Sand flies used to have a reputation for being difficult and labour-intensive to breed. Here we summarize our experience with establishment and maintenance of sand fly colonies and their use for infective experiments: techniques for collection and handling wild-caught females, rearing larvae and adults and experimental infections of sand flies by Leishmania using membrane feeding. In addition, we compare major life cycle parameters between various colonies maintained under standard laboratory conditions. The sand fly rearing is tricky but some species can be reared in large numbers with a minimum of space and equipment. Initiation of new colonies from endemic sites is a prerequisite for accurate studies on parasite-vector interaction but it is more difficult step than routine maintenance of colonies already established in laboratory for many generations. Journal of Vector Ecology 36 (Supplement 1): S1-S9. 2011.

Background: Sand fly saliva plays an important role in blood feeding and Leishmania transmission as it was shown to increase parasite virulence. On the other hand, immunity to salivary components impedes the establishment of infection. Therefore, it is most desirable to gain a deeper insight into the composition of saliva in sand fly species which serve as vectors of various forms of leishmaniases. In the present work, we focused on Phlebotomus (Adlerius) arabicus, which was recently shown to transmit Leishmania tropica, the causative agent of cutaneous leishmaniasis in Israel.

Background Phlebotomus tobbi is a vector of Leishmania infantum, and P. sergenti is a vector of Leishmania tropica. Le. infantum and Le. tropica typically cause visceral or cutaneous leishmaniasis, respectively, but Le. infantum strains transmitted by P. tobbi can cause cutaneous disease. To better understand the components and possible implications of sand fly saliva in leishmaniasis, the transcriptomes of the salivary glands (SGs) of these two sand fly species were sequenced, characterized and compared.Methodology/Principal FindingscDNA libraries of P. tobbi and P. sergenti female SGs were constructed, sequenced, and analyzed. Clones (1,152) were randomly picked from each library, producing 1,142 high-quality sequences from P. tobbi and 1,090 from P. sergenti. The most abundant, secreted putative proteins were categorized as antigen 5-related proteins, apyrases, hyaluronidases, D7-related and PpSP15-like proteins, ParSP25-like proteins, PpSP32-like proteins, yellow-related proteins, the 33-kDa salivary proteins, and the 41.9-kDa superfamily of proteins. Phylogenetic analyses and multiple sequence alignments of putative proteins were used to elucidate molecular evolution and describe conserved domains, active sites, and catalytic residues. Proteomic analyses of P. tobbi and P. sergenti SGs were used to confirm the identification of 35 full-length sequences (18 in P. tobbi and 17 in P. sergenti). To bridge transcriptomics with biology P. tobbi antigens, glycoproteins, and hyaluronidase activity was characterized.ConclusionsThis analysis of P. sergenti is the first description of the subgenus Paraphlebotomus salivary components. The investigation of the subgenus Larroussius sand fly P. tobbi expands the repertoire of salivary proteins in vectors of Le. infantum. Although P. tobbi transmits a cutaneous form of leishmaniasis, its salivary proteins are most similar to other Larroussius subgenus species transmitting visceral leishmaniasis. These transcriptomic and proteomic analyses provide a better understanding of sand fly salivary proteins across species and subgenera that will be vital in vector-pathogen and vector-host research.

Antibody (IgG) responses to the saliva of Phlebotomus argentipes were investigated using serum samples from regions of India endemic and non-endemic for visceral leishmaniasis (VL). By pre-adsorbing the sera against the saliva of the competing human-biting but non-VL vector P. papatasi, we significantly improved the specificity of a P. argentipes saliva enzyme-linked immunosorbent assay. Using this method, we observed a statistically significant correlation between antibodies to P. argenitpes saliva and the average indoor density of female sand flies. Additionally, the method was able to detect recent changes in vector exposure when sera from VL patients were assayed before, during, and after hospitalization and protected from sand fly bites under untreated bed nets. Collectively, these results highlight the utility of antibodies to P. argentipes saliva as an important tool to evaluate VL vector control programs.

Zoonotic visceral leishmaniasis (VL) caused by Leishmania infantum is transmitted from dogs to humans by sand flies and Lutzomyia longipalpis is a major vector of this disease. We studied the antibody response in dogs experimentally exposed to L. longipalpis females to characterize sand fly salivary antigens recognized by canine sera and to find out whether the level of specific anti-saliva antibodies reflects the intensity of exposure. Sera from repeatedly bitten dogs revealed up to six salivary protein bands with approximate molecular weight of 66, 55, 45, 37-39, 34, and 25 kDa in L. longipalpis salivary gland lysate. Anti-saliva immunoglobin (Ig) G and its subclasses were found to be useful markers of exposure to sand flies. Specific IgG, IgG1, and IgG2 were related to numbers of bloodfed L. longipalpis females, and increased antibody levels were detectable throughout the study, i.e. more than 6 months after the last exposure. In contrast, specific IgE response developed in some dogs only, and no correlation was observed between its level and the intensity of exposure. Screening of dog sera for specific IgG against salivary antigens of the vector is suggested as a useful epidemiological tool in VL foci. Monitoring canine antibody response to sand fly saliva also allows evaluation of the effectiveness of anti-vector campaigns.