First report of filarial nematodes in the genus Onchocerca infecting black flies (Diptera: Simuliidae) in Iran

Abstract

Black flies are blood-sucking insects of public health importance, and they are effective vectors of pathogens and parasites, such as filarial nematodes of the genus Onchocerca. Our previous surveys have shown that individuals of Simulium turgaicum are annoying pests of humans and livestock in the Aras River Basin of Iran. In the present study, adult black flies of S. turgaicum were trapped from different ecotopes of five villages in Khoda-Afarin County, Iran. By using a sensitive nested PCR assay and targeting the nuclear 18S rDNA-ITS1 marker, filarial infections were found in 38 (1.89%) of 2005 black flies. Homology exploration of 360 bp of the sequences indicated that the filarial worms are members of the family Onchocercidae, with maximum alignment scores of 93-95%. Phylogenetic analysis showed that two Iranian Onchocerca isolates were clustered in the O. fasciata-O. volvulus lineage and were well separated from other filarial nematodes. Both the entomological evidence (empty abdomen of the specimens) and climatologic data (adequate accumulated degree days for development) suggest that the filarial DNA was probably that of infective larvae of vertebrates. This is the first report of an infection by Onchocerca species in S. turgaicum and the first record of onchocercids in black flies in Iran; however, more research is required to demonstrate transmission of these filarial worms by black flies in nature.

Figure 1

Species-specific nested PCR of filarial nematodes, using the 18S rDNA-ITS1 gene. Lanes: M, 100-bp ladder (Fermentas); 1, negative control; 2, 4, Onchocerca sp. (~ 350 bp); 3, Dirofilaria sp. as positive control (~ 420 bp).

Figure 2

Maximum likelihood phylogenetic tree displaying the position of Onchocerca species identified in this study (solid circles) among other Onchocercidae filarial 18S rDNA-ITS1 sequences retrieved from GenBank. Thelazia lacrymalis was set as the outgroup. Numbers at the branch points are bootstrap values based on 1000 replicates. Bootstrap values less than 50% are not shown. The scale-bar measures evolutionary distance in substitutions per nucleotide.

Figure 3

The associated role of environmental cues for development of Onchocerca microfilariae in Simulium turgaicum in Khoda-Afarin County, East Azerbaijan Province, Iran, 2011–2021. The relationship between the accumulated degree days (ADD) generated (upper curve) and required (hatched area) for the development of the nematodes, rainfall (gray lines), and fluctuations of the black fly population (bar graph) are shown. Temperatures between the lower (16 °C) and upper (30 °C) developmental thresholds were included for ADD calculations for each day. Black and red circles represent the dates of capture of S. turgaicum with microfilarial infections.