Depletion of Lipocalin 2 (LCN2) in Mice Leads to Dysbiosis and Persistent Colonization with Segmented Filamentous Bacteria.

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.

Model-based process optimization of black soldier fly egg production.

Black soldier fly (BSF) larvae (Hermetia illucens) serve as a valuable protein source for animal feed. Limiting factors in the industrial rearing of BSF are the reproduction process and egg output. Studies indicate the potential to shorten preoviposition time and increase egg output through better utilization of environmental variables, such as temperature and light, in industrial settings. Excessive stimulation, however, can lead to stress, elevated production costs, and reduced egg numbers, emphasizing the need for a delicate balance. This study addresses these challenges by investigating controlled manipulation of environmental variables to stimulate mating and enhance egg production, thereby developing a comprehensive model encompassing the adult fly life cycle, mating, and egg production. Model parameters were fitted using literature data, and the model's plausibility was tested through simulations. Using the model and optimal control methods, the calculated dynamic trajectories for environmental variables when compared to the standard approach in a constant environment demonstrated higher output and shorter production cycles at reasonable energy costs. Applications for this model-based optimization are demonstrated for various scenarios, highlighting the practical utility and versatility of the developed model. This study contributes valuable insights for improving rearing practices of BSF through environmental stimulation, offering potential advancements in egg production efficiency and overall sustainability.

Protein- and Carbohydrate-Rich Supplements in Feeding Adult Black Soldier Flies (Hermetia illucens) Affect Life History Traits and Egg Productivity.

The black soldier fly, Hermetia illucens (BSF; Diptera: Stratiomyidae), has come into the focus of research over the past decade since its larvae are polyphagous feeders with an exceptional substrate range, making them a promising candidate for the bioconversion of various organic side streams into valuable insect protein. While larval nutritional requirements have been studied in detail, basic information on adult feeding is still lacking. The reproduction of adult flies is a bottleneck and key determinant in rearing BSF, which has extensive potential for improvement. In the present study, we examined the impact of different carbohydrate (honey and d-glucose) and protein sources (Spirulina and Chlorella powder) on a variety of life history traits using a highly standardized single pair approach. Feeding a 5% honey solution was shown to make females live 2.8 d longer, become more fecund (9 egg clutches per 10 females), lay more eggs (increasing 1.7-fold to 182.4 mg per 10 females), reduce the number of failed oviposition events 3-fold and increase multiple oviposition events from 2 to 15. Additionally, female longevity after oviposition improved 1.7-fold from 6.7 to 11.5 d. In order to further optimize adult feeding, mixtures of proteins and carbohydrates with varying ratios should be tested.

Fatty acids derived from oviposition systems guide female black soldier flies (Hermetia illucens) toward egg deposition sites.

The black soldier fly, Hermetia illucens, comes with big promises for industrial purposes since its larvae feed polyphagously on a broad spectrum of organic substrates. However, research focusing on adult flies is scarce, which is inconsistent with their reproductive relevance within the rearing cycle. In particular, directed oviposition is a challenge in artificial systems. Currently, decomposing organic matter is commonly used as oviposition substrate, which has extensive potential for improvement in view of the lack of standardization and the risk of microbial contamination. Here, we identified three fatty acids and one fatty acid methyl ester derived from the surface of old oviposition sites and targeted to elucidate their effect on preference behavior and oviposition site selection using Y-olfactometry and prepared oviposition sites, respectively. Exposure to tetradecanoic acid attracted gravid females and stimulated oviposition most strongly, while decanoic acid demonstrated a repulsive effect. Females kept in mixed-sex populations were attracted by tetradecanoic acid, resulting in a higher egg mass found in the compound box (3.0-11.4 fold), a ≥ 2.3 fold reduction of nonspecifically deposited eggs, and the highest total egg mass. Conversely, decanoic and dodecanoic acid caused females to lay a greater proportion of eggs nonspecifically outside both boxes. Our data suggest that fatty acids, especially tetradecanoic acid, are important cues for oviposition site selection in black soldier flies. In order to achieve a directed oviposition behavior, the role of further short- and long-chain fatty acids as attractants should be examined.

Isolation of Bacterial and Fungal Microbiota Associated with Hermetia illucens Larvae Reveals Novel Insights into Entomopathogenicity.

Larvae of the black soldier fly (BSF) Hermetia illucens are polyphagous feeders and show tremendous bioconversion capabilities of organic matter into high-quality insect biomass. However, the digestion of lignocellulose-rich palm oil side streams such as palm kernel meal (PKM) is a particular challenge, as these compounds are exceptionally stable and are mainly degraded by microbes. This study aimed to investigate the suitability of BSF larvae as bioconversion agents of PKM. Since the intestinal microbiota is considered to play a key role in dietary breakdown and in increasing digestibility, the bacterial and fungal communities of BSF larvae were characterized in a culture-dependent approach and screened for their putative entomopathogenicity. The lethality of six putative candidates was investigated using intracoelomal injection. In total, 93 isolates were obtained with a bacterial share of 74% that were assigned to the four phyla Actinobacteria, Bacteroidetes, Firmicutes, and Proteobacteria. Members of the genera Klebsiella, Enterococcus, and Sphingobacterium are part of the core microbiome, as they were frequently described in the gut of Hermetia larvae regardless of diet, nutritional composition, or rearing conditions. With 75%, a majority of the fungal isolates belonged to the phylum Ascomycota. We identified several taxa already published to be able to degrade lignocelluloses, including Enterococcus, Cellulomonas, Pichia yeasts, or filamentous Fusarium species. The injection assays revealed pronounced differences in pathogenicity against the larvae. While Alcaligenes faecalis caused no, Diutina rugosa weak (23.3%), Microbacterium thalassium moderate (53.3%), and Pseudomonas aeruginosa and Klebsiella pneumoniae high (≥80%) lethality, Fusarium solani injection resulted in 100% lethality.

Cottonseed Press Cake as a Potential Diet for Industrially Farmed Black Soldier Fly Larvae Triggers Adaptations of Their Bacterial and Fungal Gut Microbiota.

Black soldier fly larvae (Hermetia illucens, Diptera: Stratiomyidae) are used for the bioconversion of organic side products into valuable compounds such as proteins, lipids and chitin. However, the economic competitiveness of farmed insects compared to conventional protein production systems in agriculture and aquaculture depends on the availability of large quantities of inexpensive insect feed. Cottonseed press cake (CPC) is a side-stream of cotton production that is rich in proteins and lipids but unsuitable as feed for several farmed animals, except ruminants, due to the presence of the anti-nutritional sesquiterpenoid gossypol. Here, we tested CPC as a feed for black soldier fly larvae and studied the impact of this diet on the gut microbiome. Larvae reared on CPC developed normally and even showed a shorter life-cycle, but were smaller at the end of larval development than control larvae reared on chicken feed. The adaptability of the larvae to different diets is mediated by their versatile gut microbiome, which facilitates digestion and detoxification. We therefore used amplicon sequencing to analyze the bacterial and fungal communities associated with larvae reared on each diet, revealing differences between the larval guts and frass (residual feed substrate) as well as differences between the two diet groups. For example, Actinomycetaceae and Aspergillaceae were significantly enriched in guts of the CPC diet group and may help to metabolize compounds such as gossypol. Potentially probiotic yeasts and beneficial Enterobacteriaceae, which presumably belong to the core microbiota, were detected in high relative abundance in the gut and frass, indicating a functional role of these microbes, especially the protection against pathogens. We conclude that CPC may be suitable as an inexpensive and environmentally sustainable feed for the industrial rearing of black soldier flies.

Corrigendum: The Stand-Alone PilZ-Domain Protein MotL Specifically Regulates the Activity of the Secondary Lateral Flagellar System in Shewanella putrefaciens.

[This corrects the article DOI: 10.3389/fmicb.2021.668892.].

The Stand-Alone PilZ-Domain Protein MotL Specifically Regulates the Activity of the Secondary Lateral Flagellar System in Shewanella putrefaciens.

A number of bacterial species control the function of the flagellar motor in response to the levels of the secondary messenger c-di-GMP, which is often mediated by c-di-GMP-binding proteins that act as molecular brakes or clutches to slow the motor rotation. The gammaproteobacterium Shewanella putrefaciens possesses two distinct flagellar systems, the primary single polar flagellum and a secondary system with one to five lateral flagellar filaments. Here, we identified a protein, MotL, which specifically regulates the activity of the lateral, but not the polar, flagellar motors in response to the c-di-GMP levels. MotL only consists of a single PilZ domain binding c-di-GMP, which is crucial for its function. Deletion and overproduction analyses revealed that MotL slows down the lateral flagella at elevated levels of c-di-GMP, and may speed up the lateral flagellar-mediated movement at low c-di-GMP concentrations. In vitro interaction studies hint at an interaction of MotL with the C-ring of the lateral flagellar motors. This study shows a differential c-di-GMP-dependent regulation of the two flagellar systems in a single species, and implicates that PilZ domain-only proteins can also act as molecular regulators to control the flagella-mediated motility in bacteria.

Does Light Color Temperature Influence Aspects of Oviposition by the Black Soldier Fly (Diptera: Stratiomyidae)?

In recent years, black soldier fly, Hermetia illucens (L.), larvae have attracted increasing attention because of their high capacity for bioconversion of diverse organic material into high-quality protein and lipids. Although previous studies have focused on optimization of breeding conditions, such as the acceptance of substrates, and temperatures and moisture contents, little is known about light-dependent adult development. Artificial light sources are important to commercial H. illucens breeding, especially at latitudes with short days in autumn and winter months. We examined how 3,000, 4,000, and 6,500 K color temperatures affect aspects of oviposition. Mating occurred under all of the broad spectrum light-emitting diode panels, resulting in fertilized egg clusters. Oviposition lasted up to 15 d, while the shortest oviposition period, in the 3,000 K light treatment, was 2 d. Total oviposition performance and oviposition period were not affected by the light treatments. Oviposition peaked 1-7 d after eggs were first deposited. The time until oviposition peaked was positively correlated with increasing color temperature.