THE GUT MICROBIOME ASSOCIATED TO HONEYBEES AND WASTE-REDUCING INSECTS

Insects are the most diverse group of animals on Earth and are adapted to a wide range of habitats. They have a remarkable impact on the human life: they include not only human and animal parasites, crop pests, or vectors of human, animal and plant diseases, but also beneficial insects, such as pollinators (e.g. the honeybee Apis mellifera), insects reared to obtain products for the human benefit (e.g. silkworms) or mass-reared insects as food and feed or as biological control agents (e.g. parasitoids or predators). Moreover, since the increase of human population, the growing demand for protein for human and animal consumption is forcing the search of alternative sources: in this scenario insects have been proposed as sustainable rich-protein substrates. For instance, the black soldier fly (BSF, Hermetia illucens) is a promising candidate for the sustainable recycling of biological waste into feedstuff for livestock, poultry and aquaculture in the framework of a circular economy approach (Nguyen et al., 2015; van Huis et al., 2013). The insect evolutionary success and diversification are partially due to the symbiotic relationships that they have established with a wide range of microorganisms. These complex symbiotic interactions include commensal, parasitic and mutualistic relationships (Dale and Moran, 2006). The function commonly attributed to the microorganisms that inhabit the intestinal tract of animals is the depolymerization and breakdown of the diet components, which allow the nutrient supplementation and recycling. Moreover, they also provide detoxification of the toxic diet-components and protection against pathogens and parasites and this underlines the importance of a healthy gut microbiota for the host well-being (Dale and Moran, 2006; Engel and Moran, 2013; Hamdi et al., 2011). At the beginning of this PhD thesis an introductive chapter offers an overview of the current knowledge on the potential application of microorganisms in relation to the management of the emerging insect farming with agricultural, industrial and environmental interest. The insect gut microbiota is influenced by many factors, such as the host diet, developmental stage and genetics, and in the last years researchers have been addressed many efforts to elucidate their impact on the host gut microbiota, mainly considering insect pests, parasites or vectors of diseases (Montagna et al., 2015a; Vacchini et al., 2017). Moreover, the importance to characterize factors, such as the oxygen concentration, pH and redox potential status, existing within the insect gut compartments has been only recently recognized and, hence, not so much work has been performed so far in this direction. Indeed, the insect gut includes aerobic and anaerobic niches, passing through microaerophilic habitats, and compartments characterized by acidic, neutral or basic conditions, even in the same digestive tract (Engel and Moran, 2013). Understanding the drivers that shape the microbial diversity in the insect gut microbiota is pivotal in comprehending the symbiosis interactions and in exploiting insects for agricultural, environmental and feed applications. Since the increasing interest and limited information on this topic, the aim of this PhD thesis was to evaluate the influence of the diet, developmental stage and physicochemical conditions occurring in the different intestine compartments on the gut microbiota associated to different helpful insects. Specifically, the honeybee Apis mellifera and the waste-reducing insect H. illucens were considered. These insects are characterized by polyphagous diet, the former more specialized and the latter more variegated for the honeybee and BSF, respectively. Finally, the probiotic effect of some selected bacteria was evaluated to observe a possible bacterial contribution to the BSF performance when insects were exposed to a low-performing diet. The second chapter of my PhD was devoted to the study of the compartmentalization of the bacterial community along the honeybee intestine, taking into account the variations of the physicochemical conditions of oxygen level, pH and redox potential at a micrometer scale. Indeed, the gut microbiota of the adult honeybee workers has been deeply investigated and shown to include nine dominant bacterial phylotypes, belonging to Proteobacteria, Firmicutes and Actinobacteria (Kwong and Moran, 2016). Although much information is available on the diversity, genomic features and evolution of these phylotypes, a comprehensive study of the bacterial diversity in relation to the physicochemical conditions occurring in the gut portions was missing (Kwong and Moran, 2016). The variation of the oxygen partial pressure (pO2), redox potential and pH in the crop, midgut, ileum and rectum of honeybee foragers was measured taking advantage of microsensors and microelectrodes, while bacterial composition and co-occurrence networks were determined by 16S rRNA gene high-throughput sequencing. Bacterial abundance was measured by quantitative PCR. Results showed that a diagonal oxygen gradient was present in all the compartments that resulted anoxic in their centre, supporting that the previously reported bacterial stratification was linked to the oxygen availability. A progressive pH decrease from the crop to the rectum, presumably associated to the increasing microbial acidogenic activity, was paralleled by an increasing complexity of the network connections. Data provided the evidence of a physicochemically-driven gut compartmentalization of the bacterial communities at the level of the microbiota networking and diversity. In the third chapter the influence of diet source and developmental stage on BSF bacterial community structure and composition was investigated by applying culture-independent analysis, combining the Automated Ribosomal Intergenic Spacer Analysis (ARISA)-PCR fingerprinting and 16S rRNA high-throughput sequencing. Specifically, insects were reared on three different diets (standard, fruit-waste derived and vegetable-waste derived ones) and sampled at three different developmental stages (i.e. larval, prepupal and adult ones). According to data, the bacterial communities associated to H. illucens were characterized by three dominant phyla, Proteobacteria, Firmicutes and Bacteroidetes. Statistical analysis showed significant differences between the bacterial communities considering the three diets and the three developmental stages, except in the case of male and female adults, which did not differ statistically. Regardless the life stage and food source, 10 OTUs were shared among individuals, mainly belonging to the Enterobacteriaceae family. Moreover, by using microsensors and microelectrodes it was verified the variation of the physicochemical conditions, in terms of pO2, pH and redox potential within the gut compartments of 4th instar larvae, when reared on the three different diets. Data suggested that the variation of the physicochemical conditions, driven by the modification of the alimentary regimes, possibly affected the bacterial community structure. Once verified the effect of the diet as a driving force in shaping BSF bacterial community, the last chapter of this PhD thesis was devoted to investigate the potential bacterial contribution to BSF performance when insects were reared on a low-performing diet i.e. the fruit-waste derived one. Bacterial isolations were performed from the dissected guts of BSF larvae reared on standard diet, using selective and enrichment media, resulting in the establishment of a bacterial collection made up of 193 isolates. After the collection dereplication and the strains’ identification, the hydrolytic activities of the isolates were characterized in order to evaluate their potential metabolic contribution to the host. Data showed a potential involvement of the bacterial isolates in the breakdown of the diet components and in the nutrient supplementation. Moreover, several isolates showed the ability to produce exopolysaccharides (EPS) that could mediate the bacterial adhesion to the insect epithelia. Finally, based on the results of the metabolic profiles, two isolates were selected and administered, individually and in combination, to BSF larvae reared on nonsterile fruit-waste derived diet to assess their influence on the host performance. Results showed that the administration of selected bacteria could influence the weight gain of the larvae when reared on a nutritionally unbalanced diet. In conclusion, this work underlines that different factors can modulate the structure, composition and compartmentalization of the bacterial communities associated to non-pest and economically relevant insects. One of the innovative aspects of this study was to have evaluated the physicochemical conditions occurring in the insect gut compartments at the micrometer scale. The combination of such investigations with 16S rRNA gene high-throughput sequencing data provides useful information to describe the insect-associated microbial community to further uncover its contribution to the host. REFERENCES Dale C., and Moran N. (2006). Molecular interaction between bacterial symbionts and their hosts. Cell. 126:453-465. Engel P., Moran N.A. (2013). The gut microbiota of insects - diversity in structure and function. FEMS Microbiol. Rev. 37:699-735. Hamdi C., Balloi A., Essanaa J., Gonella E., Raddadi N., Ricci I. , Boudabous A., Borin S., Manino A., Bandi C., Alma A., Daffonchio D., Cherif A. (2011). Gut microbiome dysbiosis and honey bee health. J. Appl. Entomol. 135:524–533. Kwong W.K., Moran N.A. 2016. Gut microbial communities of social bees. Nat. Rev. Microbiol. 14:374-84. Montagna M., Chouaia, B., Mazza G., Prosdocimi E.M., Crotti, E., Mereghetti, V., Vacchini V., Giorgi A., De Biase A., Longo S., Cervo R., Lozzia C.G., Alma A., Bandi C., Daffonchio D. (2015a). Effects of the diet on the microbiota of the red palm weevil (Coleoptera: Dryophthoridae). PLoS One 10:e0117439. Nguyen T.T.X., Tomberling J.K., Vanlaerhoven S. (2015). Ability of black soldier fly (Diptera: Stratiomyidae) larvae to recycle food waste. Environ. Entomol. 44(2): 406-410. Vacchini V., Gonella E., Crotti E., Prosdocimi E.M., Mazzetto F., Chouaia B., Callegari M., Mapelli F., Mandrioli M., Alma A., Daffonchio D. (2017). Bacterial diversity shift determined by different diets in the gut of the spotted wing fly Drosophila suzukii is primarily reflected on acetic acid bacteria. Environ. Microbiol. Rep. 9(2):91-103. van Huis A. (2013). Potential of insects as food and feed in assuring food security. Annu. Rev. Entomol. 58:563-83.