Developmental stages and gut microenvironments influence gut microbiota dynamics in the invasive beetle Popillia japonica Newman (Coleoptera: Scarabaeidae).

Popillia japonica Newman (Coleoptera: Scarabaeidae) is a highly polyphagous invasive beetle originating from Japan. This insect is highly resilient and able to rapidly adapt to new vegetation. Insect-associated microorganisms can play important roles in insect physiology, helping their hosts to adapt to changing conditions and potentially contributing to an insect's invasive potential. Such symbiotic bacteria can be part of a core microbiota that is stably transmitted throughout the host's life cycle or selectively recruited from the environment at each developmental stage. The aim of this study was to investigate the origin, stability and turnover of the bacterial communities associated with an invasive population of P. japonica from Italy. Our results demonstrate that soil microbes represent an important source of gut bacteria for P. japonica larvae, but as the insect develops, its gut microbiota richness and diversity decreased substantially, paralleled by changes in community composition. Notably, only 16.75% of the soil bacteria present in larvae are maintained until the adult stage. We further identified the micro-environments of different gut sections as an important factor shaping microbiota composition in this species, likely due to differences in pH, oxygen availability and redox potential. In addition, P. japonica also harboured a stable bacterial community across all developmental stages, consisting of taxa well known for the degradation of plant material, namely the families Ruminococcacae, Christensenellaceae and Lachnospiraceae. Interestingly, the family Christensenallaceae had so far been observed exclusively in humans. However, the Christensenellaceae operational taxonomic units found in P. japonica belong to different taxonomic clades within this family.

Destabilization of the Bacterial Interactome Identifies Nutrient Restriction-Induced Dysbiosis in Insect Guts.

Stress-associated dysbiosis of microbiome can have several configurations that, under an energy landscape conceptual framework, can change from one configuration to another due to different alternating selective forces. It has been proposed-according to the Anna Karenina Principle-that in stressed individuals the microbiome are more dispersed (i.e., with a higher within-beta diversity), evidencing the grade of dispersion as indicator of microbiome dysbiosis. We hypothesize that although dysbiosis leads to different microbial communities in terms of beta diversity, these are not necessarily differently dispersed (within-beta diversity), but they form disrupted networks that make them less resilient to stress. To test our hypothesis, we select nutrient restriction (NR) stress that impairs host fitness but does not introduce overt microbiome selectors, such as toxic compounds and pathogens. We fed the polyphagous black soldier fly, Hermetia illucens, with two NR diets and a control full-nutrient (FN) diet. NR diets were dysbiotic because they strongly affected insect growth and development, inducing significant microscale changes in physiochemical conditions of the gut compartments. NR diets established new configurations of the gut microbiome compared to FN-fed guts but with similar dispersion. However, these new configurations driven by the deterministic changes induced by NR diets were reflected in rarefied, less structured, and less connected bacterial interactomes. These results suggested that while the dispersion cannot be considered a consistent indicator of the unhealthy state of dysbiotic microbiomes, the capacity of the community members to maintain network connections and stability can be an indicator of the microbial dysbiotic conditions and their incapacity to sustain the holobiont resilience and host homeostasis. IMPORTANCE Changes in diet play a role in reshaping the gut microbiome in animals, inducing dysbiotic configurations of the associated microbiome. Although studies have reported on the effects of specific nutrient contents on the diet, studies regarding the conditions altering the microbiome configurations and networking in response to diet changes are limited. Our results showed that nutrient poor diets determine dysbiotic states of the host with reduction of insect weight and size, and increase of the times for developmental stage. Moreover, the poor nutrient diets lead to changes in the compositional diversity and network interaction properties of the gut microbial communities. Our study adds a new component to the understanding of the ecological processes associated with dysbiosis, by disentangling consequences of diets on microbiome dysbiosis that is manifested with the disruption of microbiome networking properties rather than changes in microbiome dispersion and beta diversity.

Adaptation of Microbial Communities to Environmental Arsenic and Selection of Arsenite-Oxidizing Bacteria From Contaminated Groundwaters.

Arsenic mobilization in groundwater systems is driven by a variety of functionally diverse microorganisms and complex interconnections between different physicochemical factors. In order to unravel this great ecosystem complexity, groundwaters with varying background concentrations and speciation of arsenic were considered in the Po Plain (Northern Italy), one of the most populated areas in Europe affected by metalloid contamination. High-throughput Illumina 16S rRNA gene sequencing, CARD-FISH and enrichment of arsenic-transforming consortia showed that among the analyzed groundwaters, diverse microbial communities were present, both in terms of diversity and functionality. Oxidized inorganic arsenic [arsenite, As(III)] was the main driver that shaped each community. Several uncharacterized members of the genus Pseudomonas, putatively involved in metalloid transformation, were revealed in situ in the most contaminated samples. With a cultivation approach, arsenic metabolisms potentially active at the site were evidenced. In chemolithoautotrophic conditions, As(III) oxidation rate linearly correlated to As(III) concentration measured at the parental sites, suggesting that local As(III) concentration was a relevant factor that selected for As(III)-oxidizing bacterial populations. In view of the exploitation of these As(III)-oxidizing consortia in biotechnology-based arsenic bioremediation actions, these results suggest that contaminated aquifers in Northern Italy host unexplored microbial populations that provide essential ecosystem services.

Compartmentalization of bacterial and fungal microbiomes in the gut of adult honeybees.

The core gut microbiome of adult honeybee comprises a set of recurring bacterial phylotypes, accompanied by lineage-specific, variable, and less abundant environmental bacterial phylotypes. Several mutual interactions and functional services to the host, including the support provided for growth, hormonal signaling, and behavior, are attributed to the core and lineage-specific taxa. By contrast, the diversity and distribution of the minor environmental phylotypes and fungal members in the gut remain overlooked. In the present study, we hypothesized that the microbial components of forager honeybees (i.e., core bacteria, minor environmental phylotypes, and fungal members) are compartmentalized along the gut portions. The diversity and distribution of such three microbial components were investigated in the context of the physico-chemical conditions of different gut compartments. We observed that changes in the distribution and abundance of microbial components in the gut are consistently compartment-specific for all the three microbial components, indicating that the ecological and physiological interactions among the host and microbiome vary with changing physico-chemical and metabolic conditions of the gut.

Hydrolytic Profile of the Culturable Gut Bacterial Community Associated With Hermetia illucens.

Larvae of the black soldier fly (BSF) Hermetia illucens (L.) convert organic waste into high valuable insect biomass that can be used as alternative protein source for animal nutrition or as feedstock for biodiesel production. Since insect biology and physiology are influenced by the gut microbiome, knowledge about the functional role of BSF-associated microorganisms could be exploited to enhance the insect performance and growth. Although an increasing number of culture-independent studies are unveiling the microbiota structure and composition of the BSF gut microbiota, a knowledge gap remains on the experimental validation of the contribution of the microorganisms to the insect growth and development. We aimed at assessing if BSF gut-associated bacteria potentially involved in the breakdown of diet components are able to improve host nutrition. A total of 193 bacterial strains were obtained from guts of BSF larvae reared on a nutritious diet using selective and enrichment media. Most of the bacterial isolates are typically found in the insect gut, with major representatives belonging to the Gammaproteobacteria and Bacilli classes. The hydrolytic profile of the bacterial collection was assessed on compounds typically present in the diet. Finally, we tested the hypothesis that the addition to a nutritionally poor diet of the two isolates Bacillus licheniformis HI169 and Stenotrophomonas maltophilia HI121, selected for their complementary metabolic activities, could enhance BSF growth. B. licheniformis HI169 positively influenced the larval final weight and growth rate when compared to the control. Conversely, the addition of S. maltophilia HI121 to the nutritionally poor diet did not result in a growth enhancement in terms of larval weight and pupal weight and length in comparison to the control, whereas the combination of the two strains positively affected the larval final weight and the pupal weight and length. In conclusion, we isolated BSF-associated bacterial strains with potential positive properties for the host nutrition and we showed that selected isolates may enhance BSF growth, suggesting the importance to evaluate the effect of the bacterial administration on the insect performance.

Phenomics and Genomics Reveal Adaptation of Virgibacillus dokdonensis Strain 21D to Its Origin of Isolation, the Seawater-Brine Interface of the Mediterranean Sea Deep Hypersaline Anoxic Basin Discovery.

The adaptation of sporeformers to extreme environmental conditions is frequently questioned due to their capacity to produce highly resistant endospores that are considered as resting contaminants, not representing populations adapted to the system. In this work, in order to gain a better understanding of bacterial adaptation to extreme habitats, we investigated the phenotypic and genomic characteristics of the halophile Virgibacillus sp. 21D isolated from the seawater-brine interface (SBI) of the MgCl2-saturated deep hypersaline anoxic basin Discovery located in the Eastern Mediterranean Sea. Vegetative cells of strain 21D showed the ability to grow in the presence of high concentrations of MgCl2, such as 14.28% corresponding to 1.5 M. Biolog phenotype MicroArray (PM) was adopted to investigate the strain phenotype, with reference to carbon energy utilization and osmotic tolerance. The strain was able to metabolize only 8.4% of 190 carbon sources provided in the PM1 and PM2 plates, mainly carbohydrates, in accordance with the low availability of nutrients in its habitat of origin. By using in silico DNA-DNA hybridization the analysis of strain 21D genome, assembled in one circular contig, revealed that the strain belongs to the species Virgibacillus dokdonensis. The genome presented compatible solute-based osmoadaptation traits, including genes encoding for osmotically activated glycine-betaine/carnitine/choline ABC transporters, as well as ectoine synthase enzymes. Osmoadaptation of the strain was then confirmed with phenotypic assays by using the osmolyte PM9 Biolog plate and growth experiments. Furthermore, the neutral isoelectric point of the reconstructed proteome suggested that the strain osmoadaptation was mainly mediated by compatible solutes. The presence of genes involved in iron acquisition and metabolism indicated that osmoadaptation was tailored to the iron-depleted saline waters of the Discovery SBI. Overall, both phenomics and genomics highlighted the potential capability of V. dokdonensis 21D vegetative cells to adapt to the environmental conditions in Discovery SBI.

Bacterial diversity shift determined by different diets in the gut of the spotted wing fly Drosophila suzukii is primarily reflected on acetic acid bacteria.

The pivotal role of diet in shaping gut microbiota has been evaluated in different animal models, including insects. Drosophila flies harbour an inconstant microbiota among which acetic acid bacteria (AAB) are important components. Here, we investigated the bacterial and AAB components of the invasive pest Drosophila suzukii microbiota, by studying the same insect population separately grown on fruit-based or non-fruit artificial diet. AAB were highly prevalent in the gut under both diets (90 and 92% infection rates with fruits and artificial diet respectively). Fluorescent in situ hybridization and recolonization experiments with green fluorescent protein (Gfp)-labelled strains showed AAB capability to massively colonize insect gut. High-throughput sequencing on 16S rRNA gene indicated that the bacterial microbiota of guts fed with the two diets clustered separately. By excluding AAB-related OTUs from the analysis, insect bacterial communities did not cluster separately according to the diet, suggesting that diet-based diversification of the community is primarily reflected on the AAB component of the community. Diet influenced also AAB alpha-diversity, with separate OTU distributions based on diets. High prevalence, localization and massive recolonization, together with AAB clustering behaviour in relation to diet, suggest an AAB role in the D. suzukii gut response to diet modification.