Interactions Between Black Soldier Fly Larvae and Foodborne Pathogens: Implications for Growth, Welfare, and Bioconversion Efficiency

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Ogbon, Daniel Dzepe, Eugenie Famou, Farid Abdel-Kader Baba-Moussa, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5388328/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract This study investigates the interactions between black soldier fly (BSF) larvae ( Hermetia illucens ) and foodborne pathogens, specifically Salmonella spp and Escherichia coli ( E. coli ), to assess their impact on larval growth, welfare, and bioconversion efficiency. BSF larvae were reared on substrates inoculated with varying combinations of these pathogens and compared to a control group. Results indicated that larvae exposed to individual treatments of Salmonella spp or E. coli exhibited significantly slower growth rates, achieving only about half the weight of control larvae by Day 9. Notably, Salmonella spp exposure shortened the larval stage while prolonging the prepupal stage, suggesting metabolic stress. In contrast, the combination of both pathogens enhanced bioconversion rates, indicating complex microbial interactions that may benefit waste processing. The dynamics of pathogen persistence revealed that E. coli remained detectable in substrates for up to nine days, while Salmonella spp was only present for three days, highlighting the larvae's potential to mitigate pathogen levels in organic waste. Despite the resilience of BSF larvae to varying microbial loads, exposure to these pathogens negatively affected adult emergence rates, raising concerns about population sustainability and overall health. These findings underscore the importance of optimizing rearing conditions and implementing stringent quality control measures to minimize pathogen risks in BSF production systems. Black soldier fly Foodborne pathogens Salmonella spp Escherichia coli Bioconversion efficiency Figures Figure 1 Figure 2 Figure 3 Figure 4 Introduction The increasing global emphasis on sustainable waste management and food production has underscored the potential of black soldier fly (BSF) larvae ( Hermetia illucens ) as a transformative solution. BSF larvae efficiently convert organic waste into high-quality protein and fat, positioning them as valuable resources in animal feed and other applications [ 1 ]. However, their interaction with foodborne pathogens such as Salmonella spp and E. coli raises critical questions about their health and safety in these roles. Recent studies have demonstrated that BSF larvae possess antimicrobial properties that can inhibit pathogenic bacteria, thereby reducing the risk of zoonotic disease transmission [ 2 ]. For instance, research indicates that BSF larvae can significantly lower populations of Salmonella spp and E. coli in contaminated substrates [ 3 – 6 ] suggesting their potential role in bioconversion processes involving organic waste with high microbial loads [ 7 ]. The gut microbiota of BSF larvae is integral to this antimicrobial activity, with specific gut microbes identified as suppressors of pathogen growth [ 8 ]. Despite these promising findings, significant gaps remain in our understanding of how BSF larvae interact with Salmonella spp and E. coli across different life stages. Studies report inconsistent outcomes regarding pathogen reduction; some indicate effective suppression while others show minimal impact [ 6 , 9 ]. This inconsistency highlights the need for comprehensive research to elucidate the mechanisms underlying these interactions and assess their implications for food and feed production. As BSF production scales up, concerns about potential pathogen risks associated with industrial rearing conditions also arise. Although BSF larvae generally exhibit resistance to many entomopathogens, emerging diseases could pose substantial challenges in large-scale operations [ 10 ]. Furthermore, the persistence of Salmonella spp and E. coli in insect hosts poses significant food safety risks. Research indicates that Salmonella spp can survive for extended periods within various insect species, including up to 29 days in hosts like the black blow fly ( Phormia regina ) [ 11 ]. Similarly, E. coli has been shown to persist in insects like house flies ( Musca domestica ) and German cockroaches ( Blatella germanica ), raising concerns about potential contamination in insect-based food products [ 12 ]. While BSF larvae have demonstrated potential for reducing pathogen loads in contaminated substrates, this antimicrobial effect is not universally applicable and varies based on environmental conditions and specific strains involved [ 9 ]. Understanding how Salmonella spp and E. coli affect the well-being of BSF larvae is essential for optimizing their use in sustainable waste management and food production systems. This study seeks to address existing knowledge gaps by investigating the impact of Salmonella spp and E. coli on the health of BSF larvae while evaluating their efficacy in reducing microbial loads in contaminated substrates. By understanding these dynamics, we aim to enhance the safety and effectiveness of using BSF larvae in waste management and food production systems. Methodology Materials The Salmonella spp and Escherichia coli strains used in this study were sourced from the National University Hospital Centre of Benin (CNHU). They were transported on agar culture medium in a sterile container to the microbiology laboratory at the International Institute of Tropical Agriculture (IITA) in Benin, where they were confirmed using the API 20E gallery (BioMérieux, France). Suspensions of each bacterial strain were prepared according to the protocol described by Nordentoft [ 13 ] and incubated for 18 hours at 37°C before being stored at -20°C for subsequent use. The larvae used for the experiment were obtained from a BSF mass-rearing unit established at IITA-Benin. Freshly laid BSF eggs were collected and incubated for 2–3 days, following a protocol by Dzepe et al. [ 1 ]. After hatching, neonate larvae were reared on commercial chicken feed (composed of 80% corn, 10% soybean, 3% oyster shell, 5% concentrate, 1.7% wheat bran, and 0.3% salt) for five days before being used in the experiment. Experimental design The experiment was conducted in 12 plastic containers (10 cm × 17 cm × 6 cm) at room temperature (~ 30°C), using pasteurized commercial chicken feed mixed with sterilized tap water in a 1:2 (w/v) ratio, serving as the substrate. Each container was filled with 225 g of substrate and grouped into four sets of three containers. The first group acted as a control and did not receive any bacteria. The second and third groups were inoculated at 1% with prepared suspensions of Salmonella spp and Escherichia coli , respectively, while the fourth group was inoculated at 1% with a combination of both suspensions (1:1). The control group received sterilized distilled water instead of bacterial suspensions. Each treatment was incubated for 6 hours at 37°C after inoculation to ensure effective substrate contamination before being subjected to BSF larvae. Three hundred five-day-old BSF larvae were introduced into each treatment container previously filled with 225 g of commercial chicken feed, resulting in a feed load of 0.75 g per larva (wet weight) and a larval density of 1.2 larvae/cm². They were maintained in the rearing containers until reaching the pupal stage, at which point they were collected and subsequently transferred to adult cages for emergence. Each adult cage (29 cm × 29 cm × 29 cm), made of mosquito net, was equipped with a drinker to provide hydration for the adult flies and an egg-laying nest to collect their eggs. Data collection and calculations The effect of different microbial treatments on the welfare of BSF during the experimental period was evaluated using various growth parameters, including larval weight, larval survival rate (Eq. 1 ), development time, and emergence success of adult flies. Additionally, recycling parameters such as bioconversion efficiency (Eq. 2 ) and material reduction efficiency (Eq. 3 ) were assessed as suggested by Dzepe et al. [ 14 ]. Larval weight : Ten larvae were collected from each treatment container every three days, cleaned, then weighed on a high-precision electronic balance (accuracy ± 0.001 g). Larval survival rate : The survival rate of the larvae was calculated using the formula: $$\:Survival\:Rate\:\left(\%\right)=\left(\frac{{n}_{prepupae}}{i{n}_{larvae}\:-\:{ns}_{larvae}}\right)\times\:100$$ 1 Where n prepupae is the total number of BSF prepupae at the end of the larval stage; in larvae is the initial number of BSF larvae at the start of the experiment; and ns larvae the total number of larvae sampled for microbial analyses. Development time : For each stage, development time was defined as the number of days required for 50% of the individuals in the treatment unit to move to the next stage [ 15 ]. Emergence success : The emergence rate was considered as the percentage of prepupae transformed into adult BSF flies [ 16 ]. Bioconversion rate : The bioconversion efficiency of BSF larvae was calculated on a dry matter basis using the following formula: $$\:Bioconversion\:rate\:\left(\%\right)=\left(\frac{{m}_{larvae}\:\times\:\:{DM}_{larvae}}{{(m}_{substrate}-{ms}_{substrate})\:\times\:\:{DM}_{substrate}}\right)\times\:100$$ 2 Where m larvae is the biomass of larvae harvested at the end of the larval stage, m substrate is the initial masse of the substrate added in the treatment unit, ms substrate is the total mass of the substrate sampled for microbial analyses, and DM larvae and DM substrate are the dry mater content of larvae and substrate, respectively. Material reduction rate : The material reduction efficiency was also calculated on a dry matter basis using the following formula : $$\:Reduction\:rate\:\left(\%\right)=\left(\frac{{(m}_{substrate}-{ms}_{substrate})\:\times\:\:{DM}_{substrate}-\:{m}_{residue}\times\:{DM}_{residue}}{{(m}_{substrate}-{ms}_{substrate})\:\times\:\:{DM}_{substrate}}\right)\times\:100$$ 3 Where m substrate is the initial masse of the substrate added in the treatment unit, ms substrate is the total mass of the substrate sampled for microbial analyses, m residue is the total mass of the residual substrate collected in the treatment unit at the end of the larval stage, and DM substrate and DM residue are the dry mater content of substrate and residue, respectively. The dry matter content (DM) was determined by drying samples at 70°C for 48 h in an oven [ 17 ]. Throughout the experimental period, 10 larvae and 2 g of substrates were sampled every three days for microbial analyses. Prepupae, pupae, adult flies, and their eggs were also sampled for the same analyses. Microbial analyses The samples were prepared according to Bessa et al. [ 18 ]. After sampling, larvae, prepupae, pupae, and adult flies were rinsed with saline and distilled water to remove any dirt or external contamination from their bodies and then crushed using a sterilized pestle and mortar. Substrates and eggs were also sampled using a sterilized spatula and crushed following the same procedure. Microbiological analyses were carried out according to the methodology described by Erickson et al. [ 19 ]. To 1 g of each sample, 9 ml of nutrient broth was added to create the initial suspension, which was incubated for 24 h at 37°C. After incubation, the initial suspension was diluted to 1/100, 1/1,000, and 1/10,000; then 50 µL of each dilution was inoculated onto selective agar plates in Petri dishes (Hektoen for Salmonella spp and MacConkey for Escherichia coli ), and reincubated at 37°C for an additional 24 h for observation and counting. Salmonella spp counts were identified by black colonies on Hektoen medium, while E. coli counts were identified by brick-red colonies on MacConkey medium. The identified colonies were confirmed using the API 20E gallery. The concentration of microbial agents (CFU/mL) were calculated after enumeration of colony forming units (CFU) on the plates as suggested by Lichtenberg et al. [ 20 ]: $$\:\text{C}\text{F}\text{U}/\text{m}\text{L}\:=\left(\frac{\:{n}_{\text{C}\text{F}\text{U}}\:\times\:\:\text{d}\text{i}\text{l}\text{u}\text{t}\text{i}\text{o}\text{n}\:\text{f}\text{a}\text{c}\text{t}\text{o}\text{r}}{{\text{V}}_{culture\:plate}}\right)$$ , Where n CFU was the enumerated CFU on the selected plate, and V culture plate was the volume of the sample solution on the plate. Statistical analyses The analysis of the effect of microbial treatments on the growth performance and bioconversion efficiency of BSF was conducted using a systematic approach to ensure robust statistical evaluation. Descriptive statistics were calculated to summarize data distribution across different treatments. The Shapiro-Wilk test was used to check for normality, revealing that while some measurements were normally distributed, others were not. Levene's test confirmed the homogeneity of variances among treatments. For metrics with normally distributed data and homogeneous variances, a one-way ANOVA (analysis of variance) was performed to evaluate the overall effect of different microbial treatments. For measurements that did not meet these assumptions, the Kruskal-Wallis test was employed as a non-parametric alternative. If significant differences were found (p < 0.05), post-hoc analysis using Tukey's HSD (Honestly Significant Difference) test was used to identified specific treatment pairs with significant differences. To analyse the dynamics of Salmonella spp and E. coli in the substrate and larvae, linear regression analysis conducted to evaluate trends in bacterial concentrations over time, and finally, a t-test was performed to compare the concentrations of both bacteria. All analyses were carried out using Python 3.12.4. Results Growth Performance Figure 1 illustrates the effects of Salmonella spp and E. coli treatments on BSF larval weight over a 15-day period, revealing significant differences in growth patterns compared to the control treatment. The control substrate supports a steep growth curve, peaking around Day 9 before experiencing a slight decline and eventual stabilization. Interestingly, larvae subjected to the combined Salmonella spp and E. coli treatment exhibit a similar growth trajectory. In contrast, larvae exposed to the individual Salmonella spp or E. coli treatments demonstrate a slower, more gradual increase in weight, reaching only about half the weight of those in the control group by Day 9. There were significant variations in BSF developmental stages across different bacterial treatments. Notably, the presence of Salmonella spp shortened the larval stage compared to both the control and E. coli treatments. Interestingly, while the prepupal stage is generally brief, it was uniquely prolonged by Salmonella spp treatment. In terms of pupal development, variability was most pronounced; both E. coli and Salmonella spp individually extended this stage, whereas their combination did not yield the same effect. The adult fly stage exhibited minimal variation across treatments (Fig. 2 ). Notably, the Salmonella spp treatment resulted in the longest overall development time due to extended prepupal and pupal stages. Furthermore, the combination of Salmonella spp and E. coli often produced different outcomes compared to individual treatments. Process efficiency Figure 3 provides a comprehensive overview of the effects of Salmonella spp and E. coli treatments on BSF larval survival, bioconversion efficiency, material reduction efficiency, and adult emergence success. Larval survival shows minimal variation among treatments. In contrast, bioconversion rates exhibit notable differences. Specifically, the combination of Salmonella spp and E. coli in the substrate results in a higher bioconversion rate compared to the control group. However, individual applications of Salmonella spp or E. coli slow down bioconversion efficiency relative to the control. Material reduction efficiency was also significantly affected, with Salmonella spp treatment resulting in a lower reduction rate, followed by E. coli . Both bacterial species negatively impact the emergence success of adult BSF flies, with Salmonella spp treatment leading to the lowest emergence rate, followed by E. coli , and then the combination treatment compared to the control. Salmonella spp and E. coli dynamics The analysis of Salmonella spp and E. coli dynamics in both substrate and larvae revealed distinct patterns for each bacterial species. In the substrate, both bacteria exhibited significant decreasing trends over time, as indicated by negative slopes in linear regression models ( Salmonella spp : -0.5592; E. coli : -0.5931), with high statistical significance (p < 0.0001 for both). The models explained a substantial portion of the variance, with R 2 values of 0.6711 for Salmonella spp and 0.7631 for E. coli (Fig. 4 ). Notably, E. coli demonstrated longer persistence in the substrate, remaining detectable up to day 9, while Salmonella spp was only detectable until day 3. In BSF larvae, E. coli maintained a relatively stable presence throughout the larval stage, with counts clustered between approximately 5 and 8 log10 CFU/g; its regression line displayed a slight downward slope, indicating minimal decrease over time. In contrast, Salmonella spp started with lower initial concentrations (between 5 and 7 log10 CFU/g) and exhibited a more pronounced decline, with no counts detected after day 6 (Fig. 4 ). When comparing the dynamics of both pathogens in substrate and larvae environments, Salmonella spp shows a significant decline in both contexts but with a steeper decline in the substrate. Conversely, while E. coli declines rapidly in the substrate, it remains stable within BSF larvae, suggesting more favourable conditions for its survival in this environment. Discussion The findings regarding the effects of Salmonella spp and E. coli on BSF larval weight over the experimental period reveal significant implications for larval welfare. In this study, the control group demonstrated robust growth, peaking around Day 9. In contrast, larvae exposed to individual treatments of Salmonella spp or E. coli exhibited markedly slower growth, achieving only about half the weight of their control counterparts by the same time point. This stark difference underscores the detrimental impact both bacterial species have on larval development [ 21 ]. The slower growth rates associated with pathogen exposure can lead to heightened stress levels and increased mortality rates, raising concerns about the overall well-being of BSF larvae [ 22 ]. Additionally, the presence of these pathogens may compromise the larvae’s immune systems, rendering them more susceptible to other diseases and environmental stressors [ 23 ]. Interestingly, while BSF larvae possess some capacity to reduce pathogen loads through their gut microbiota, this protective effect appears insufficient to counteract the adverse impacts on their growth and health [ 24 ]. Notably, Salmonella spp significantly shortened the larval stage compared to both control and E. coli treatments, suggesting that pathogen exposure can accelerate development at the potential expense of health and vitality [ 21 ]. This rapid transition may induce increased stress levels, adversely affecting overall well-being and survival rates [ 22 ]. While the prepupal stage is typically brief, it was uniquely prolonged by Salmonella spp , indicating that this pathogen disrupts normal developmental processes and may induce metabolic stress [ 7 ]. Pupal development exhibited considerable variability; both E. coli and Salmonella spp individually extended this stage, yet their combination did not produce the same effect. This highlights complex microbial interactions that can significantly influence BSF growth [ 25 ]. The adult fly stage showed minimal variation across treatments, suggesting that bacterial presence has a limited impact during this phase. However, the extended overall development time associated with Salmonella spp , primarily due to prolonged prepupal and pupal stages, raises concerns about resource allocation during critical growth periods [ 26 ]. These findings emphasize the necessity for optimized rearing conditions that minimize pathogen exposure to enhance BSF welfare. The minimal variation in larval survival across treatments indicates that BSF larvae can thrive in environments with varying microbial loads, underscoring their potential as a sustainable solution for organic waste processing [ 27 ]. Furthermore, the observed increase in bioconversion rates when both Salmonella spp and E. coli were present in the substrate suggests a complex interaction that may enhance metabolic processes beneficial for waste conversion, despite individual applications of these bacteria slowing down efficiency [ 28 ]. This complexity points to the larvae's adaptive mechanisms, including an efficient immune response capable of rapidly reducing high E. coli counts in their substrate [ 29 ]. However, the negative impact on material reduction efficiency; particularly with Salmonella spp , raises concerns about potential stressors that could compromise larval health and productivity [ 30 ]. The detrimental effect of Salmonella spp on adult emergence rates was particularly significant. Reduced emergence can lead to lower population sustainability, which is vital for maintaining effective bioconversion processes. Studies have shown that exposure to pathogens can hinder the reproductive potential of BSF populations, emphasizing the need for careful management of microbial exposure to optimize both welfare and recycling performance [ 31 , 32 ]. Overall, while BSF larvae exhibit resilience to pathogens, these findings underscore the importance of understanding the dynamics between microbial exposure and BSF larvae in treatment units. The dynamics of Salmonella spp and E. coli in both substrate and BSF larvae environments provides valuable insights into the behaviour and persistence of these pathogens, which are crucial for understanding their implications for waste management and food safety. The observed significant decreasing trends for both bacteria in the substrate, supported by robust statistical models, indicate the potential of BSF larvae to mitigate pathogen levels in organic waste [ 27 , 33 , 34 ]. Notably, E. coli demonstrated longer persistence, remaining detectable up to day 9, while Salmonella spp was only detectable until day 3. This aligns with existing literature that highlights the ability of pathogenic strains of E. coli to survive in various environments due to their capacity to form biofilms and adapt to environmental stressors [ 35 , 36 ]. In the larvae environment, the stability of E. coli throughout the larval stage suggests that BSF larvae may provide a favourable habitat for this pathogen, allowing it to thrive despite challenging conditions [ 6 , 29 ]. Conversely, the pronounced decline of Salmonella spp , with no counts detected after day 6, can likely be attributed to the antimicrobial properties of BSF larvae and their gut microbiota dynamics that favour the suppression of certain pathogens [ 7 ]. This is consistent with findings that demonstrate BSF larvae's ability to inhibit zoonotic pathogens through various mechanisms, including antimicrobial peptide expression and beneficial gut microorganisms [ 37 ]. When comparing the dynamics of both pathogens across environments, it is evident that while Salmonella spp shows significant declines in both substrate and larvae contexts, its decline is steeper in the substrate. In contrast, although E. coli declines rapidly in the substrate, it remains stable within BSF larvae, suggesting that the larval environment offers conditions conducive to its survival [ 32 ]. This differential behaviour underscores the importance of understanding microbial interactions within BSF systems, as they can significantly influence both larval health and the efficacy of bioconversion processes. Moreover, these findings emphasize the need for stringent quality control measures when rearing BSF larvae on organic waste substrates. The potential for pathogenic transmission from substrate to larvae necessitates thorough microbiological screening and appropriate treatment methods to ensure safety [ 38 – 40 ]. Thermal pretreatments have been suggested as effective strategies for reducing microbial loads in substrates before they are introduced into BSF rearing systems [ 41 ]. Overall, understanding these dynamics is essential for optimizing BSF larvae's role in sustainable waste management while minimizing health risks associated with foodborne pathogens. Conclusion This study highlights the complex interactions between BSF larvae and foodborne pathogens, specifically Salmonella spp and E. coli , revealing significant implications for larval growth, welfare, and bioconversion efficiency. The findings demonstrate that while BSF larvae exhibit resilience to varying microbial loads, exposure to these pathogens adversely affects their growth rates, developmental stages, and overall health. Notably, the presence of Salmonella spp not only accelerated larval development but also prolonged certain stages, indicating potential metabolic stress. Conversely, the combination of both pathogens in the substrate enhanced bioconversion rates, suggesting a nuanced relationship that warrants further investigation. The differential persistence of E. coli and Salmonella spp within BSF larvae environments underscores the importance of microbial dynamics in optimizing waste management processes. These insights emphasize the necessity for stringent quality control measures in BSF rearing systems to mitigate pathogen risks while maximizing their potential as sustainable solutions for organic waste processing and animal feed production. Declarations Ethics approval and consent to participate: Not applicable Consent for publication: Not applicable Availability of data and material: The datasets and materials used and/or analyzed during the current study are available from the corresponding author on reasonable request. Competing interests: The authors declare no competing interests. Funding: The research received no specific grant from any funding agency in public, commercial, or non-profit sectors. Authors' contributions: Eyitayo Azaratou Ogbon: Conceptualization, data collection, formal analysis, methodology, writing original draft, writing, review and editing. Daniel Dzepe: Conceptualization, project administration, methodology, writing, review and editing. Eugenie Famou: data collection, formal analysis. Farid Abdel-Kader Baba-Moussa: writing, review and editing. Justin G. Behanzin: supervision, review and editing. Rousseau Djouaka: supervision, project administration, review and editing. Acknowledgements: The authors thank AgroEcoHealth unit (IITA) for technical support on the experimental work and the Islamic Development Bank (IsDB) for the scholarship of the PhD student Eyitayo Azaratou Ogbon. Authors' information (optional): Not applicable. Conflicts of Interest: The authors declare no conflict of interest. References Dzepe D, Nana P, Fotso A, Tchuinkam T, Djouaka R. Influence of larval density, substrate moisture content and feedstock ratio on life history traits of black soldier fly larvae. Journal of Insects as Food and Feed. 2020;6(2):133–140. https://doi.org/10.3920/JIFF2019.0034 . Zabulionė A, Šalaševičienė A, Makštutienė N, Šarkinas A. 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Naser El Deen S, van Rozen K, Elissen H, van Wikselaar P, Fodor I, van der Weide R, Hoek-van den Hil EF, Rezaei Far A, Veldkamp T. Bioconversion of Different Waste Streams of Animal and Vegetal Origin and Manure by Black Soldier Fly Larvae Hermetia illucens L. (Diptera: Stratiomyidae). Insects. 2023;14:204. https://doi.org/10.3390/insects14020204 . Tanga CM, Waweru JW, Tola YH, Onyoni AA, Khamis FM, Ekesi S, Paredes JC. Organic Waste Substrates Induce Important Shifts in Gut Microbiota of Black Soldier Fly ( Hermetia illucens L.): Coexistence of Conserved, Variable, and Potential Pathogenic Microbes. Front Microbiol. 2021;12(12):635881. https://doi.org/10.3389/fmicb.2021.635881 . Amrul NF, Kabir Ahmad I, Ahmad Basri NE, Suja F, Abdul Jalil NA, Azman NA. A Review of Organic Waste Treatment Using Black Soldier Fly ( Hermetia illucens ). Sustainability. 2022 ;14:4565. https://doi.org/10.3390/su14084565 . Kudva IT, Blanch K, Hovde CJ. Analysis of Escherichia coli O157: H7 survival in ovine or bovine manure and manure slurry. Applied and environmental microbiology. 1998;64(9):3166–3174. https://doi.org/10.1128/AEM.64.9.3166-3174 . van Elsas JD, Semenov AV, Costa R, Trevors JT. Survival of Escherichia coli in the environment: fundamental and public health aspects. ISME J. 2011;5(2): 367. https://doi.org/10.1038/ismej.2010.80 . Shao M, Zhao X, Rehman KU, Cai M, Zheng L, Huang F, Zhang J. Synergistic bioconversion of organic waste by black soldier fly ( Hermetia illucens ) larvae and thermophilic cellulose-degrading bacteria. Frontiers in Microbiology. 2023; 14:1288227. https://doi.org/10.3389/fmicb.2023.1288227 . Gold M, Tomberlin JK, Diener S, Zurbrügg C, Mathys A. Decomposition of biowaste macronutrients, microbes, and chemicals in black soldier fly larval treatment: a review. Waste Management. 2018;82:302–318. Larouche J, Deschamps MH, Saucier L, Lebeuf Y, Doyen A, Vandenberg GW. 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Ogbon","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA2UlEQVRIiWNgGAWjYBACNgbmA8xAWo6BgbGBOC38DGwJIC3GxGuRbOAxAGlJJFI9EBgc4DF+XVBxJ72/f3Hj5woGm3x5B8JazKxnnHmWO+PGw2bJMwxplhsPEKHFmLftcO4GiYMNkg0Mhw0MCTnRHqol3UDiYPNPorSA/PIYqCXBgL+xDWyLPAEdDAaH2dKYZ5w5bDjjBmObZYNBmoEBQS3Hmw9/Lqg4LM/ff/zxzYYKGwN5Qg5jYGZgkwAzJBJAJoCcSkgLUNMHMMUPVUrYllEwCkbBKBhpAADZl0LTmZhc1wAAAABJRU5ErkJggg==","orcid":"","institution":"University of Abomey-Calavi","correspondingAuthor":true,"prefix":"","firstName":"Eyitayo","middleName":"A.","lastName":"Ogbon","suffix":""},{"id":377387836,"identity":"73583be5-8cf9-483d-a19f-ad192e96952a","order_by":1,"name":"Daniel Dzepe","email":"","orcid":"","institution":"International Institute of Tropical Agriculture","correspondingAuthor":false,"prefix":"","firstName":"Daniel","middleName":"","lastName":"Dzepe","suffix":""},{"id":377387837,"identity":"3f39dfa9-1714-4ee6-ae05-4371d4f25fbb","order_by":2,"name":"Eugenie Famou","email":"","orcid":"","institution":"University of Abomey-Calavi","correspondingAuthor":false,"prefix":"","firstName":"Eugenie","middleName":"","lastName":"Famou","suffix":""},{"id":377387838,"identity":"8b1a8ba7-e5cc-4c07-8d92-1e541d52a7a7","order_by":3,"name":"Farid Abdel-Kader Baba-Moussa","email":"","orcid":"","institution":"University of Abomey-Calavi","correspondingAuthor":false,"prefix":"","firstName":"Farid","middleName":"Abdel-Kader","lastName":"Baba-Moussa","suffix":""},{"id":377387839,"identity":"001cce3c-4327-47bd-8fc0-20a47102ea85","order_by":4,"name":"Justin G. Behanzin","email":"","orcid":"","institution":"University of Abomey-Calavi","correspondingAuthor":false,"prefix":"","firstName":"Justin","middleName":"G.","lastName":"Behanzin","suffix":""},{"id":377387840,"identity":"be7443f8-442d-4569-808b-3927bbab9290","order_by":5,"name":"Rousseau Djouaka","email":"","orcid":"","institution":"International Institute of Tropical Agriculture","correspondingAuthor":false,"prefix":"","firstName":"Rousseau","middleName":"","lastName":"Djouaka","suffix":""}],"badges":[],"createdAt":"2024-11-04 13:08:19","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5388328/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5388328/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":69906281,"identity":"d49ca6d0-0ce9-40a4-9415-77a7339bf64f","added_by":"auto","created_at":"2024-11-26 12:58:06","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":289316,"visible":true,"origin":"","legend":"\u003cp\u003eDaily evolution of the average weight of BSF larvae according to the substrate treatment\u003c/p\u003e","description":"","filename":"image1.png","url":"https://assets-eu.researchsquare.com/files/rs-5388328/v1/3cb34f8f60c91a46132b88bf.png"},{"id":69906280,"identity":"ab077978-b467-4dc7-bad5-f02a89520f26","added_by":"auto","created_at":"2024-11-26 12:58:06","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":108151,"visible":true,"origin":"","legend":"\u003cp\u003eDuration of each stage of BSF development according to the substrate treatment\u003c/p\u003e","description":"","filename":"image2.png","url":"https://assets-eu.researchsquare.com/files/rs-5388328/v1/28b00684f3257b0142218267.png"},{"id":69906571,"identity":"10e654fd-5a10-4682-b010-c55fa95e7eb7","added_by":"auto","created_at":"2024-11-26 13:06:06","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":259833,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of \u003cem\u003eSalmonella spp\u003c/em\u003eand \u003cem\u003eE. coli\u003c/em\u003e treatments on BSF larval survival, bioconversion efficiency, material reduction efficiency and adult emergence success.\u003c/p\u003e","description":"","filename":"image3.png","url":"https://assets-eu.researchsquare.com/files/rs-5388328/v1/df30e4a49a5de9fd21f8fdb5.png"},{"id":69906283,"identity":"accb22a9-b64a-47f2-8a29-37020077e565","added_by":"auto","created_at":"2024-11-26 12:58:06","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":229352,"visible":true,"origin":"","legend":"\u003cp\u003eNumber of colony-forming units of \u003cem\u003eSalmonella spp\u003c/em\u003e and \u003cem\u003eE. coli\u003c/em\u003e counted in the substrate and larvae.\u003c/p\u003e","description":"","filename":"image4.png","url":"https://assets-eu.researchsquare.com/files/rs-5388328/v1/c6568f4847bc866fffb33de7.png"},{"id":73004810,"identity":"3acfa94a-3406-4dd5-a4d6-e4a16d8ae58d","added_by":"auto","created_at":"2025-01-05 19:01:23","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1413616,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5388328/v1/4797c47b-010b-4373-82c1-4915fc89d647.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Interactions Between Black Soldier Fly Larvae and Foodborne Pathogens: Implications for Growth, Welfare, and Bioconversion Efficiency","fulltext":[{"header":"Introduction","content":"\u003cp\u003eThe increasing global emphasis on sustainable waste management and food production has underscored the potential of black soldier fly (BSF) larvae (\u003cem\u003eHermetia illucens\u003c/em\u003e) as a transformative solution. BSF larvae efficiently convert organic waste into high-quality protein and fat, positioning them as valuable resources in animal feed and other applications [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. However, their interaction with foodborne pathogens such as \u003cem\u003eSalmonella spp\u003c/em\u003e and \u003cem\u003eE. coli\u003c/em\u003e raises critical questions about their health and safety in these roles. Recent studies have demonstrated that BSF larvae possess antimicrobial properties that can inhibit pathogenic bacteria, thereby reducing the risk of zoonotic disease transmission [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. For instance, research indicates that BSF larvae can significantly lower populations of \u003cem\u003eSalmonella spp\u003c/em\u003e and \u003cem\u003eE. coli\u003c/em\u003e in contaminated substrates [\u003cspan additionalcitationids=\"CR4 CR5\" citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e] suggesting their potential role in bioconversion processes involving organic waste with high microbial loads [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. The gut microbiota of BSF larvae is integral to this antimicrobial activity, with specific gut microbes identified as suppressors of pathogen growth [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eDespite these promising findings, significant gaps remain in our understanding of how BSF larvae interact with \u003cem\u003eSalmonella spp\u003c/em\u003e and \u003cem\u003eE. coli\u003c/em\u003e across different life stages. Studies report inconsistent outcomes regarding pathogen reduction; some indicate effective suppression while others show minimal impact [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. This inconsistency highlights the need for comprehensive research to elucidate the mechanisms underlying these interactions and assess their implications for food and feed production. As BSF production scales up, concerns about potential pathogen risks associated with industrial rearing conditions also arise. Although BSF larvae generally exhibit resistance to many entomopathogens, emerging diseases could pose substantial challenges in large-scale operations [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eFurthermore, the persistence of \u003cem\u003eSalmonella spp\u003c/em\u003e and \u003cem\u003eE. coli\u003c/em\u003e in insect hosts poses significant food safety risks. Research indicates that \u003cem\u003eSalmonella spp\u003c/em\u003e can survive for extended periods within various insect species, including up to 29 days in hosts like the black blow fly (\u003cem\u003ePhormia regina\u003c/em\u003e) [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Similarly, \u003cem\u003eE. coli\u003c/em\u003e has been shown to persist in insects like house flies (\u003cem\u003eMusca domestica\u003c/em\u003e) and German cockroaches (\u003cem\u003eBlatella germanica\u003c/em\u003e), raising concerns about potential contamination in insect-based food products [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. While BSF larvae have demonstrated potential for reducing pathogen loads in contaminated substrates, this antimicrobial effect is not universally applicable and varies based on environmental conditions and specific strains involved [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Understanding how \u003cem\u003eSalmonella spp\u003c/em\u003e and \u003cem\u003eE. coli\u003c/em\u003e affect the well-being of BSF larvae is essential for optimizing their use in sustainable waste management and food production systems.\u003c/p\u003e \u003cp\u003eThis study seeks to address existing knowledge gaps by investigating the impact of \u003cem\u003eSalmonella spp\u003c/em\u003e and \u003cem\u003eE. coli\u003c/em\u003e on the health of BSF larvae while evaluating their efficacy in reducing microbial loads in contaminated substrates. By understanding these dynamics, we aim to enhance the safety and effectiveness of using BSF larvae in waste management and food production systems.\u003c/p\u003e"},{"header":"Methodology","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eMaterials\u003c/h2\u003e \u003cp\u003eThe \u003cem\u003eSalmonella spp\u003c/em\u003e and \u003cem\u003eEscherichia coli\u003c/em\u003e strains used in this study were sourced from the National University Hospital Centre of Benin (CNHU). They were transported on agar culture medium in a sterile container to the microbiology laboratory at the International Institute of Tropical Agriculture (IITA) in Benin, where they were confirmed using the API 20E gallery (BioM\u0026eacute;rieux, France). Suspensions of each bacterial strain were prepared according to the protocol described by Nordentoft [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e] and incubated for 18 hours at 37\u0026deg;C before being stored at -20\u0026deg;C for subsequent use.\u003c/p\u003e \u003cp\u003eThe larvae used for the experiment were obtained from a BSF mass-rearing unit established at IITA-Benin. Freshly laid BSF eggs were collected and incubated for 2\u0026ndash;3 days, following a protocol by Dzepe et al. [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. After hatching, neonate larvae were reared on commercial chicken feed (composed of 80% corn, 10% soybean, 3% oyster shell, 5% concentrate, 1.7% wheat bran, and 0.3% salt) for five days before being used in the experiment.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eExperimental design\u003c/h3\u003e\n\u003cp\u003eThe experiment was conducted in 12 plastic containers (10 cm \u0026times; 17 cm \u0026times; 6 cm) at room temperature (~\u0026thinsp;30\u0026deg;C), using pasteurized commercial chicken feed mixed with sterilized tap water in a 1:2 (w/v) ratio, serving as the substrate. Each container was filled with 225 g of substrate and grouped into four sets of three containers. The first group acted as a control and did not receive any bacteria. The second and third groups were inoculated at 1% with prepared suspensions of \u003cem\u003eSalmonella spp\u003c/em\u003e and \u003cem\u003eEscherichia coli\u003c/em\u003e, respectively, while the fourth group was inoculated at 1% with a combination of both suspensions (1:1). The control group received sterilized distilled water instead of bacterial suspensions. Each treatment was incubated for 6 hours at 37\u0026deg;C after inoculation to ensure effective substrate contamination before being subjected to BSF larvae.\u003c/p\u003e \u003cp\u003eThree hundred five-day-old BSF larvae were introduced into each treatment container previously filled with 225 g of commercial chicken feed, resulting in a feed load of 0.75 g per larva (wet weight) and a larval density of 1.2 larvae/cm\u0026sup2;. They were maintained in the rearing containers until reaching the pupal stage, at which point they were collected and subsequently transferred to adult cages for emergence. Each adult cage (29 cm \u0026times; 29 cm \u0026times; 29 cm), made of mosquito net, was equipped with a drinker to provide hydration for the adult flies and an egg-laying nest to collect their eggs.\u003c/p\u003e\n\u003ch3\u003eData collection and calculations\u003c/h3\u003e\n\u003cp\u003eThe effect of different microbial treatments on the welfare of BSF during the experimental period was evaluated using various growth parameters, including larval weight, larval survival rate (Eq.\u0026nbsp;\u003cspan refid=\"Equ1\" class=\"InternalRef\"\u003e1\u003c/span\u003e), development time, and emergence success of adult flies. Additionally, recycling parameters such as bioconversion efficiency (Eq.\u0026nbsp;\u003cspan refid=\"Equ2\" class=\"InternalRef\"\u003e2\u003c/span\u003e) and material reduction efficiency (Eq.\u0026nbsp;\u003cspan refid=\"Equ3\" class=\"InternalRef\"\u003e3\u003c/span\u003e) were assessed as suggested by Dzepe et al. [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e].\u003c/p\u003e \u003cp\u003e \u003cul\u003e \u003cli\u003e \u003cp\u003e \u003cb\u003eLarval weight\u003c/b\u003e: Ten larvae were collected from each treatment container every three days, cleaned, then weighed on a high-precision electronic balance (accuracy\u0026thinsp;\u0026plusmn;\u0026thinsp;0.001 g).\u003c/p\u003e \u003c/li\u003e \u003cli\u003e \u003cp\u003e \u003cb\u003eLarval survival rate\u003c/b\u003e: The survival rate of the larvae was calculated using the formula:\u003c/p\u003e \u003c/li\u003e \u003c/ul\u003e \u003cdiv id=\"Equ1\" class=\"Equation\"\u003e \u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equ1\" name=\"EquationSource\"\u003e\n$$\\:Survival\\:Rate\\:\\left(\\%\\right)=\\left(\\frac{{n}_{prepupae}}{i{n}_{larvae}\\:-\\:{ns}_{larvae}}\\right)\\times\\:100$$\u003c/div\u003e \u003cdiv class=\"EquationNumber\"\u003e1\u003c/div\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eWhere \u003cem\u003en\u003c/em\u003e\u003csub\u003e\u003cem\u003eprepupae\u003c/em\u003e\u003c/sub\u003e is the total number of BSF prepupae at the end of the larval stage; \u003cem\u003ein\u003c/em\u003e\u003csub\u003e\u003cem\u003elarvae\u003c/em\u003e\u003c/sub\u003e is the initial number of BSF larvae at the start of the experiment; and \u003cem\u003ens\u003c/em\u003e\u003csub\u003e\u003cem\u003elarvae\u003c/em\u003e\u003c/sub\u003e the total number of larvae sampled for microbial analyses.\u003c/p\u003e \u003cp\u003e \u003cul\u003e \u003cli\u003e \u003cp\u003e \u003cb\u003eDevelopment time\u003c/b\u003e: For each stage, development time was defined as the number of days required for 50% of the individuals in the treatment unit to move to the next stage [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e].\u003c/p\u003e \u003c/li\u003e \u003cli\u003e \u003cp\u003e \u003cb\u003eEmergence success\u003c/b\u003e: The emergence rate was considered as the percentage of prepupae transformed into adult BSF flies [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e].\u003c/p\u003e \u003c/li\u003e \u003cli\u003e \u003cp\u003e \u003cb\u003eBioconversion rate\u003c/b\u003e: The bioconversion efficiency of BSF larvae was calculated on a dry matter basis using the following formula:\u003c/p\u003e \u003c/li\u003e \u003c/ul\u003e \u003cdiv id=\"Equ2\" class=\"Equation\"\u003e \u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equ2\" name=\"EquationSource\"\u003e\n$$\\:Bioconversion\\:rate\\:\\left(\\%\\right)=\\left(\\frac{{m}_{larvae}\\:\\times\\:\\:{DM}_{larvae}}{{(m}_{substrate}-{ms}_{substrate})\\:\\times\\:\\:{DM}_{substrate}}\\right)\\times\\:100$$\u003c/div\u003e \u003cdiv class=\"EquationNumber\"\u003e2\u003c/div\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eWhere \u003cem\u003em\u003c/em\u003e\u003csub\u003e\u003cem\u003elarvae\u003c/em\u003e\u003c/sub\u003e is the biomass of larvae harvested at the end of the larval stage, \u003cem\u003em\u003c/em\u003e\u003csub\u003esubstrate\u003c/sub\u003e is the initial masse of the substrate added in the treatment unit, \u003cem\u003ems\u003c/em\u003e\u003csub\u003e\u003cem\u003esubstrate\u003c/em\u003e\u003c/sub\u003e is the total mass of the substrate sampled for microbial analyses, and \u003cem\u003eDM\u003c/em\u003e\u003csub\u003e\u003cem\u003elarvae\u003c/em\u003e\u003c/sub\u003e and \u003cem\u003eDM\u003c/em\u003e\u003csub\u003e\u003cem\u003esubstrate\u003c/em\u003e\u003c/sub\u003e are the dry mater content of larvae and substrate, respectively.\u003c/p\u003e \u003cp\u003e \u003cul\u003e \u003cli\u003e \u003cp\u003e \u003cb\u003eMaterial reduction rate\u003c/b\u003e: The material reduction efficiency was also calculated on a dry matter basis using the following formula :\u003c/p\u003e \u003c/li\u003e \u003c/ul\u003e \u003cdiv id=\"Equ3\" class=\"Equation\"\u003e \u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equ3\" name=\"EquationSource\"\u003e\n$$\\:Reduction\\:rate\\:\\left(\\%\\right)=\\left(\\frac{{(m}_{substrate}-{ms}_{substrate})\\:\\times\\:\\:{DM}_{substrate}-\\:{m}_{residue}\\times\\:{DM}_{residue}}{{(m}_{substrate}-{ms}_{substrate})\\:\\times\\:\\:{DM}_{substrate}}\\right)\\times\\:100$$\u003c/div\u003e \u003cdiv class=\"EquationNumber\"\u003e3\u003c/div\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eWhere \u003cem\u003em\u003c/em\u003e\u003csub\u003esubstrate\u003c/sub\u003e is the initial masse of the substrate added in the treatment unit, \u003cem\u003ems\u003c/em\u003e\u003csub\u003e\u003cem\u003esubstrate\u003c/em\u003e\u003c/sub\u003e is the total mass of the substrate sampled for microbial analyses, \u003cem\u003em\u003c/em\u003e\u003csub\u003e\u003cem\u003eresidue\u003c/em\u003e\u003c/sub\u003e is the total mass of the residual substrate collected in the treatment unit at the end of the larval stage, and \u003cem\u003eDM\u003c/em\u003e\u003csub\u003e\u003cem\u003esubstrate\u003c/em\u003e\u003c/sub\u003e and \u003cem\u003eDM\u003c/em\u003e\u003csub\u003e\u003cem\u003eresidue\u003c/em\u003e\u003c/sub\u003e are the dry mater content of substrate and residue, respectively.\u003c/p\u003e \u003cp\u003eThe dry matter content (DM) was determined by drying samples at 70\u0026deg;C for 48 h in an oven [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Throughout the experimental period, 10 larvae and 2 g of substrates were sampled every three days for microbial analyses. Prepupae, pupae, adult flies, and their eggs were also sampled for the same analyses.\u003c/p\u003e\n\u003ch3\u003eMicrobial analyses\u003c/h3\u003e\n\u003cp\u003eThe samples were prepared according to Bessa et al. [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. After sampling, larvae, prepupae, pupae, and adult flies were rinsed with saline and distilled water to remove any dirt or external contamination from their bodies and then crushed using a sterilized pestle and mortar. Substrates and eggs were also sampled using a sterilized spatula and crushed following the same procedure. Microbiological analyses were carried out according to the methodology described by Erickson et al. [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. To 1 g of each sample, 9 ml of nutrient broth was added to create the initial suspension, which was incubated for 24 h at 37\u0026deg;C. After incubation, the initial suspension was diluted to 1/100, 1/1,000, and 1/10,000; then 50 \u0026micro;L of each dilution was inoculated onto selective agar plates in Petri dishes (Hektoen for \u003cem\u003eSalmonella spp\u003c/em\u003e and MacConkey for \u003cem\u003eEscherichia coli\u003c/em\u003e), and reincubated at 37\u0026deg;C for an additional 24 h for observation and counting. \u003cem\u003eSalmonella spp\u003c/em\u003e counts were identified by black colonies on Hektoen medium, while \u003cem\u003eE. coli\u003c/em\u003e counts were identified by brick-red colonies on MacConkey medium. The identified colonies were confirmed using the API 20E gallery.\u003c/p\u003e \u003cp\u003eThe concentration of microbial agents (CFU/mL) were calculated after enumeration of colony forming units (CFU) on the plates as suggested by Lichtenberg et al. [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]:\u003cdiv id=\"Equa\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equa\" name=\"EquationSource\"\u003e\n$$\\:\\text{C}\\text{F}\\text{U}/\\text{m}\\text{L}\\:=\\left(\\frac{\\:{n}_{\\text{C}\\text{F}\\text{U}}\\:\\times\\:\\:\\text{d}\\text{i}\\text{l}\\text{u}\\text{t}\\text{i}\\text{o}\\text{n}\\:\\text{f}\\text{a}\\text{c}\\text{t}\\text{o}\\text{r}}{{\\text{V}}_{culture\\:plate}}\\right)$$\u003c/div\u003e\u003c/div\u003e,\u003c/p\u003e \u003cp\u003eWhere \u003cem\u003en\u003c/em\u003e\u003csub\u003eCFU\u003c/sub\u003e was the enumerated CFU on the selected plate, and V\u003csub\u003e\u003cem\u003eculture plate\u003c/em\u003e\u003c/sub\u003e was the volume of the sample solution on the plate.\u003c/p\u003e\n\u003ch3\u003eStatistical analyses\u003c/h3\u003e\n\u003cp\u003eThe analysis of the effect of microbial treatments on the growth performance and bioconversion efficiency of BSF was conducted using a systematic approach to ensure robust statistical evaluation. Descriptive statistics were calculated to summarize data distribution across different treatments. The Shapiro-Wilk test was used to check for normality, revealing that while some measurements were normally distributed, others were not. Levene's test confirmed the homogeneity of variances among treatments. For metrics with normally distributed data and homogeneous variances, a one-way ANOVA (analysis of variance) was performed to evaluate the overall effect of different microbial treatments. For measurements that did not meet these assumptions, the Kruskal-Wallis test was employed as a non-parametric alternative. If significant differences were found (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05), post-hoc analysis using Tukey's HSD (Honestly Significant Difference) test was used to identified specific treatment pairs with significant differences. To analyse the dynamics of \u003cem\u003eSalmonella spp\u003c/em\u003e and \u003cem\u003eE. coli\u003c/em\u003e in the substrate and larvae, linear regression analysis conducted to evaluate trends in bacterial concentrations over time, and finally, a t-test was performed to compare the concentrations of both bacteria. All analyses were carried out using Python 3.12.4.\u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eGrowth Performance\u003c/h2\u003e \u003cp\u003eFigure \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e illustrates the effects of \u003cem\u003eSalmonella spp\u003c/em\u003e and \u003cem\u003eE. coli\u003c/em\u003e treatments on BSF larval weight over a 15-day period, revealing significant differences in growth patterns compared to the control treatment. The control substrate supports a steep growth curve, peaking around Day 9 before experiencing a slight decline and eventual stabilization. Interestingly, larvae subjected to the combined \u003cem\u003eSalmonella spp\u003c/em\u003e and \u003cem\u003eE. coli\u003c/em\u003e treatment exhibit a similar growth trajectory. In contrast, larvae exposed to the individual \u003cem\u003eSalmonella spp\u003c/em\u003e or \u003cem\u003eE. coli\u003c/em\u003e treatments demonstrate a slower, more gradual increase in weight, reaching only about half the weight of those in the control group by Day 9. There were significant variations in BSF developmental stages across different bacterial treatments. Notably, the presence of \u003cem\u003eSalmonella spp\u003c/em\u003e shortened the larval stage compared to both the control and \u003cem\u003eE. coli\u003c/em\u003e treatments. Interestingly, while the prepupal stage is generally brief, it was uniquely prolonged by \u003cem\u003eSalmonella spp\u003c/em\u003e treatment. In terms of pupal development, variability was most pronounced; both \u003cem\u003eE. coli\u003c/em\u003e and \u003cem\u003eSalmonella spp\u003c/em\u003e individually extended this stage, whereas their combination did not yield the same effect. The adult fly stage exhibited minimal variation across treatments (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Notably, the \u003cem\u003eSalmonella spp\u003c/em\u003e treatment resulted in the longest overall development time due to extended prepupal and pupal stages. Furthermore, the combination of \u003cem\u003eSalmonella spp\u003c/em\u003e and \u003cem\u003eE. coli\u003c/em\u003e often produced different outcomes compared to individual treatments.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eProcess efficiency\u003c/h3\u003e\n\u003cp\u003eFigure \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e provides a comprehensive overview of the effects of \u003cem\u003eSalmonella spp\u003c/em\u003e and \u003cem\u003eE. coli\u003c/em\u003e treatments on BSF larval survival, bioconversion efficiency, material reduction efficiency, and adult emergence success. Larval survival shows minimal variation among treatments. In contrast, bioconversion rates exhibit notable differences. Specifically, the combination of \u003cem\u003eSalmonella spp\u003c/em\u003e and \u003cem\u003eE. coli\u003c/em\u003e in the substrate results in a higher bioconversion rate compared to the control group. However, individual applications of \u003cem\u003eSalmonella spp\u003c/em\u003e or \u003cem\u003eE. coli\u003c/em\u003e slow down bioconversion efficiency relative to the control. Material reduction efficiency was also significantly affected, with \u003cem\u003eSalmonella spp\u003c/em\u003e treatment resulting in a lower reduction rate, followed by \u003cem\u003eE. coli\u003c/em\u003e. Both bacterial species negatively impact the emergence success of adult BSF flies, with \u003cem\u003eSalmonella spp\u003c/em\u003e treatment leading to the lowest emergence rate, followed by \u003cem\u003eE. coli\u003c/em\u003e, and then the combination treatment compared to the control.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eSalmonella spp\u003c/b\u003e \u003cb\u003eand\u003c/b\u003e \u003cb\u003eE. coli\u003c/b\u003e \u003cb\u003edynamics\u003c/b\u003e\u003c/p\u003e \u003cp\u003eThe analysis of \u003cem\u003eSalmonella spp\u003c/em\u003e and \u003cem\u003eE. coli\u003c/em\u003e dynamics in both substrate and larvae revealed distinct patterns for each bacterial species. In the substrate, both bacteria exhibited significant decreasing trends over time, as indicated by negative slopes in linear regression models (\u003cem\u003eSalmonella spp\u003c/em\u003e: -0.5592; \u003cem\u003eE. coli\u003c/em\u003e: -0.5931), with high statistical significance (p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001 for both). The models explained a substantial portion of the variance, with R\u003csup\u003e2\u003c/sup\u003e values of 0.6711 for \u003cem\u003eSalmonella spp\u003c/em\u003e and 0.7631 for \u003cem\u003eE. coli\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). Notably, \u003cem\u003eE. coli\u003c/em\u003e demonstrated longer persistence in the substrate, remaining detectable up to day 9, while \u003cem\u003eSalmonella spp\u003c/em\u003e was only detectable until day 3. In BSF larvae, \u003cem\u003eE. coli\u003c/em\u003e maintained a relatively stable presence throughout the larval stage, with counts clustered between approximately 5 and 8 log10 CFU/g; its regression line displayed a slight downward slope, indicating minimal decrease over time. In contrast, \u003cem\u003eSalmonella spp\u003c/em\u003e started with lower initial concentrations (between 5 and 7 log10 CFU/g) and exhibited a more pronounced decline, with no counts detected after day 6 (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). When comparing the dynamics of both pathogens in substrate and larvae environments, \u003cem\u003eSalmonella spp\u003c/em\u003e shows a significant decline in both contexts but with a steeper decline in the substrate. Conversely, while \u003cem\u003eE. coli\u003c/em\u003e declines rapidly in the substrate, it remains stable within BSF larvae, suggesting more favourable conditions for its survival in this environment.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe findings regarding the effects of \u003cem\u003eSalmonella spp\u003c/em\u003e and \u003cem\u003eE. coli\u003c/em\u003e on BSF larval weight over the experimental period reveal significant implications for larval welfare. In this study, the control group demonstrated robust growth, peaking around Day 9. In contrast, larvae exposed to individual treatments of \u003cem\u003eSalmonella spp\u003c/em\u003e or \u003cem\u003eE. coli\u003c/em\u003e exhibited markedly slower growth, achieving only about half the weight of their control counterparts by the same time point. This stark difference underscores the detrimental impact both bacterial species have on larval development [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. The slower growth rates associated with pathogen exposure can lead to heightened stress levels and increased mortality rates, raising concerns about the overall well-being of BSF larvae [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. Additionally, the presence of these pathogens may compromise the larvae\u0026rsquo;s immune systems, rendering them more susceptible to other diseases and environmental stressors [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. Interestingly, while BSF larvae possess some capacity to reduce pathogen loads through their gut microbiota, this protective effect appears insufficient to counteract the adverse impacts on their growth and health [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eNotably, \u003cem\u003eSalmonella spp\u003c/em\u003e significantly shortened the larval stage compared to both control and \u003cem\u003eE. coli\u003c/em\u003e treatments, suggesting that pathogen exposure can accelerate development at the potential expense of health and vitality [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. This rapid transition may induce increased stress levels, adversely affecting overall well-being and survival rates [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. While the prepupal stage is typically brief, it was uniquely prolonged by \u003cem\u003eSalmonella spp\u003c/em\u003e, indicating that this pathogen disrupts normal developmental processes and may induce metabolic stress [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Pupal development exhibited considerable variability; both \u003cem\u003eE. coli\u003c/em\u003e and \u003cem\u003eSalmonella spp\u003c/em\u003e individually extended this stage, yet their combination did not produce the same effect. This highlights complex microbial interactions that can significantly influence BSF growth [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. The adult fly stage showed minimal variation across treatments, suggesting that bacterial presence has a limited impact during this phase. However, the extended overall development time associated with \u003cem\u003eSalmonella spp\u003c/em\u003e, primarily due to prolonged prepupal and pupal stages, raises concerns about resource allocation during critical growth periods [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. These findings emphasize the necessity for optimized rearing conditions that minimize pathogen exposure to enhance BSF welfare.\u003c/p\u003e \u003cp\u003eThe minimal variation in larval survival across treatments indicates that BSF larvae can thrive in environments with varying microbial loads, underscoring their potential as a sustainable solution for organic waste processing [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. Furthermore, the observed increase in bioconversion rates when both \u003cem\u003eSalmonella spp\u003c/em\u003e and \u003cem\u003eE. coli\u003c/em\u003e were present in the substrate suggests a complex interaction that may enhance metabolic processes beneficial for waste conversion, despite individual applications of these bacteria slowing down efficiency [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. This complexity points to the larvae's adaptive mechanisms, including an efficient immune response capable of rapidly reducing high \u003cem\u003eE. coli\u003c/em\u003e counts in their substrate [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. However, the negative impact on material reduction efficiency; particularly with \u003cem\u003eSalmonella spp\u003c/em\u003e, raises concerns about potential stressors that could compromise larval health and productivity [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. The detrimental effect of \u003cem\u003eSalmonella spp\u003c/em\u003e on adult emergence rates was particularly significant. Reduced emergence can lead to lower population sustainability, which is vital for maintaining effective bioconversion processes. Studies have shown that exposure to pathogens can hinder the reproductive potential of BSF populations, emphasizing the need for careful management of microbial exposure to optimize both welfare and recycling performance [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. Overall, while BSF larvae exhibit resilience to pathogens, these findings underscore the importance of understanding the dynamics between microbial exposure and BSF larvae in treatment units.\u003c/p\u003e \u003cp\u003eThe dynamics of \u003cem\u003eSalmonella spp\u003c/em\u003e and \u003cem\u003eE. coli\u003c/em\u003e in both substrate and BSF larvae environments provides valuable insights into the behaviour and persistence of these pathogens, which are crucial for understanding their implications for waste management and food safety. The observed significant decreasing trends for both bacteria in the substrate, supported by robust statistical models, indicate the potential of BSF larvae to mitigate pathogen levels in organic waste [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. Notably, \u003cem\u003eE. coli\u003c/em\u003e demonstrated longer persistence, remaining detectable up to day 9, while \u003cem\u003eSalmonella spp\u003c/em\u003e was only detectable until day 3. This aligns with existing literature that highlights the ability of pathogenic strains of \u003cem\u003eE. coli\u003c/em\u003e to survive in various environments due to their capacity to form biofilms and adapt to environmental stressors [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. In the larvae environment, the stability of \u003cem\u003eE. coli\u003c/em\u003e throughout the larval stage suggests that BSF larvae may provide a favourable habitat for this pathogen, allowing it to thrive despite challenging conditions [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. Conversely, the pronounced decline of \u003cem\u003eSalmonella spp\u003c/em\u003e, with no counts detected after day 6, can likely be attributed to the antimicrobial properties of BSF larvae and their gut microbiota dynamics that favour the suppression of certain pathogens [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. This is consistent with findings that demonstrate BSF larvae's ability to inhibit zoonotic pathogens through various mechanisms, including antimicrobial peptide expression and beneficial gut microorganisms [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eWhen comparing the dynamics of both pathogens across environments, it is evident that while \u003cem\u003eSalmonella spp\u003c/em\u003e shows significant declines in both substrate and larvae contexts, its decline is steeper in the substrate. In contrast, although \u003cem\u003eE. coli\u003c/em\u003e declines rapidly in the substrate, it remains stable within BSF larvae, suggesting that the larval environment offers conditions conducive to its survival [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. This differential behaviour underscores the importance of understanding microbial interactions within BSF systems, as they can significantly influence both larval health and the efficacy of bioconversion processes. Moreover, these findings emphasize the need for stringent quality control measures when rearing BSF larvae on organic waste substrates. The potential for pathogenic transmission from substrate to larvae necessitates thorough microbiological screening and appropriate treatment methods to ensure safety [\u003cspan additionalcitationids=\"CR39\" citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. Thermal pretreatments have been suggested as effective strategies for reducing microbial loads in substrates before they are introduced into BSF rearing systems [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. Overall, understanding these dynamics is essential for optimizing BSF larvae's role in sustainable waste management while minimizing health risks associated with foodborne pathogens.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eThis study highlights the complex interactions between BSF larvae and foodborne pathogens, specifically \u003cem\u003eSalmonella spp\u003c/em\u003e and \u003cem\u003eE. coli\u003c/em\u003e, revealing significant implications for larval growth, welfare, and bioconversion efficiency. The findings demonstrate that while BSF larvae exhibit resilience to varying microbial loads, exposure to these pathogens adversely affects their growth rates, developmental stages, and overall health. Notably, the presence of \u003cem\u003eSalmonella spp\u003c/em\u003e not only accelerated larval development but also prolonged certain stages, indicating potential metabolic stress. Conversely, the combination of both pathogens in the substrate enhanced bioconversion rates, suggesting a nuanced relationship that warrants further investigation. The differential persistence of \u003cem\u003eE. coli\u003c/em\u003e and \u003cem\u003eSalmonella spp\u003c/em\u003e within BSF larvae environments underscores the importance of microbial dynamics in optimizing waste management processes. These insights emphasize the necessity for stringent quality control measures in BSF rearing systems to mitigate pathogen risks while maximizing their potential as sustainable solutions for organic waste processing and animal feed production.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003eEthics approval and consent to participate: Not applicable\u003c/p\u003e\n\u003cp\u003eConsent for publication: Not applicable\u003c/p\u003e\n\u003cp\u003eAvailability of data and material: The datasets and materials used and/or analyzed during the current study are available from the corresponding author on reasonable request.\u003c/p\u003e\n\u003cp\u003eCompeting interests: The authors declare no competing interests.\u003c/p\u003e\n\u003cp\u003eFunding: The research received no specific grant from any funding agency in public, commercial, or non-profit sectors.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAuthors\u0026apos; contributions: Eyitayo Azaratou Ogbon: Conceptualization, data collection, formal analysis, methodology, writing original draft, writing, review and editing. Daniel Dzepe: Conceptualization, project administration, methodology, writing, review and editing. Eugenie Famou: data collection, formal analysis. Farid Abdel-Kader Baba-Moussa: writing, review and editing. Justin G. Behanzin: supervision, review and editing. Rousseau Djouaka: supervision, project administration, review and editing.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAcknowledgements: The authors thank AgroEcoHealth unit (IITA) for technical support on the experimental work and the Islamic Development Bank (IsDB) for the scholarship of the PhD student Eyitayo Azaratou Ogbon.\u003c/p\u003e\n\u003cp\u003eAuthors\u0026apos; information (optional): Not applicable.\u003c/p\u003e\n\u003cp\u003eConflicts of Interest: The authors declare no conflict of interest.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eDzepe D, Nana P, Fotso A, Tchuinkam T, Djouaka R. 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Animals. 2019;9:182. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3390/ani9040182\u003c/span\u003e\u003cspan address=\"10.3390/ani9040182\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eShelomi M. Mitigation Strategies against Food Safety Contaminant Transmission from Black Soldier Fly Larva Bioconversion. Animals. 2024;14:1590. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3390/ani14111590\u003c/span\u003e\u003cspan address=\"10.3390/ani14111590\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWynants E, Frooninckx L, Crauwels S, Verreth C, De Smet J, Sandrock C, Van Campenhout L. Assessing the Microbiota of Black Soldier Fly Larvae (\u003cem\u003eHermetia illucens\u003c/em\u003e) Reared on Organic Waste Streams on Four Different Locations at Laboratory and Large Scale. Microb Ecol. 2019;77:913\u0026ndash;930. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1007/s00248-018-1286-x\u003c/span\u003e\u003cspan address=\"10.1007/s00248-018-1286-x\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Black soldier fly, Foodborne pathogens, Salmonella spp, Escherichia coli, Bioconversion efficiency","lastPublishedDoi":"10.21203/rs.3.rs-5388328/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5388328/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThis study investigates the interactions between black soldier fly (BSF) larvae (\u003cem\u003eHermetia illucens\u003c/em\u003e) and foodborne pathogens, specifically \u003cem\u003eSalmonella spp\u003c/em\u003e and \u003cem\u003eEscherichia coli\u003c/em\u003e (\u003cem\u003eE. coli\u003c/em\u003e), to assess their impact on larval growth, welfare, and bioconversion efficiency. BSF larvae were reared on substrates inoculated with varying combinations of these pathogens and compared to a control group. Results indicated that larvae exposed to individual treatments of \u003cem\u003eSalmonella spp\u003c/em\u003e or \u003cem\u003eE. coli\u003c/em\u003e exhibited significantly slower growth rates, achieving only about half the weight of control larvae by Day 9. Notably, \u003cem\u003eSalmonella spp\u003c/em\u003e exposure shortened the larval stage while prolonging the prepupal stage, suggesting metabolic stress. In contrast, the combination of both pathogens enhanced bioconversion rates, indicating complex microbial interactions that may benefit waste processing. The dynamics of pathogen persistence revealed that \u003cem\u003eE. coli\u003c/em\u003e remained detectable in substrates for up to nine days, while \u003cem\u003eSalmonella spp\u003c/em\u003e was only present for three days, highlighting the larvae's potential to mitigate pathogen levels in organic waste. Despite the resilience of BSF larvae to varying microbial loads, exposure to these pathogens negatively affected adult emergence rates, raising concerns about population sustainability and overall health. These findings underscore the importance of optimizing rearing conditions and implementing stringent quality control measures to minimize pathogen risks in BSF production systems.\u003c/p\u003e","manuscriptTitle":"Interactions Between Black Soldier Fly Larvae and Foodborne Pathogens: Implications for Growth, Welfare, and Bioconversion Efficiency","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-11-26 12:58:01","doi":"10.21203/rs.3.rs-5388328/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"8a597bf9-7796-42f2-975b-a1df9b07bcfa","owner":[],"postedDate":"November 26th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-01-05T18:53:12+00:00","versionOfRecord":[],"versionCreatedAt":"2024-11-26 12:58:01","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-5388328","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5388328","identity":"rs-5388328","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}