Surveillance along the Rio Grande during the 2020 Vesicular Stomatitis Outbreak Reveals Spatio-Temporal Dynamics of and Viral RNA Detection in Black Flies

Vesicular stomatitis virus (VSV) emerges periodically from its focus of endemic transmission in southern Mexico to cause epizootics in livestock in the US. The ecology of VSV involves a diverse, but largely undefined, repertoire of potential reservoir hosts and invertebrate vectors. As part of a larger program to decipher VSV transmission, we conducted a study of the spatiotemporal dynamics of Simulium black flies, a known vector of VSV, along the Rio Grande in southern New Mexico, USA from March to December 2020. Serendipitously, the index case of VSV-Indiana (VSIV) in the USA in 2020 occurred at a central point of our study. Black flies appeared soon after the release of the Rio Grande’s water from an upstream dam in March 2020. Two-month and one-year lagged precipitation, maximum temperature, and vegetation greenness, measured as Normalized Difference Vegetation Index (NDVI), were associated with increased black fly abundance. We detected VSIV RNA in 11 pools comprising five black fly species using rRT-PCR; five pools yielded a VSIV sequence. To our knowledge, this is the first detection of VSV in the western US from vectors that were not collected on premises with infected domestic animals.

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 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 CO2-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

Potential phenotypic plasticity within Simulium nigrimanum Macquart, 1838 (Diptera: Simuliidae) larvae

Black fly larvae (Diptera: Simuliidae) are suspension filter-feeders which strongly depend on water velocity for proper feeding. Black fly species feature different microhabitat preferences. Studies of Holarctic black fly larvae revealed their phenotypic plasticity in response to water current velocity variation, but such studies have been rarely undertaken with Neotropical black flies. The current work presents results on the phenotypic plasticity of the black fly species Simulium nigrimanum Macquart. Twelve last instar larvae, sampled from the Brazilian Cerrado, were photographed under a stereoscopic microscope and measured using the CMEIAS Image tool software. Linear regressions with water velocity as the independent variable were performed, indicating that while body size and anal disk diameter correlated positively with water velocity, labral fan length correlated negatively. The observed relationships between water velocity and labral fan length and anal disk diameter were consistent with the literature, while the pattern of body size variation partially corroborated previous studies. The present work results suggest that potential phenotypic plasticity can be observed in black fly larvae within one population distributed in different microhabitats of the same stream section, as opposed toprevious reports implying that such variation is only found among population from different streams.

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 CO2-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.

Atypical behavior of a black fly species connects cavity-nesting birds with generalist blood parasites in an arid area of Spain

Background The feeding behavior of bloodsucking insects determines the transmission, distribution, host spectrum and evolution of blood parasites in the wild. Conventional wisdom suggests that some vector groups (e.g. black flies, family Simuliidae) are consistently exophagous daytime biters. We aimed to understand more about the exceptions to this pattern by combining targeted trapping and molecular identification of parasites in vectors. Methods In this study, we collected black flies in nest boxes used by European rollers Coracias garrulus in southeastern Spain. We molecularly analyzed 434 individual insects, identifying the black fly species caught in the nest boxes, their potential vertebrate blood meals, and the haemosporidian parasite lineages that they carried. Results Only one black fly species, Simulium rubzovianum, appeared to enter the nest boxes of rollers. Among the trapped specimens, 15% contained vertebrate DNA, which always belonged to rollers, even though only half of those specimens were visibly engorged. Furthermore, 15% of all black flies contained Leucocytozoon lineages, indicating previous feeding on avian hosts but probably not on infected adult rollers. The known vertebrate hosts of the recorded Leucocytozoon lineages suggested that large and/or abundant birds are their hosts. Particularly represented were cavity-nesting species breeding in the vicinity, such as pigeons, corvids and owls. Open-nesting species such as thrushes and birds of prey were also represented. Conclusions Our data strongly suggest that S. rubzovianum bites uninfected roller nestlings and infected individuals of other species, potentially incubating adults, inside nest boxes and natural cavities. This simuliid does not appear to have a strong preference for specific host clades. Contrary to the general pattern for the group, and possibly enhanced by the harsh environmental conditions in the study area, this black fly appeared to intensively use and may even have a preference for confined spaces such as cavities for feeding and resting. Preferences of vectors for atypical microhabitat niches where hosts are less mobile may enable social and within-family transmission and parasite speciation in the long term. At the same time, a lack of host preference in concentrated multispecies communities can lead to host switches. Both processes may be underappreciated driving forces in the evolution of avian blood parasites. Graphical abstract