaceus \nyes positive 72,8 mg ± 9,7 mg 3 89,7 mg ± 10,8 mg  3 no \nZhong et al. \n2017 (Zhong et \nal., 2017) \n27 Tenebrio \nmolitor \nBifidobacterium \nbifidum, Clostridium \nbutyricum, Bacillus \nsubtilis, Bacillus \nlicheniformis \nno positive 8,5 g ± 0,3 g 6 9,00 g ± 0,16g 6 no \nYu et al. 2011 \n(Yu et al., 2011) \n28 Hermetia \nillucens \nBacillus subtilis S15 no positive 0,008 g ± 0,012 g  3 0,095 g ± 0,015 g 3 manu\nre \nYu et al. 2011 \n(Yu et al., 2011) \n29 Hermetia \nillucens \nBacillus subtilis S16 no positive 0,008 g ± 0,012 g  3 0,092 g ± 0,013 g 3 manu\nre \nYu et al. 2011 \n(Yu et al., 2011) \n30 Hermetia \nillucens \nBacillus subtilis S19 no positive 0,008 g ± 0,012 g  3 0,087 g ± 0,017 g 3 manu\nre \nYu et al. 2011 \n(Yu et al., 2011) \n31 Hermetia \nillucens \nBacillus natto D1 no positive 0,008 g ± 0,012 g  3 0,0848 g ± 0,0014 \ng \n3 manu\nre \nKooienga et al. \n2020 \n(Kooienga et \nal., 2020) \n32 Hermetia \nillucens \nArthrobacter no positive 10,2 g ± 2,12 g 4 9,6 g ± 0,4 g 4 no \nKooienga et al. \n2020 \n(Kooienga et \nal., 2020) \n33 Hermetia \nillucens \nBifidobacterium breve no positive 14,3 g ± 2,00 g 3 1,05 g ± 0,13 g 3 no \nKooienga et al. \n2020  \n(Kooienga et \nal., 2020) \n34 Hermetia \nillucens \nRhodococcus \nrhodochrous \nno positive 10,2 g ± 2,15 g 4 9,0 g ± 0,403 g 4 no \n.CC-BY 4.0 International licenseavailable under a \nwas not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprint (whichthis version posted April 4, 2025. ; https://doi.org/10.1101/2025.04.04.647199doi: bioRxiv preprint \n\n 37\nKooienga et al. \n2020  \n(Kooienga et \nal., 2020) \n35 Hermetia \nillucens \nArthrobacter no positive 2,88 g ± 0,13 g 3 2,54 g ± 0,11 g 3 no \nMoustafa et al. \n2019 (Moustafa \nand Soliman, \n2019) \n36 Bombyx \nmori \nLactobacillus \nrhamnosus \nyes positive 2,325 g ± 0,114 g  3 3,51 g ± 0,31 g 3 no \nMoustafa et al. \n2019 (Moustafa \nand Soliman, \n2019) \n37 Bombyx \nmori \nBifidobacterium \nbifidum \nno positive 2,325 g ± 0,114 g  3 3,775 g ± 0,18 g 3 no \nZeng et al. \n2024 (Zeng et \nal., 2024) \n38 Bombyx \nmori \nBacillus spp. no positive 1,5 g ± 0,2 g 3 1,55 g ± 0,09 g 3 no \nZeng et al. \n2024 (Zeng et \nal., 2024) \n39 Bombyx \nmori \nBacillus cereus no positive 1,5 g ± 0,2 g 3 1,50 g ± 0,08 g 3 no \nZeng et al. \n2024 (Zeng et \nal., 2024) \n40 Bombyx \nmori \nBacillus huizhouensi no positive 1,5 g ± 0,2 g 3 1,19 g ± 0,09 g 3 no \nZeng et al. \n2024 (Zeng et \nal., 2024) \n41 Bombyx \nmori \nEnterococcus \ncasseliflavus  \nno positive 1,5 g ± 0,2 g 3 1,68 g ± 0,06 g 3 no \nZeng et al. \n2024 (Zeng et \nal., 2024) \n42 \nBombyx \nmori \nEnterococcus mundtii \n75-4 \nno positive 1,5 g ± 0,2 g 3 1,59 g ± 0,07 g 3 no \nZeng et al. \n2024 (Zeng et \n43 Bombyx \nmori \nEnterococcus mundtii \nX-2 \nno positive 1,5 g ± 0,2 g 3 1,29 g ± 0,07 g 3 no \n.CC-BY 4.0 International licenseavailable under a \nwas not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprint (whichthis version posted April 4, 2025. ; https://doi.org/10.1101/2025.04.04.647199doi: bioRxiv preprint \n\n 38\nal., 2024) \nZeng et al. \n2024 (Zeng et \nal., 2024) \n44 Bombyx \nmori \nPediococcus \npentosaceus \nyes positive 2,325 g ± 0,114 g  3 3,78 g ± 0,18 g 3 no \nZeng et al. \n2024 (Zeng et \nal., 2024) \n45 Bombyx \nmori \nEnterococcus faecium \nNL5-5 \nno positive 1,5 g ± 0,2 g 3 1,36 g ± 0,08 g 3 no \nZeng et al. \n2024 (Zeng et \nal., 2024) \n46 Bombyx \nmori \nEnterococcus faecium \nML21 \nno positive 1,5 g ± 0,2 g 3 1,41 g ± 0,03 g 3 no \nZeng et al. \n2024 (Zeng et \nal., 2024) \n47 Bombyx \nmori \nEnterococcus faecium \nCA01 \nno positive 1,5 g ± 0,2 g 3 1,48 g ± 0,03 g 3 no \nUnban et al. \n2022 (Unban et \nal., 2022) \n48 Bombyx \nmori \nEnterococcus hirae no positive 4,2 g ± 0,04 g 3 4,53 g ± 0,07 g 3 no \nRehman et al. \n2019 (Rehman \net al., 2019) \n49 Hermetia \nillucens \nPaenibacillus \npolymyxa \nno positive 90,46 g ± 0,89 g 3 97,69 g ± 0,22 g 3 manu\nre \nRehman et al. \n2019 Rehman \net al., 2019) \n50 Hermetia \nillucens \nBacillus spp SMO1 no positive 90,46 g ± 0,89 g 3 105,61 g ± 0,56 g 3 manu\nre \nRehman et al. \n2019 Rehman \net al., 2019) \n51 Hermetia \nillucens \nBacillus spp. SMO\n2 no positive 90,46 g ± 0,89 g 3 102,48 g ± 0,66 g 3 manu\nre \nRehman et al. \n2019 Rehman \net al., 2019) \n52 Hermetia \nillucens \nBacillus spp. MRO2 no positive 90,46 g ± 0,89 g 3 112,49 g ± 0,69 g 3 manu\nre \n.CC-BY 4.0 International licenseavailable under a \nwas not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprint (whichthis version posted April 4, 2025. ; https://doi.org/10.1101/2025.04.04.647199doi: bioRxiv preprint \n\n 39\nRehman et al. \n2019 Rehman \net al., 2019) \n53 Hermetia \nillucens \nBacillus spp. SMO4 no positive 90,46 g ± 0,89 g 3 101,94 g ± 1,01 g 3 manu\nre \nFranks et al. \n2021 (Franks et \nal., 2021) \n54 \nHermetia \nillucens \nRhodococcus \nrhodochrous \nno positive 882.9 mg  ± 29,59 \nmg \n3 1498,1 mg ± 52,62 \nmg \n3 no \nMazza et al. \n2020 (Mazza et \nal., 2020) \n55 Hermetia \nillucens \nBacillus subtilis no positive 0,089 g ± 0,004 g\n 3 0,118 g ± 0,004 g 3 manu\nre \nMazza et al. \n2020  (Mazza \net al., 2020) \n56 Hermetia \nillucens \nKocuria marina no positive 0,089 g ± 0,004 g  3 0,113 g ± 0,005 g 3 manu\nre \nMazza et al. \n2020  (Mazza \net al., 2020) \n57 Hermetia \nillucens \nMicrococcus luteus no positive 0,089 g ± 0,004 g  3 0,114 g ± 0,006 g 3 manu\nre \nMazza et al. \n2020  (Mazza \net al., 2020) \n58 Hermetia \nillucens \nEnterococcus faecalis no positive 0,089 g ± 0,004 g  3 0,110 g ± 0,010 g 3 manu\nre \nMazza et al. \n2020  (Mazza \net al., 2020) \n59 Hermetia \nillucens \nLysinibacillus \nboronitolerans \nno positive 0,089 g ± 0,004 g  3 0,118 g ± 0,012 g 3 manu\nre \nMazza et al. \n2020  (Mazza \net al., 2020) \n60 Hermetia \nillucens \nSporosarcina \nkoreensis \nno positive 0,089 g ± 0,004 g\n 3 0,103 g ± 0,008 g 3 manu\nre \nMazza et al. \n2020  (Mazza \net al., 2020) \n61 Hermetia \nillucens \nGordonia sihwensis no positive 0,089 g ± 0,004 g  3 0,111 g ± 0,003 g 3 manu\nre \nMazza et al. \n2020  (Mazza \n62 Hermetia \nillucens \nEnterobacter spp. no negative 0,089 g ± 0,004 g  3 0,104 g ± 0,003 g 3 manu\nre \n.CC-BY 4.0 International licenseavailable under a \nwas not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprint (whichthis version posted April 4, 2025. ; https://doi.org/10.1101/2025.04.04.647199doi: bioRxiv preprint \n\n 40\net al., 2020) \nMazza et al. \n2020  (Mazza \net al., 2020) \n63 Hermetia \nillucens \nProteus mirabilis no negative 0,089 g ± 0,004 g  3 0,113 g ± 0,013 g 3 manu\nre \nMazza et al. \n2020  (Mazza \net al., 2020) \n64 Hermetia \nillucens \nBacillus subtilis no positive 0,089 g ± 0,004 g  3 0,105 g ± 0,002 g 3 manu\nre \nLi et al. 2023 \n(Li et al., 2023) \n65 Hermetia \nillucens \nProvidencia spp. no negative 0,364 g ± 0,001 g  3 0,335 g ± 0,004 g 3 no \nLi et al. 2023 \n(Li et al., 2023) \n66 Hermetia \nillucens \nCitrobacter spp. no negative 0,364 g ± 0,001 g  3 0,344 g ± 0,002 g 3 no \nLi et al. 2023 \n(Li et al., 2023) \n67 Hermetia \nillucens \nKlebsiella spp. no negative 0,364 g ± 0,001 g  3 0,360 g ± 0,001 g 3 no \nLi et al. 2023 \n(Li et al., 2023) \n68 Hermetia \nillucens \nProteus spp. no negative 0,364 g ± 0,001 g  3 0,321 g ± 0,003 g 3 no \nLi et al. 2023 \n(Li et al., 2023) \n69 Hermetia \nillucens \nGordonia sihwensis \nspp. \nno positive 0,364 g ± 0,001 g  3 0,351 g ± 0,003 g 3 no \nLi et al. 2023 \n(Li et al., 2023) \n70 Hermetia \nillucens \nDysgonomonas spp. no negative 0,364 g ± 0,001 g  3 0,336 g ± 0,001 g 3 no \nPei et al. 2022 \n(Pei et al., \n2022) \n71 Hermetia \nillucens \nBacillus velezensis  no postive 0,81 g ± 0,024 g  3 0,941 g ± 0,021 g 3 no \n \n \n Table 2. Included studies and relevant information on the insect studies included in the second meta-analysis comparing Shannon \nindices as an indication of bacterial community diversity. \n.CC-BY 4.0 International licenseavailable under a \nwas not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprint (whichthis version posted April 4, 2025. ; https://doi.org/10.1101/2025.04.04.647199doi: bioRxiv preprint \n\n 41\nReference Insect species Bacterial species Shannon \ncontrol \nSE \ncontrol \nN \ncontrol \nShannon \nprobiotic \nSE \nprobiotic \nN \nprobiotic \nLecoq et al. 2021 \n(Lecocq et al., 2021) Tenebrio molitor Pediococcus pentoceus 0,73 ± 0,18 8 1,19 ± 0,07 8 \nSavio et al. 2024 \n(Savio et al., 2024a) Tenebrio molitor Lactiplantibacillus plantarum 1,31 ± 0,34 3 0,82 ± 0,10 3 \nSavio et al. 2024  \n(Savio et al., 2024a) Tenebrio molitor Pediococcus pentoceus 1,31 ± 0,34 3 1,68 ± 0,05 3 \nMeng et al. 2023 \n(Meng et al., 2023) Hermetia illucens Rhodopseudomonas palustris 1,71 ± 0,04 6 1,83 ± 0,03 6 \nMeng et al. 2023 \n(Meng et al., 2023) Hermetia illucens Rhodopseudomonas palustris 1,71 ± 0,04 6 2,49 ± 0,09 6 \nKooienga et al. 2020 \n(Kooienga et al., \n2020) \nHermetia illucens Arthrobacter 2,79 ± 0,01 4 2,62 ± 0,01 4 \nKooienga et al. 2020 \n(Kooienga et al., \n2020) \nHermetia illucens Rhodococcus rhodochrous 2,79 ± 0,01 4 2,75 ± 0,01 4 \nYuan et al. 2023 \n(Yuan et al., 2023) Bombyx mori Bacillus subtilis 1,04 ± 0,20 5 1,33 ± 0,15 5 \nYuan et al. 2023 \n(Yuan et al., 2023) Bombyx mori Bacillus subtilis 0,47 ± 0,13 5 0,81 ± 0,19 5 \nPei et al. 2022 (Pei et \nal., 2022) Hermetia illucens Bacillus velezensis 2,61 ± 0,18 3 1,93 ± 0,06 3 \n.CC-BY 4.0 International licenseavailable under a \nwas not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprint (whichthis version posted April 4, 2025. ; https://doi.org/10.1101/2025.04.04.647199doi: bioRxiv preprint \n\n 42\n \n.CC-BY 4.0 International licenseavailable under a \nwas not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprint (whichthis version posted April 4, 2025. ; https://doi.org/10.1101/2025.04.04.647199doi: bioRxiv preprint \n\nData set\nEffect size \nInsect species \nApis mellifera\nBombyx mori\nHermetia illucens\nTenebrio molitor\n.CC-BY 4.0 International licenseavailable under a \nwas not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprint (whichthis version posted April 4, 2025. ; https://doi.org/10.1101/2025.04.04.647199doi: bioRxiv preprint \n\nA B\n.CC-BY 4.0 International licenseavailable under a \nwas not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprint (whichthis version posted April 4, 2025. ; https://doi.org/10.1101/2025.04.04.647199doi: bioRxiv preprint \n\nInsect species \nBombyx mori\nHermetia illucens\nTenebrio molitor\nData set\nEffect size \n.CC-BY 4.0 International licenseavailable under a \nwas not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprint (whichthis version posted April 4, 2025. ; https://doi.org/10.1101/2025.04.04.647199doi: bioRxiv preprint","source_license":"CC-BY-4.0","license_restricted":false}