水島 大貴

Phlebotomine sand flies are vectors of the protozoan parasite Leishmania spp. Although the intestinal microbiota is involved in a wide range of biological and physiological processes and has the potential to alter vector competence, little is known about the impact of host species and environment on the gut microbiome. To address this issue, a comparative analysis of the microbiota of sand fly vector populations of Leishmania major and L. tropica in a mixed focus of cutaneous leishmaniasis in Tunisia was performed. Bacterial 16S rRNA gene amplification and Illumina MiSeq sequencing were used to characterize and compare the overall bacterial and fungal composition of field-collected sand flies: Phlebotomus papatasi, Ph. perniciosus, Ph. riouxi, and Ph. sergenti. Thirty-eight bacterial genera belonging to five phyla were identified in 117 female specimens. The similarities and differences between the microbiome data from different samples collected from three collections were determined using principal coordinate analysis (PCoA). Substantial variations in the bacterial composition were found between geographically distinct populations of the same sand fly species, but not between different species at the same location, suggesting that the microbiota content was structured according to environmental factors rather than host species. These findings suggest that host phylogeny may play a minor role in determining the insect gut microbiota, and its potential to affect the transmission of the Leishmania parasite appear to be very low. These results highlight the need for further studies to decode sand fly Leishmania-microbiota interactions, as even the same bacterial species, such as Enterococcus faecalis, can exert completely opposite effects when confronted with different pathogens within various host insects and vice versa.

BACKGROUND: Blood-sucking phlebotomine sand flies are vectors of the protozoan parasites Leishmania spp. Although the intestinal microbiota is involved in a wide range of biological and physiological processes and has the potential to alter vector competence, little is known about the factors that modify the gut microbiota composition of sand flies. As a key step toward addressing this issue, we investigated the impact of host species on the gut bacterial composition in Phlebotomus and Lutzomyia sand flies reared under the same conditions. METHODS: Bacterial 16S rRNA gene amplification and Illumina MiSeq sequencing were used to characterize the overall bacterial composition of three laboratory-reared sandflies: Phlebotomus papatasi, Ph. duboscqi, and Lutzomyia longipalpis. RESULTS: Our results showed that the larvae of the three sand fly species harbored almost the same microbes but had different relative abundances. Adult Ph. papatasi and Ph. duboscqi revealed similar microbiome compositions, which were distinct from that of adult Lu. longipalpis. Furthermore, we showed that Ph. papatasi and Ph. duboscqi are hosts for different bacterial genera. The experiment was repeated twice to improve accuracy and increase reliability of the data, and the same results were obtained even when a distinct composition of the microbiome among the same species was identified probably because of the use of different larvae food batch. CONCLUSIONS: The present study provides key insights into the role of host species in the gut microbial content of different sand fly species reared under the same conditions, which may influence their susceptibility to Leishmania infection.

A rare sugar, allose, was reported to inhibit the development of Plasmodium parasites in Anopheles mosquitoes; however, the mechanism remains unknown. The present study addressed the inhibitory mechanism of allose on the development of the Plasmodium parasite by connecting it with bacteria involvement in the midgut. In addition, further inhibitory sugars against Plasmodium infection in mosquitoes were explored. Antibiotic-treated and antibiotic-untreated Anopheles stephensi were fed fructose with or without allose. The mosquitoes were infected with luciferase-expressing Plasmodium berghei, and parasite development was evaluated by luciferase activity. Bacterial composition analysis in gut of their mosquitoes was performed with comprehensive 16S ribosomal RNA sequencing. As the result, allose inhibited the development of oocysts in mosquitoes regardless of prior antibiotic treatment. Microbiome analysis showed that the midgut bacterial composition in mosquitoes before and after blood feeding was not affected by allose. Although allose inhibited transient growth of the midgut microbiota of mosquitoes after blood feeding, neither toxic nor inhibitory effects of allose on the dominant midgut bacteria were observed. Ookinete development in the mosquito midgut was also not affected by allose feeding. Additional 15 sugars including six monosaccharides, four polyols, and five polysaccharides were tested; however, no inhibitory effect against Plasmodium development in mosquitoes was observed. These results indicated that allose inhibits parasite development in midgut stage of the mosquito independently of midgut microbiota. Although further studies are needed, our results suggest that allose may be a useful material for the vector control of malaria as a "transmission-blocking sugar."

Transcriptome analysis of the salivary gland cDNA library from a phlebotomine sand fly, Lutzomyia ayacuchensis, identified a transcript coding for the PpSP15/SL1 family protein as the second most abundant salivary component. In the present study, a recombinant protein of the PpSP15/SL1 family protein, designated ayaconin, was expressed in Escherichia coli, and its biological activity was characterized. The recombinant ayaconin purified from the soluble fraction of E. coli lysate efficiently inhibited the intrinsic but not extrinsic blood coagulation pathway. When the target of ayaconin was evaluated using fluorescent substrates of coagulation factors, ayaconin inhibited factor XIIa (FXIIa) activity more efficiently in a dose-dependent manner, suggesting that FXII is the primary target of ayaconin. In addition, incubation of ayaconin with FXII prior to activation effectively inhibited FXIIa activity, whereas such inhibition was not observed when ayaconin was mixed after the production of FXIIa, indicating that ayaconin inhibits the activation process of FXII to produce FXIIa, but not the enzymatic activity of FXIIa. Moreover, ayaconin was shown to bind to FXII, suggesting that the binding of ayaconin to FXII is involved in the inhibitory mechanism against FXII activation. These results suggest that ayaconin plays an important role in the blood-sucking of Lu. ayacuchensis.

Sand flies are a significant public health concern in many parts of the world where they are known to transmit agents of several zoonotic diseases to humans, such as leishmaniasis. Vector control remains a key component of many anti-leishmaniasis programs and probably will remain so until an effective vaccine becomes available. The sand fly gut microbiota has recently emerged as an encouraging field for the exploration of vector-based disease control. In particular, the gut microbiome was previously reported to either enhance or inhibit parasite activity depending on the species of bacteria and, thus, has the potential to alter vector competence. Here, we describe the technological advances that are currently expanding our understanding of microbiota composition in sand flies. The acquisition and composition of microbiomes are influenced by several abiotic and biotic factors, including host immunity, genetics, and the environment. Therefore, the microbiomes of sand flies can vary substantially between individuals, life stages, species, and over geographical space, and this variation likely contributes to differences in host phenotypes, highlighting opportunities for novel vector control strategies.