Presence of a Cryptic Onchocerca Species in Black flies of Northern California, USA

Background: Black flies (Diptera: Simuliidae) serve as arthropod vectors for various species of Onchocerca (Nematoda: Onchocercidae) that may be associated with disease in humans, domestic animals, and wildlife. The emergence of zoonotic Onchocerca lupi in North America and reports of cervid-associated zoonotic onchocerciasis highlight the need for increased entomological surveillance. In addition, there is mounting evidence that Onchocerca diversity in North America is far greater than previously thought, currently regarded as Onchocerca cervipedis species complex. This study reports new geographic records and black fly vector associations of an uncharacterized Onchocerca species. Methods: To better understand the biodiversity and geographic distribution of Onchocerca , 485 female black flies (2015: 150, 2016: 335) were collected using CO 2 -baited traps from February to October, 2015-2016 in Lake County, northern California, USA. Individual flies were morphologically identified and pooled (£ 10 individuals) by species, collection date, and trap location. Black fly pools were processed for DNA extraction, and subsequent PCR and sequencing targeting of the NADH dehydrogenase subunit 5 gene. Results: Among the pools of black flies, there were: 158 individuals of S. tescorum (2015: 57, 2016: 101), 302 individuals of S. vittatum (s.l.) (2015: 82, 2016: 220), 16 individuals of S. clarum “black” phenotype (2015: 5, 2016: 11), and 13 individuals of S. clarum “orange” phenotype (2015: 6, 2016: 7). PCR analysis revealed the percentage of positive pools in 2015-16 were 7.50% (n=3) for S. tescorum , 3.75% (n=3) for S. vittatum (s.l.; likely S. tribulatum ), 7.69% (n=1) for S. clarum “black” phenotype, and no positives for S. clarum “orange” phenotype. Genetic distance and phylogenetic analyses suggest that the northern California Onchocerca isolates belong to the same species reported in black flies from southern California (average pairwise comparison: 0.32%), and seems closely related to Onchocerca isolates of white-tailed deer from upstate New York (average pairwise comparison: 2.31%). Conclusion: Onchocerca cervipedis is part of larger, continentally-distributed species complex rather than a single described species of North America. It is unclear how many isolates of Onchocerca cervipedis exists at this time and more data sampling will be required before a scientific consensus can be determined. In addition, there are at least three putative vectors of black flies ( S. clarum , S. tescorum , S. vittatum ) associated with this cryptic Onchocerca species. A comprehensive reassessment of North American Onchocerca biodiversity, host, and geographic range is necessary.