Wild specimens of sand fly phlebotomine Lutzomyia evansi, vector of leishmaniasis, show high abundance of Methylobacterium and natural carriage of Wolbachia and Cardinium types in the midgut microbiome

Abstract

Phlebotomine sand flies are remarkable vectors of several etiologic agents (virus, bacterial, trypanosomatid Leishmania), posing a heavy health burden for human populations mainly located at developing countries. Their intestinal microbiota is involved in a wide range of biological and physiological processes, and could exclude or facilitate such transmission of pathogens. In this study, we investigated the Eubacterial microbiome from digestive tracts of Lu. evansi adults structure using 16S rRNA gene sequence amplicon high throughput sequencing (Illumina MiSeq) obtained from digestive tracts of Lu. evansi adults. The samples were collected at two locations with high incidence of the disease in humans: peri-urban and forest ecosystems from the department of Sucre, Colombia. 289,068 quality-filtered reads of V4 region of 16S rRNA gene were obtained and clustered into 1,762 operational taxonomic units (OTUs) with 97% similarity. Regarding eubacterial diversity, 14 bacterial phyla and 2 new candidate phyla were found to be consistently associated with the gut microbiome content. Proteobacteria, Firmicutes, and Bacteroidetes were the most abundant phyla in all the samples and the core microbiome was particularly dominated by Methylobacterium genus. Methylobacterium species, are known to have mutualistic relationships with some plants and are involved in shaping the microbial community in the phyllosphere. As a remarkable feature, OTUs classified as Wolbachia spp. were found abundant on peri-urban ecosystem samples, in adult male (OTUs n = 776) and unfed female (OTUs n = 324). Furthermore, our results provide evidence of OTUs classified as Cardinium endosymbiont in relative abundance, notably higher with respect to Wolbachia. The variation in insect gut microbiota may be determined by the environment as also for the type of feeding. Our findings increase the richness of the microbiota associated with Lu. evansi. In this study, OTUs of Methylobacterium found in Lu. evansi was higher in engorged females, suggesting that there are interactions between microbes from plant sources, blood nutrients and the parasites they transmit during the blood intake.

Effects of larval rearing substrates on some life-table parameters of Lutzomyia longipalpis sand flies

“…Our results showing the presence of Methylobacterium in females from colony and field substrate after the infection with L . infantum support the suggestion of [ 50 ], that Methylobacteriaceae remains in the sand fly midgut in the presence of Leishmania infections. Methylobacteriacae were exceptionally extant in poorly developing larvae in sand flies reared on cashew leaf litter, so it would be worthwhile to explore whether the harmful effect is attributable to Methylobacterium or innate chemical properties of cashew leaves.…”

Host Species Determines the Composition of the Prokaryotic Microbiota in Phlebotomus Sandflies

“…The distinct composition of the bacterial community of P. papatasi was driven by three OTUs belonging to Spiroplasma and Wolbachia . These are common facultative endosymbionts of Phlebotomus [ 28 , 44 ], Lutzomyia [ 30 , 31 ] and other insects; it is now estimated that Wolbachia and Spiroplasma endosymbionts are present in up to 30% of all insects [ 45 ]. They are maternally inherited, and they affect host ecology, physiology and fitness [ 15 , 46 , 47 ].…”

“…Sandfly gut microbiota can be modulated (i) by their feeding habits: larvae feeding on soil dead organic matter, while female adults get blood and sugar meals (plant-fed) and (ii) through interactions with transmitting parasites [ 26 , 27 ]. To date the gut microbiota of Phlebotomus sandflies has been studied using culture-dependent approaches [ 28 , 29 ], while recent studies on Lutzomyia longipalpis have used culture-independent approaches [ 30 , 31 ]. The gut bacterial community of sandflies is dominated by Proteobacteria and Firmicutes.…”

Comparative Analysis of Bacterial Communities in Lutzomyia ayacuchensis Populations with Different Vector Competence to Leishmania Parasites in Ecuador and Peru

“…It is important to note that previous findings on gut bacterial communities in female sand flies from South America were obtained using classic techniques for molecular ecology, such as denaturing gradient gel electrophoresis (DGGE), except for a recent study in which a microbiome analysis of the digestive tracts of Lu. evansi adults was performed using 16S rRNA gene sequence amplicon high-throughput sequencing (Illumina MiSeq) [ 44 ]. The use of high-throughput technology enables a large number of reads in a single run, thereby providing a greater sampling depth than other techniques [ 45 ].…”

“…These results are partly in agreement with the findings of a recent meta-analysis of sand fly-associated bacteria, which revealed that more than 57% and 47% of identified bacteria belonged to the Proteobacteria phylum in New and Old World sand fly species, respectively [ 46 ]. Actinobacteria showed lower abundance [ 11 , 44 , 47 , 48 , 49 ]. Previous studies reported that the phylum Proteobacteria contributed to host nutrition by fixing atmospheric nitrogen [ 50 ].…”