Metatranscriptomic analysis of the gut microbiome of black soldier fly larvae reared on lignocellulose-rich fiber diets unveils key lignocellulolytic enzymes.

Recently, interest in the black soldier fly larvae (BSFL) gut microbiome has received increased attention primarily due to their role in waste bioconversion. However, there is a lack of information on the positive effect on the activities of the gut microbiomes and enzymes (CAZyme families) acting on lignocellulose. In this study, BSFL were subjected to lignocellulose-rich diets: chicken feed (CF), chicken manure (CM), brewers' spent grain (BSG), and water hyacinth (WH). The mRNA libraries were prepared, and RNA-Sequencing was conducted using the PCR-cDNA approach through the MinION sequencing platform. Our results demonstrated that BSFL reared on BSG and WH had the highest abundance of Bacteroides and Dysgonomonas. The presence of GH51 and GH43_16 enzyme families in the gut of BSFL with both α-L-arabinofuranosidases and exo-alpha-L-arabinofuranosidase 2 were common in the BSFL reared on the highly lignocellulosic WH and BSG diets. Gene clusters that encode hemicellulolytic arabinofuranosidases in the CAZy family GH51 were also identified. These findings provide novel insight into the shift of gut microbiomes and the potential role of BSFL in the bioconversion of various highly lignocellulosic diets to fermentable sugars for subsequent value-added products (bioethanol). Further research on the role of these enzymes to improve existing technologies and their biotechnological applications is crucial.

Loss and Gain of Gut Bacterial Phylotype Symbionts in Afrotropical Stingless Bee Species (Apidae: Meliponinae).

Stingless bees (Apidae: Meliponini) are the most diverse group of corbiculate bees and are important managed and wild pollinators distributed in the tropical and subtropical regions of the globe. However, little is known about their associated beneficial microbes that play major roles in host nutrition, detoxification, growth, activation of immune responses, and protection against pathogens in their sister groups, honeybees and bumble bees. Here, we provide an initial characterization of the gut bacterial microbiota of eight stingless bee species from sub-Saharan Africa using 16S rRNA amplicon sequencing. Our findings revealed that Firmicutes, Actinobacteria, and Proteobacteria were the dominant and conserved phyla across the eight stingless bee species. Additionally, we found significant geographical and host intra-species-specific bacterial diversity. Notably, African strains showed significant phylogenetic clustering when compared with strains from other continents, and each stingless bee species has its own microbial composition with its own dominant bacterial genus. Our results suggest host selective mechanisms maintain distinct gut communities among sympatric species and thus constitute an important resource for future studies on bee health management and host-microbe co-evolution and adaptation.

Organic Waste Substrates Induce Important Shifts in Gut Microbiota of Black Soldier Fly (Hermetia illucens L.): Coexistence of Conserved, Variable, and Potential Pathogenic Microbes.

The sustainable utilization of black soldier fly (BSF) for recycling organic waste into nutrient-rich biomass, such as high-quality protein additive, is gaining momentum, and its microbiota is thought to play important roles in these processes. Several studies have characterized the BSF gut microbiota in different substrates and locations; nonetheless, in-depth knowledge on community stability, consistency of member associations, pathogenic associations, and microbe-microbe and host-microbe interactions remains largely elusive. In this study, we characterized the bacterial and fungal communities of BSF larval gut across four untreated substrates (brewers' spent grain, kitchen food waste, poultry manure, and rabbit manure) using 16S and ITS2 amplicon sequencing. Results demonstrated that substrate impacted larval weight gain from 30 to 100% gain differences among diets and induced an important microbial shift in the gut of BSF larvae: fungal communities were highly substrate dependent with Pichia being the only prevalent genus across 96% of the samples; bacterial communities also varied across diets; nonetheless, we observed six conserved bacterial members in 99.9% of our samples, namely, Dysgonomonas, Morganella, Enterococcus, Pseudomonas, Actinomyces, and Providencia. Among these, Enterococcus was highly correlated with other genera including Morganella and Providencia. Additionally, we showed that diets such as rabbit manure induced a dysbiosis with higher loads of the pathogenic bacteria Campylobacter. Together, this study provides the first comprehensive analysis of bacterial and fungal communities of BSF gut across untreated substrates and highlights conserved members, potential pathogens, and their interactions. This information will contribute to the establishment of safety measures for future processing of BSF larval meals and the creation of legislation to regulate their use in animal feeds.

Characterization of the Kenyan Honey Bee (Apis mellifera) Gut Microbiota: A First Look at Tropical and Sub-Saharan African Bee Associated Microbiomes.

Gut microbiota plays important roles in many physiological processes of the host including digestion, protection, detoxification, and development of immune responses. The honey bee (Apis mellifera) has emerged as model for gut-microbiota host interaction studies due to its gut microbiota being highly conserved and having a simple composition. A key gap in this model is understanding how the microbiome differs regionally, including sampling from the tropics and in particular from Africa. The African region is important from the perspective of the native diversity of the bees, and differences in landscape and bee management. Here, we characterized the honey bee gut microbiota in sub-Saharan Africa using 16S rRNA amplicon sequencing. We confirm the presence of the core gut microbiota members and highlight different compositions of these communities across regions. We found that bees from the coastal regions harbor a higher relative abundance and diversity on core members. Additionally, we showed that Gilliamella, Snodgrassella, and Frischella dominate in all locations, and that altitude and humidity affect Gilliamella abundance. In contrast, we found that Lactobacillus was less common compared temperate regions of the world. This study is a first comprehensive characterization of the gut microbiota of honey bees from sub-Saharan Africa and underscores the need to study microbiome diversity in other indigenous bee species and regions.

Blind killing of both male and female Drosophila embryos by a natural variant of the endosymbiotic bacterium Spiroplasma poulsonii.

Spiroplasma poulsonii is a vertically transmitted endosymbiont of Drosophila melanogaster that causes male-killing, that is the death of infected male embryos during embryogenesis. Here, we report a natural variant of S. poulsonii that is efficiently vertically transmitted yet does not selectively kill males, but kills rather a subset of all embryos regardless of their sex, a phenotype we call 'blind-killing'. We show that the natural plasmid of S. poulsonii has an altered structure: Spaid, the gene coding for the male-killing toxin, is deleted in the blind-killing strain, confirming its function as a male-killing factor. Then we further investigate several hypotheses that could explain the sex-independent toxicity of this new strain on host embryos. As the second non-male-killing variant isolated from a male-killing original population, this new strain raises questions on how male-killing is maintained or lost in fly populations. As a natural knock-out of Spaid, which is unachievable yet by genetic engineering approaches, this variant also represents a valuable tool for further investigations on the male-killing mechanism.

Complete Reference Genome Assembly for Commensalibacter sp. Strain AMU001, an Acetic Acid Bacterium Isolated from the Gut of Honey Bees.

We report here the genome sequence of a Commensalibacter sp. strain (AMU001) isolated from honey bees (Apis mellifera) from Seychelles. By combining long- and short-read sequencing technologies, we produced the first complete reference genome assembly for the Commensalibacter genus. We anticipate that this will aid future comparative and functional genomic studies.

The Role of Lipid Competition for Endosymbiont-Mediated Protection against Parasitoid Wasps in Drosophila.

UNLABELLED: Insects commonly harbor facultative bacterial endosymbionts, such as Wolbachia and Spiroplasma species, that are vertically transmitted from mothers to their offspring. These endosymbiontic bacteria increase their propagation by manipulating host reproduction or by protecting their hosts against natural enemies. While an increasing number of studies have reported endosymbiont-mediated protection, little is known about the mechanisms underlying this protection. Here, we analyze the mechanisms underlying protection from parasitoid wasps in Drosophila melanogaster mediated by its facultative endosymbiont Spiroplasma poulsonii Our results indicate that S. poulsonii exerts protection against two distantly related wasp species, Leptopilina boulardi and Asobara tabida S. poulsonii-mediated protection against parasitoid wasps takes place at the pupal stage and is not associated with an increased cellular immune response. In this work, we provide three important observations that support the notion that S. poulsonii bacteria and wasp larvae compete for host lipids and that this competition underlies symbiont-mediated protection. First, lipid quantification shows that both S. poulsonii and parasitoid wasps deplete D. melanogaster hemolymph lipids. Second, the depletion of hemolymphatic lipids using the Lpp RNA interference (Lpp RNAi) construct reduces wasp success in larvae that are not infected with S. poulsonii and blocks S. poulsonii growth. Third, we show that the growth of S. poulsonii bacteria is not affected by the presence of the wasps, indicating that when S. poulsonii is present, larval wasps will develop in a lipid-depleted environment. We propose that competition for host lipids may be relevant to endosymbiont-mediated protection in other systems and could explain the broad spectrum of protection provided. IMPORTANCE: Virtually all insects, including crop pests and disease vectors, harbor facultative bacterial endosymbionts. They are vertically transmitted from mothers to their offspring, and some protect their host against pathogens. Here, we studied the mechanism of protection against parasitoid wasps mediated by the Drosophila melanogaster endosymbiont Spiroplasma poulsonii Using genetic manipulation of the host, we provide strong evidence supporting the hypothesis that competition for host lipids underlies S. poulsonii-mediated protection against parasitoid wasps. We propose that lipid competition-based protection may not be restricted to Spiroplasma bacteria but could also apply other endosymbionts, notably Wolbachia bacteria, which can suppress human disease-causing viruses in insect hosts.

Genome sequence of the Drosophila melanogaster male-killing Spiroplasma strain MSRO endosymbiont.

UNLABELLED: Spiroplasmas are helical and motile members of a cell wall-less eubacterial group called Mollicutes. Although all spiroplasmas are associated with arthropods, they exhibit great diversity with respect to both their modes of transmission and their effects on their hosts; ranging from horizontally transmitted pathogens and commensals to endosymbionts that are transmitted transovarially (i.e., from mother to offspring). Here we provide the first genome sequence, along with proteomic validation, of an endosymbiotic inherited Spiroplasma bacterium, the Spiroplasma poulsonii MSRO strain harbored by Drosophila melanogaster. Comparison of the genome content of S. poulsonii with that of horizontally transmitted spiroplasmas indicates that S. poulsonii has lost many metabolic pathways and transporters, demonstrating a high level of interdependence with its insect host. Consistent with genome analysis, experimental studies showed that S. poulsonii metabolizes glucose but not trehalose. Notably, trehalose is more abundant than glucose in Drosophila hemolymph, and the inability to metabolize trehalose may prevent S. poulsonii from overproliferating. Our study identifies putative virulence genes, notably, those for a chitinase, the H2O2-producing glycerol-3-phosphate oxidase, and enzymes involved in the synthesis of the eukaryote-toxic lipid cardiolipin. S. poulsonii also expresses on the cell membrane one functional adhesion-related protein and two divergent spiralin proteins that have been implicated in insect cell invasion in other spiroplasmas. These lipoproteins may be involved in the colonization of the Drosophila germ line, ensuring S. poulsonii vertical transmission. The S. poulsonii genome is a valuable resource to explore the mechanisms of male killing and symbiont-mediated protection, two cardinal features of many facultative endosymbionts. IMPORTANCE: Most insect species, including important disease vectors and crop pests, harbor vertically transmitted endosymbiotic bacteria. These endosymbionts play key roles in their hosts' fitness, including protecting them against natural enemies and manipulating their reproduction in ways that increase the frequency of symbiont infection. Little is known about the molecular mechanisms that underlie these processes. Here, we provide the first genome draft of a vertically transmitted male-killing Spiroplasma bacterium, the S. poulsonii MSRO strain harbored by D. melanogaster. Analysis of the S. poulsonii genome was complemented by proteomics and ex vivo metabolic experiments. Our results indicate that S. poulsonii has reduced metabolic capabilities and expresses divergent membrane lipoproteins and potential virulence factors that likely participate in Spiroplasma-host interactions. This work fills a gap in our knowledge of insect endosymbionts and provides tools with which to decipher the interaction between Spiroplasma bacteria and their well-characterized host D. melanogaster, which is emerging as a model of endosymbiosis.

Insect endosymbiont proliferation is limited by lipid availability.

Spiroplasma poulsonii is a maternally transmitted bacterial endosymbiont that is naturally associated with Drosophila melanogaster. S. poulsonii resides extracellularly in the hemolymph, where it must acquire metabolites to sustain proliferation. In this study, we find that Spiroplasma proliferation specifically depletes host hemolymph diacylglyceride, the major lipid class transported by the lipoprotein, Lpp. RNAi-mediated knockdown of Lpp expression, which reduces the amount of circulating lipids, inhibits Spiroplasma proliferation demonstrating that bacterial proliferation requires hemolymph-lipids. Altogether, our study shows that an insect endosymbiont acquires specific lipidic metabolites from the transport lipoproteins in the hemolymph of its host. In addition, we show that the proliferation of this endosymbiont is limited by the availability of hemolymph lipids. This feature could limit endosymbiont over-proliferation under conditions of host nutrient limitation as lipid availability is strongly influenced by the nutritional state.

Vertical transmission of a Drosophila endosymbiont via cooption of the yolk transport and internalization machinery.

UNLABELLED: Spiroplasma is a diverse bacterial clade that includes many vertically transmitted insect endosymbionts, including Spiroplasma poulsonii, a natural endosymbiont of Drosophila melanogaster. These bacteria persist in the hemolymph of their adult host and exhibit efficient vertical transmission from mother to offspring. In this study, we analyzed the mechanism that underlies their vertical transmission, and here we provide strong evidence that these bacteria use the yolk uptake machinery to colonize the germ line. We show that Spiroplasma reaches the oocyte by passing through the intercellular space surrounding the ovarian follicle cells and is then endocytosed into oocytes within yolk granules during the vitellogenic stages of oogenesis. Mutations that disrupt yolk uptake by oocytes inhibit vertical Spiroplasma transmission and lead to an accumulation of these bacteria outside the oocyte. Impairment of yolk secretion by the fat body results in Spiroplasma not reaching the oocyte and a severe reduction of vertical transmission. We propose a model in which Spiroplasma first interacts with yolk in the hemolymph to gain access to the oocyte and then uses the yolk receptor, Yolkless, to be endocytosed into the oocyte. Cooption of the yolk uptake machinery is a powerful strategy for endosymbionts to target the germ line and achieve vertical transmission. This mechanism may apply to other endosymbionts and provides a possible explanation for endosymbiont host specificity. IMPORTANCE: Most insect species, including important disease vectors and crop pests, harbor vertically transmitted endosymbiotic bacteria. Studies have shown that many facultative endosymbionts, including Spiroplasma, confer protection against different classes of parasites on their hosts and therefore are attractive tools for the control of vector-borne diseases. The ability to be efficiently transmitted from females to their offspring is the key feature shaping associations between insects and their inherited endosymbionts, but to date, little is known about the mechanisms involved. In oviparous animals, yolk accumulates in developing eggs and serves to meet the nutritional demands of embryonic development. Here we show that Spiroplasma coopts the yolk transport and uptake machinery to colonize the germ line and ensure efficient vertical transmission. The uptake of yolk is a female germ line-specific feature and therefore an attractive target for cooption by endosymbionts that need to maintain high-fidelity maternal transmission.

Negative regulation by amidase PGRPs shapes the Drosophila antibacterial response and protects the fly from innocuous infection.

Peptidoglycan recognition proteins (PGRPs) are key regulators of insect immune responses. In addition to recognition PGRPs, which activate the Toll and Imd pathways, the Drosophila genome encodes six catalytic PGRPs with the capacity to scavenge peptidoglycan. We have performed a systematic analysis of catalytic PGRP function using deletions, separately and in combination. Our findings support the role of PGRP-LB as a negative regulator of the Imd pathway and brought to light a synergy of PGRP-SCs with PGRP-LB in the systemic response. Flies lacking all six catalytic PGRPs were still viable but exhibited deleterious immune responses to innocuous gut infections. Together with recent studies on mammalian PGRPs, our study uncovers a conserved role for PGRPs in gut homeostasis. Analysis of the immune phenotype of flies lacking all catalytic PGRPs and the Imd regulator Pirk reveals that the Imd-mediated immune response is highly constrained by the existence of multiple negative feedbacks.

Drosophila immunity: analysis of PGRP-SB1 expression, enzymatic activity and function.

Peptidoglycan is an essential and specific component of the bacterial cell wall and therefore is an ideal recognition signature for the immune system. Peptidoglycan recognition proteins (PGRPs) are conserved from insects to mammals and able to bind PGN (non-catalytic PGRPs) and, in some cases, to efficiently degrade it (catalytic PGRPs). In Drosophila, several non-catalytic PGRPs function as selective peptidoglycan receptors upstream of the Toll and Imd pathways, the two major signalling cascades regulating the systemic production of antimicrobial peptides. Recognition PGRPs specifically activate the Toll pathway in response to Lys-type peptidoglycan found in most Gram-positive bacteria and the Imd pathway in response to DAP-type peptidoglycan encountered in Gram-positive bacilli-type bacteria and in Gram-negative bacteria. Catalytic PGRPs on the other hand can potentially reduce the level of immune activation by scavenging peptidoglycan. In accordance with this, PGRP-LB and PGRP-SC1A/B/2 have been shown to act as negative regulators of the Imd pathway. In this study, we report a biochemical and genetic analysis of PGRP-SB1, a catalytic PGRP. Our data show that PGRP-SB1 is abundantly secreted into the hemolymph following Imd pathway activation in the fat body, and exhibits an enzymatic activity towards DAP-type polymeric peptidoglycan. We have generated a PGRP-SB1/2 null mutant by homologous recombination, but its thorough phenotypic analysis did not reveal any immune function, suggesting a subtle role or redundancy of PGRP-SB1/2 with other molecules. Possible immune functions of PGRP-SB1 are discussed.