Of maggots and microbes

The black soldier fly, Hermetia illucens (L.) (BSF; Diptera: Stratiomyidae), is one of the most promising insect species to provide an alternative protein source for animal feed. Insects reared on organic side streams can be a more sustainable alternative protein source than soymeal or fishmeal. This is important because global meat production would need to increase by an annual 200 million tons to meet projected global food demand by 2050. BSF larvae can survive and grow on a wide range of organic side streams and by-products, but the nutritional quality and performance of the larvae depend on the type of feed substrate. Besides, substrate-associated microorganisms and chemical contaminants may introduce microbial and chemical safety risks to the use of BSF larvae as a feed ingredient. Microorganisms can colonize the larval gut from the environment (including feed substrate) and insect eggs. The larval gut exerts selection pressures on ingested and resident microbiota and excretes a microbiota of altered community composition into the substrate. Foraging in aggregations, the larvae may thus alter the substrate microbiota and physicochemical properties. However, the relative contributions of substrate, eggs, and larval aggregations on larval performance and microbiota have not been investigated to date. Therefore, this thesis focused on studying and understanding the performance of BSF larvae and their interactions with microbial communities in organic side streams.

In Chapter 2, BSF larval performance was investigated on diets of chicken feed with partial substitution by oilseed by-products. The oilseed crops crambe and camelina produce oils rich in erucic acid and n-3 polyunsaturated fatty acids (PUFA), respectively. After oil pressing, a protein-rich seed cake remains, which can be defatted to seed meal. These by-products have limited value as livestock feed because they contain glucosinolates which are enzymatically broken down into toxic isothiocyanates and nitriles. I tested the effects of 25%, 50% and 100% oilseed by-product inclusion in the diet on survival, development, biomass production and fatty acid composition of BSF larvae. Larvae fed up to 50% camelina by-product or 25% crambe by-product performed similarly to larvae fed control diet (chicken feed), and performance decreased with higher inclusion percentages. The n-6 : n-3 PUFA ratio decreased with increasing proportion of by-product, especially on camelina diets. These findings indicate that BSF larvae successfully grow on diets with camelina or crambe oilseed by-products, and that the resulting larval n-6 : n-3 PUFA ratio is favourable for animal feed. However, the fate of glucosinolates and their derivatives remains to be determined to warrant chemical safety of the larvae for use in animal feed.

BSF larvae interact with a rich microbial community of bacteria and fungi, which strongly depends on the type of substrate. These microorganisms impact the larval microbiota, but the larvae can also alter substrate microbiota. Chapter 3 aimed to investigate the relative importance of substrate type and larval density on bacterial community dynamics. Four larval densities (0 (control), 50, 100, or 200 larvae per container) and three feed substrates (chicken feed, chicken manure, and camelina substrate (50% chicken feed, 50% camelina oilseed press cake) were investigated and bacterial communities of substrates and larvae were sampled at three time points over 15 days. BSF larvae altered bacterial community composition over time in all three feed substrates and substrate type was the strongest driver of bacterial community composition. The impact of the larvae depended on substrate and larval density. Larval and substrate microbiota differed for chicken manure and camelina, whereas they overlapped in chicken feed. These findings demonstrate the flexibility of the association between BSF larvae and bacteria and support the substrate-dependent impact of BSF larvae on bacteria both within the larvae and in the substrate.

In Chapter 4, I aimed to quantify the relative importance of substrate-associated and egg-associated microorganisms on BSF larval performance, bacterial abundance, and microbiota composition, when larvae were fed with chicken feed or chicken manure. For this we inactivated substrate-associated microorganisms by autoclaving, or disinfected BSF eggs. Larval survival, biomass, and proportion of prepupae were determined on day 15. We collected substrate and larval samples on days 0 and 15 and performed 16S rRNA gene-targeted qPCR and amplicon sequencing. In both chicken feed and chicken manure, egg disinfection did not cause any difference in larval performance or overall microbiota composition. In contrast, in chicken manure, substrate-associated microorganisms increased larval biomass and sterilizing the substrate caused major shifts in microbiota. Thus, substrate-associated microorganisms not only impact the larval microbiota but also larval performance, whereas egg-associated microorganisms have a minor role at the densities present.

The findings of this thesis were discussed in a community ecology context in Chapter 5. This approach facilitates future identification of key players and processes in the community dynamics and functions of the microbiota of the BSF gut and feed substrate during bioconversion. Both the impact of larval density on substrate microbiota and the impact of egg-associated microorganisms on gut microbiota were subordinate to the contribution of the feed substrate microorganisms. This suggests that potentially vertically transmitted microorganisms play a minor role in larval gut microbiota and performance, compared to environmentally acquired microorganisms. Intrinsic and extrinsic disturbances during larval development suggest it is unlikely that the BSF larval gut microbiota reaches a stable composition. The most drastic disturbance of larval microbiota may be the common practice of transferring larvae from a nursery diet to a biowaste substrate. Substrate-associated microorganisms and chemicals may exert additional selection pressures on the host and resident gut microbiota. Microbial community recovery after disturbance depends on the competitive strength of the resident microbiota relative to ingested microbiota, the host immune response, the combined digestive capabilities of host and microbiota, and the transmission of microorganisms between larvae. In order to predict and control host health, microbial safety, and successful application of microbial inocula, future research should prioritize quantifying the resilience of the larval gut microbiome to disturbances and identifying key contributors and conserved functions.