SYMBIOSIS BETWEEN ARTHROPODS AND FUNGI: THE CASE OF PHLEBOTOMUS PERNICIOSUS, THE VECTOR OF VISCERAL AND CANINE LEISHMANIASIS

Introduction Vector borne diseases (VBDs) are one of the greatest public health problems worldwide and their control represents a key global public health challenge. In particular, leishmaniases are parasitic VBDs endemic in 98 countries from five continents distributed in tropical, subtropical and temperate zones. During the last twenty years, their distribution have undergone an expansion, also due to climate and environmental changes, the reason why they are considered re-emerging diseases. Italy is traditionally endemic for cutaneous and visceral leishmaniasis caused by the protozoan parasite Leishmania infantum, whose reservoir is the dog. In the Old World, the natural vectors of the protozoa Leishmania spp. are several species of sand flies belonging to the genus Phlebotomus. To control VBDs, the World Health Organization promotes the rational use of insecticides and integrated strategies for vector control through chemical and biological strategies. The "symbiotic control" is one of the biological control strategies that exploits microorganisms that live in symbiosis with the vector, in order to reduce or interfere with the pathogen transmission. The aim of my thesis project was to increase the knowledge on killer yeasts, in general, and in the arthropod Phlebotomus, in particular, in order to open the way toward their development as tools for the biological/integrated control of vector-borne diseases. Article 1 My first work, here presented, reports the investigation of the fungal community of the sand fly Phlebotomus perniciosus, the main vector of leishmaniasis in the western Mediterranean area and the most widespread sand fly in Italy, with a focus on the dominant yeast species Meyerozyma guilliermondii. The yeast displayed higher prevalence in the reared population than in the analyzed wild individuals, letting us hypothesize a contribution of this yeast to the diet of reared sand flies with nutrients that are on the other side easily available in nature. The localization of the yeast, found in the midgut of adults of both sexes and larvae and in Malpighian tubules of female sand flies, in addition to the genome analysis, let us speculate that the yeast may confer the additional advantage of contributing to the removal of the excess of nitrogenous wastes after the blood meal of the insect host. Article 2 Whole-genome sequencing, de novo assemby and phylogenomic analysis of one strain of M. guilliermondii isolated from P. perniciosus and of other yeast strains of the M. guilliermondii complex isolated from arthropods were the goals of another research paper here presented. This work led to publish the first four genomes of isolates of Meyerozyma caribbica and to better understand evolutionary relationships within the clade. Articles 3-5 The other works presented in this document were focused on the yeast Wickerhamomyces anomalus, here studied as a yeast candidate for symbiotic VBDs control. In one of these papers (Article 3) the isolation and phylogenetic characterization of a killer strain of W. anomalus from the sand fly P. perniciosus is reported. The association between the yeast and sand flies seems to be facultative. The killer toxin produced by the isolated W. anomalus strain was purified, characterized and tested against fungal pathogens in another work (Article 4). This toxin is a β-1,3-glucanase that possesses in vitro activity against Candida glabrata isolates, independently from their drug-resistance phenotypes. The obtained results suggest a potential application of this toxin against fungal infections at skin and mucosal membrane levels provoked by C. glabrata and may be useful for the design of new therapeutic molecules and the study of yeast resistance mechanisms. Finally, W. anomalus is widely distuributed in nature and is considered by EFSA as a non-pathogenic agent, completely safe for healthy individuals (level of bio-security 1/QPS). Nevertheless, in the last paper I investigated the circulation of W. anomalus by qPCR screening examining a large panel of immunocompromised and / or intensive care patients, as well as healthy donors as controls (Article 5). The screening revealed only one qPCR-positive subject, out of over 500 analyzed samples. Considering the few published cases of clinical infection by this yeast, in immunocompromised patients, we can conclude that this microorganism is a rather uncommon opportunistic pathogen. In conclusion, the study of the mycobiota associated with sand flies could contribute to the understanding of the biology these insects, and offers the potential of discovering novel microrganisms for future application in the symbiotic control of leishmaniasis.