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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 by  Onchocerca jakutensis  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 of filarioids.    Results  Among the pools of black flies, there were 158 individuals of  Simulium tescorum  (2015: 57, 2016: 101), 302 individuals of  Simulium vittatum  ( sensu lato  [s.l.]) (2015: 82, 2016: 220), 16 individuals of  Simulium 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 filarioid-positive pools 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 seem closely related to  Onchocerca  isolates of white-tailed deer from upstate New York (average pairwise comparison: 2.31%).    Conclusion  A cryptic  Onchocerca  species was found in Lake County, California, and may be a part of a larger, continentally distributed species complex rather than a single described species of North America. 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.    Graphical abstract

Presence of a cryptic Onchocerca species in black flies of northern California, USA