Black soldier fly larvae (BSFL) are fast-growing, resilient insects that can break down a variety of organic substrates and convert them into valuable proteins and lipids for applications in the feed industry. Decomposition is mediated by an abundant and versatile gut microbiome, which has been studied for more than a decade. However, little is known about the phylogeny, properties and functions of bacterial isolates from the BSFL gut. We therefore characterized the BSFL gut microbiome in detail, evaluating bacterial diversity by culture-dependent methods and amplicon sequencing of the 16S rRNA gene. Redundant strains were identified by genomic fingerprinting and 105 non-redundant isolates were then tested for their ability to inhibit pathogens. We cultivated representatives of 26 genera, covering 47% of the families and 33% of the genera detected by amplicon sequencing. Among these isolates, we found several representatives of the most abundant genera: Morganella, Enterococcus, Proteus and Providencia. We also isolated diverse members of the less-abundant phylum Actinobacteria, and a novel genus of the order Clostridiales. We found that 15 of the isolates inhibited at least one of the tested pathogens, suggesting a role in helping to prevent colonization by pathogens in the gut. The resulting culture collection of unique BSFL gut bacteria provides a promising resource for multiple industrial applications.

Insects offer a promising alternative source of protein to mitigate the environmental consequences of conventional livestock farming. Larvae of the black soldier fly (Hermetia illucens; Linnaeus, 1758) efficiently convert a variety of organic side streams and residues into valuable proteins, lipids, and chitin. Here, we evaluated the suitability of two palm oil industry side streams—empty fruit bunches (EFB) and palm kernel meal (PKM)—as larval feed, and their impact on the larval gut microbiome. Among 69 fungal species we screened, Marasmius palmivorus, Irpex consors, and Bjerkandera adusta achieved the fastest growth and lignin degradation, so these fungi were used for the pretreatment of 7:3 mixtures of EFB and PKM. Larvae reared on the mixture pretreated with B. adusta (BAD) developed significantly more quickly and reached a higher final weight than those reared on the other pretreatments or the non-fermented reference (NFR). Amplicon sequencing of the BAD and NFR groups revealed major differences in the larval gut microbiome. The NFR group was dominated by facultatively anaerobic Enterobacteriaceae (typical of H. illucens larvae) whereas the BAD group favored obligately anaerobic, cellulolytic bacteria (Ruminococcaceae and Lachnospiraceae). We hypothesize that fungal lignin degradation led to an accumulation of mycelia and subsequent cellulolytic breakdown of fiber residues, thus improving substrate digestibility.

Lipocalin 2 (LCN2) mediates key roles in innate immune responses. It has affinity for many lipophilic ligands and binds various siderophores, thereby limiting bacterial growth by iron sequestration. Furthermore, LCN2 protects against obesity and metabolic syndrome by interfering with the composition of gut microbiota. Consequently, complete or hepatocyte-specific ablation of the Lcn2 gene is associated with higher susceptibility to bacterial infections. In the present study, we comparatively profiled microbiota in fecal samples of wild type and Lcn2 null mice and show, in contrast to previous reports, that the quantity of DNA in feces of Lcn2 null mice is significantly lower than that in wild type mice (p < 0.001). By using the hypervariable V4 region of the 16S rDNA gene and Next-Generation Sequencing methods, we found a statistically significant change in 16 taxonomic units in Lcn2^-/- mice, including eight gender-specific deviations. In particular, members of Clostridium, Escherichia, Helicobacter, Lactococcus, Prevotellaceae_UCG-001 and Staphylococcus appeared to expand in the intestinal tract of knockout mice. Interestingly, the proportion of Escherichia (200-fold) and Staphylococcus (10-fold) as well as the abundance of intestinal bacteria encoding the LCN2-sensitive siderphore enterobactin (entA) was significantly increased in male Lcn2 null mice (743-fold, p < 0.001). This was accompanied by significant higher immune cell infiltration in the ileum as demonstrated by increased immunoreactivity against the pan-leukocyte protein CD45, the lymphocyte transcription factor MUM-1/IRF4, and the macrophage antigen CD68/Macrosialin. In addition, we found a higher expression of mucosal mast cell proteases indicating a higher number of those innate immune cells. Finally, the ileum of Lcn2 null mice displayed a high abundance of segmented filamentous bacteria, which are intimately associated with the mucosal cell layer, provoking epithelial antimicrobial responses and affecting T-helper cell polarization.

Animal venoms are a rich source of novel biomolecules with potential applications in medicine and agriculture. Ants are one of the most species-rich lineages of venomous animals. However, only a fraction of their biodiversity has been studied so far. Here, we investigated the venom components of two myrmicine (subfamily Myrmicinae) ants: Myrmica rubra and Myrmica ruginodis. We applied a venomics workflow based on proteotranscriptomics and found that the venoms of both species are composed of several protein classes, including venom serine proteases, cysteine-rich secretory protein, antigen 5 and pathogenesis-related 1 (CAP) superfamily proteins, Kunitz-type serine protease inhibitors and venom acid phosphatases. Several of these protein classes are known venom allergens, and for the first time we detected phospholipase A1 in the venom of M. ruginodis. We also identified two novel epidermal growth factor (EGF) family toxins in the M. ruginodis venom proteome and an array of additional EGF-like toxins in the venom gland transcriptomes of both species. These are similar to known toxins from the related myrmicine ant, Manica rubida, and the myrmecine (subfamily Myrmeciinae) Australian red bulldog ant Myrmecia gullosa, and are possibly deployed as weapons in defensive scenarios or to subdue prey. Our work suggests that M.rubra and M. ruginodis venoms contain many enzymes and other high-molecular-weight proteins that cause cell damage. Nevertheless, the presence of EGF-like toxins suggests that myrmicine ants have also recruited smaller peptide components into their venom arsenal. Although little is known about the bioactivity and function of EGF-like toxins, their presence in myrmicine and myrmecine ants suggests they play a key role in the venom systems of the superfamily Formicoidea. Our work adds to the emerging picture of ant venoms as a source of novel bioactive molecules and highlights the need to incorporate such taxa in future venom bioprospecting programs.

The venoms of ants (Formicidae) are a promising source of novel bioactive molecules with potential for clinical and agricultural applications. However, despite the rich diversity of ant species, only a fraction of this vast resource has been thoroughly examined in bioprospecting programs. Previous studies focusing on the venom of Central European ants (subfamily Myrmicinae) identified a number of short linear decapeptides and nonapeptides resembling antimicrobial peptides (AMPs). Here, we describe the in silico approach and bioactivity profiling of 10 novel AMP-like peptides from the fellow Central European myrmicine ants Myrmica rubra and Myrmica ruginodis. Using the sequences of known ant venom peptides as queries, we screened the venom gland transcriptomes of both species. We found transcripts of nine novel decapeptides and one novel nonapeptide. The corresponding peptides were synthesized for bioactivity profiling in a broad panel of assays consisting of tests for cytotoxicity as well as antiviral, insecticidal, and antimicrobial activity. U-MYRTX-Mrug5a showed moderately potent antimicrobial effects against several bacteria, including clinically relevant pathogens such as Listeria monocytogenes and Staphylococcus epidermidis, but high concentrations showed negligible cytotoxicity. U-MYRTX-Mrug5a is, therefore, a probable lead for the development of novel peptide-based antibiotics.

Black soldier fly larvae (BSFL) have attracted attention due to their ability to upcycle various biological side streams into valuable biomass, such as proteins, lipids, and chitin. In this study, we investigated the impact of high-fiber diets on larval growth performance and the shift of microbes in the gut. We tested empty fruit bunches (EFB), potato pulp (PP), and cottonseed press cake (CPC), with chicken feed (CF) used as a control diet. We found that larvae reared on the EFB, PP, and CPC were smaller than control larvae at the end of development due to the low nutritional value of the diets. However, survival rates of more than 90% were observed regardless of the diet. We used a cultivation-dependent approach to analyze the microbial community in the gut of BSFL, isolated, and identified a total of 329 bacterial strains. Bacillaceae were most frequently isolated from larvae reared on the high-fiber EFB diet. These isolates were predicted to degrade cellulose in silico and this was subsequently confirmed in vitro using the Congo Red assay. Whereas the members of Enterobacteriaceae and Morganellaceae were mostly found in guts of larvae reared on the high-protein diets CPC and CF. We conclude that the gut microbiome plays a crucial role in the digestion of fiber-rich plant organic material, thereby enabling the BSFL to successfully complete their life cycle also on substrates with low nutritional value. As a result, BSFL convert industrial side streams into valuable biomass, reducing waste and promoting sustainability. IMPORTANCE: Organic side streams from various industries pose a challenge to the environment. They are often present in huge amounts and are mostly discarded, incinerated, used for biogas production, or as feed for ruminant animals. Many plant-based side streams contain difficult-to-digest fiber as well as anti-nutritional or even insecticidal compounds that could harm the animals. These challenges can be addressed using black soldier fly larvae, which are known to degrade various organic substrates and convert them into valuable biomass. This will help mitigate agro-industrial side streams via efficient waste management and will contribute to the more economical and sustainable farming of insects.