Molecular detection of Dirofilaria spp. and host blood-meal identification in the Simulium turgaicum complex (Diptera: Simuliidae) in the Aras River Basin, northwestern Iran

Background: Blackflies (Diptera: Simuliidae) are known as effective vectors of human and animal pathogens, worldwide. We have already indicated that some individuals in the Simulium turgaicum complex are annoying pests of humans and livestock in the Aras River Basin, Iran. However, there is no evidence of host preference and their possible vectorial role in the region. This study was conducted to capture the S. turgaicum (s.l.), to identify their host blood-meals, and to examine their potential involvement in the circulation of zoonotic microfilariae in the study areas. Methods: Adult blackflies of the S. turgaicum complex were bimonthly trapped with insect net in four ecotopes (humans/animals outdoors, irrigation canals, lands along the river, as well as rice and alfalfa farms) of ten villages (Gholibaiglou, Gungormaz, Hamrahlou, Hasanlou, Khetay, Khomarlou, Larijan, Mohammad Salehlou, Parvizkhanlou and Qarloujeh) of the Aras River Basin. A highly sensitive and specific nested PCR assay was used for detection of filarial nematodes in S. turgaicum (s.l.), using nuclear 18S rDNA-ITS1 markers. The sources of blood meals of engorged specimens were determined using multiplex and conventional cytb PCR assays. Results: A total of 2754 females of S. turgaicum (s.l.) were collected. The DNA of filarial parasites was detected in 6 (0.62%) of 960 randomly examined individuals. Sequence analysis of 420 base pairs of 18S rDNA-ITS1 genes identified Dirofilaria spp. including 5 D. immitis and 1 D. repens. Importantly, all filarial positive specimens have been captured from humans and animals outdoors. Cytb-PCR assays showed that in all ecotypes studied, members of the S. turgaicum complex had preferably fed on humans, dogs, bovids, and birds, respectively. Conclusions: To the best of our knowledge, this is the first report of D. immitis/D. repens detection in blackflies. Results showed that S. turgaicum (s.l.) was the most abundant (97%) and anthropophilic (45%) blackfly in all studied ecotypes/villages and that DNA of Dirofilaria spp. was detected in the flies taken from six villages. Dirofilariasis is a common zoonosis between humans and carnivores, with mosquitoes (Culicidae) as the principal vectors. Further investigations are needed to demonstrate that blackflies are actual vectors of Dirofilaria in the studied region.