MICROBIAL ECOLOGY OF THE SPOTTED WING FLY DROSOPHILA SUZUKII

Abstract Symbiotic relationships between arthropods and microorganisms are widespread in nature. In the last years these interactions are received considerable attention, as many microorganisms may play relevant roles in the biology and lifecycle of insects (Dale and Moran, 2006; Moran et al., 2008). In this perspective, researchers are directing many efforts to depict the interactions that shape the symbiosis. Furthermore, considering the importance that microorganisms play for their hosts, the modification of the microbiome structure in the insect body could support the development of sustainable strategies, alternative to chemical pesticides. To achieve the development of these methods, the knowledge and the identification of the symbionts associated to the pest of interest, is a mandatory requirement. Recent studies documented the evidence of stable associations between acetic acid bacteria (AAB) and insects characterized by a sugar-based diet. These include Diptera, Hymenoptera and Hemiptera orders (Crotti et al., 2010). It was reported that AAB are essential in the modulation of the immune homeostasis as well as metabolism and larval development. These capacities have been demonstrated in Drosophila (Ryu et al., 2008; Shin et al., 2011), but have been recently confirmed in other models, like Anopheles (Chouaia et al. 2012, Hughes et al., 2014). Along with bacteria, drosophilid flies establish a mutualistic relationship with yeasts, in particular with those belonging to the Saccharomycetaceae family: these microorganisms represent the main nutritional source for the flies, as they provide proteins, vitamins and other nutrients. Yeasts are vectored by Drosophila, from which they are dispersed, favoring the colonization of new habitats (Christiaens et al., 2014). Moreover, they can affect the fly development and fitness in terms of susceptibility to parasitism (Anagnostou et al. 2010). Yeasts share the same environments with AAB, supporting the hypothesis of possible microbe-microbe interactions. The aim of my PhD project was the characterization of the microbiome associated to the spotted wing fly Drosophila suzukii Matsumura (Diptera: Drosophilidae), an economically damaging pest of healthy soft summer fruits, rapidly spreading in many countries from South-East Asia (Lee et al., 2011). In particular, targets of the research were AAB and yeasts symbionts. Results revealed that AAB were a major component of D. suzukii bacterial community. Members of Gluconobacter, Gluconacetobacter and Acetobacter genera were the main representatives, as shown by culture-dependent (isolation by using specific media, dereplication with ITS-PCR and isolate identification through partial 16S rRNA gene sequencing) and -independent analyses (16S rRNA barcoding and Denaturing Gradient Gel Electrophoresis-PCR). The investigation was performed on specimens of different developmental stages (larvae, pupae and adults), reared on two feeding substrates (fruit or an artificial diet). The plasmid pHM2(Gfp) was introduced by electroporation in three selected AAB isolates, Gluconobacter oxydans DSF1C.9A, Acetobacter tropicalis BYea.1.23 and Acetobacter indonesiensis BTa1.1.44 to label them with Green fluorescent protein (Gfp). After oral administration to the insects, Gfp-tagged strains were visualized in the host by fluorescence microscopy. The symbionts were able to successfully reach and colonize the epithelium of the insect crop, proventriculus and midgut. Tests performed on bacterial cultures grown in liquid media showed that several AAB isolates are able to produce an extracellular matrix in which the cells are entrapped and that presumably is implicated in the bacterial adhesion to the insect epithelia and maintenance in the digestive system. By using probes specific for AAB (Texas red-labelled probe AAB455) and Eubacteria (Texas red-labelled universal Eubacterial probe Eub338), fluorescent in situ hybridization (FISH) experiments on the host dissected tissues from D. suzukii, detected AAB within the peritrophic membrane of the midgut and proventriculus. Probes specific for Gluconobacter (Cy5-labelled probes Go615 and Go618) were also designed, and used to confirm the presence of this species within the intestinal tract of the fly. Due to the abundance and intimate connection of these symbionts with D. suzukii tissues and organs, I predict that AAB have important roles in the lifecycle of the host. The capacity of D. suzukii-selected AAB isolates to emit microbial volatile compounds for flies’ specific attraction was subsequently analysed. Microbial volatiles are known to attract or repel insects, inhibit or stimulate the plant growth. For example, acetic acid was described to be an attractant molecule for Drosophila flies (Cha et al., 2014). With the aim to evaluate the different attraction capabilities of some selected AAB on D. sukuzii, a two-choice olfactometer assay was developed and attraction experiments were carried out. After the first evaluation of the bacterial growth to set up the experimental conditions, flies were exposed to volatile organic compounds (VOCs) produced by AAB isolates in comparison to a control, represented by the growth medium without bacteria. Higher attractiveness for flies than the other bacteria was obtained with Gluconobacter oxydans DSF1C.9A, Gluconobacter kanchanaburiensis L2.1.A.16 and Gluconacetobacter saccharivorans DSM1A.65A strains. Since currently traps for flies are composed by vinegar and baker’s yeast, the analyses of the best attractive molecules released by specific microorganisms might provide novel tools for D. suzukii biocontrol and the assembly of baits specifically targeted for this pest. Flies at different developmental stages (larvae, pupae and adults) reared on different food sources (fruit or artificial diet) were analyzed through cultivation-independent (DGGE-PCR, 16S rRNA 454-pyrosequencing) and -dependent (isolation trials, and isolate identification) techniques to investigate the yeast community. Most of the analyzed sequences obtained from the excised DGGE bands and pyrosequncng data had close similarity with sequences assigned to Saccharomycetales, in particular Candida, Geotrichum and Pichia genera. These yeasts comprise specialist colonizers of rotten and fermenting fruits, and the skin of intact fruits eaten by Drosophila. A collection of 237 yeast isolates were obtained from the isolation trials, with the purpose to explore the community diversity in individuals of different life stages, and reared on the two-abovementioned food sources. Identification of the yeast species was carried out using RFLP (Restriction Fragment Length Polymorphism) analysis of the ITS1-ITS2 region of the fungal rRNA gene, of all the isolates. Restriction patterns were obtained through the use of HaeIII, HinfI and TaqI endonucleases. After the analysis of the generated digestion patterns, the representative isolates of each RFLP profile were submitted to sequencing analyses of both the D1-D2 region of the large subunit (LSU) of the fungal rRNA gene and the ITS1-ITS2 region of the fungal rRNA gene. Phylogenetic trees completed the analysis. The results strengthened and enlarged the molecular data, and showed that the most abundant species recorded, i.e. Pichia occidentalis, Saccharomycopsis craetegensis and Arthroascus schoenii, were the only species isolated from all of the tree life stages (larvae, pupae, and adults). Insects reared on fruits were characterized by a higher diversity in terms of yeast species. In particular, it was also recorded the presence of Hanseniaspora uvarum, which was described in previous works as a dominant yeast genus associated to different Drosophila species, including D. suzukii (Chandler et al., 2012, Hamby et al., 2012). Data obtained from the yeast community characterization, as well as from the bacterial community one, were exploited for a screening of the possible interactions among symbionts. In particular, some yeast strains are able to compete for their own ecological niche and nutrients by producing an array of compounds named “killer toxins” (Woods and Bevan, 1968). Since the production of killer toxins could be highly affected by the culture conditions, in terms of pH, temperature and carbon source content, then the optimal ones were developed for the growth of selected yeast isolates, and subsequently antagonistic activity tests were performed. The results highlighted the capacity of a specific yeast isolate, Candida stellimalicola AF4.1.P.268, to limit the growth of several yeast and AAB isolates, by creating inhibition haloes. This feature might have a role in a pest management perspective. In conclusion, this project indicates that AAB and yeast communities establish an intimate association with D. suzukii, as they were found stably and abundantly in individuals of different stages and fed on different diets. Recolonization trials performed with AAB strains suggest, in particular, their importance for the biology of this pest. 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