Simulium Vittatum Research Articles

Community composition of black flies during and after the 2020 vesicular stomatitis virus outbreak in Southern New Mexico, USA

BackgroundVesicular stomatitis virus (VSV), a vector-borne pathogen of livestock, emerges periodically in the western US. In New Mexico (NM), US, most cases occur close to the Rio Grande River, implicating black flies (Simulium spp.) as a possible vector. In 2020, VS cases were reported in NM from April to May, although total black fly abundance remained high until September. We investigated the hypothesis that transience of local VSV transmission results from transient abundance of key, competent black fly species. Additionally, we investigated whether irrigation canals in southern NM support a different community of black flies than the main river. Lastly, to gain insight into the source of local black flies, in 2023 we collected black fly larvae prior to the release of water into the Rio Grande River channel.MethodsWe randomly sub-sampled adult black flies collected along the Rio Grande during and after the 2020 VSV outbreak. We also collected black fly adults along the river in 2021 and 2022 and at southern NM farms and irrigation canals in 2022. Black fly larvae were collected from dams in the area in 2023. All collections were counted, and individual specimens were subjected to molecular barcoding for species identification.ResultsDNA barcoding of adult black flies detected four species in 2020: Simulium meridionale (N = 158), S. mediovittatum (N = 83), S. robynae (N = 26) and S. griseum/notatum (N = 1). Simulium robynae was only detected during the VSV outbreak period, S. meridionale showed higher relative abundance, but lower absolute abundance, during the outbreak than post-outbreak period, and S. mediovittatum was rare during the outbreak period but predominated later in the summer. In 2022, relative abundance of black fly species did not differ significantly between the Rio Grande sites and farm and irrigation canals. Intriguingly, 63 larval black flies comprised 56% Simulium vittatum, 43% S. argus and 1% S. encisoi species that were either extremely rare or not detected in previous adult collections.ConclusionsOur results suggest that S. robynae and S. meridionale could be shaping patterns of VSV transmission in southern NM. Thus, field studies of the source of these species as well as vector competence studies are warranted.Graphical

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

BackgroundBlack 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.MethodsTo 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.ResultsAmong 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%).ConclusionA 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

Light Polarization by Biological Nanocoatings.

Light plays paramount functions for living beings in nature. In addition to color, the polarization of light is used by many animals for navigation and communication. In this study, we describe the light polarizing role of special nanostructures coating cuticular surfaces of diverse arthropods. These structures are built as parallel nanoscale ridges covering the eyes of the sunlight-navigating spider Drassodes lapidosus and of the water pond-swarming black fly Simulium vittatum, as well as the light-emitting abdominal lantern of the firefly Aquatica lateralis. Exact topography and dimensions of the parallel nanoridges provide different light polarizing efficiencies and wavelength sensitivity. Optical modeling confirms that the nanoscale ridges are responsible for the spectral polarization dependency. Co-opting from our recent work on the self-assembly of Drosophila corneal nanostructures, we engineer arthropod-like parallel nanoridges on artificial surfaces, which recapitulate the light polarization effects. Our work highlights the fundamental importance of nanocoatings in arthropods for the light polarization management and provides a new biomimetic approach to produce ordered nanostructures under mild conditions.

Contrasting responses of black fly species (Diptera: Simuliidae) to experimental whole‐stream warming

Abstract As global temperatures continue to rise, assessment of how species within ecological communities respond to shifts in temperature has become increasingly important. However, such assessments require detailed long‐term observations or ecosystem‐level manipulations that allow for interactions among species and the potential for species dispersal and exchange with the regional species pool. We examined the effects of experimental whole‐stream warming on a larval black fly assemblage in southwest Iceland. We used a paired‐catchment design, in which we studied the warmed stream and a nearby reference stream for 1 year prior to warming and 2 years during warming and estimated population abundance, biomass, secondary production, and growth rates for larvae of three black fly species. Experimental warming by 3.8°C had contrasting effects on the three black fly species in the assemblage. The abundance, biomass, growth, and production of Prosimulium ursinum decreased in the experimental stream during the warming manipulation. Despite increasing in the reference stream, the abundance, biomass, and production of another species, Simulium vernum, decreased in the experimental stream during warming. In contrast, warming had an overall positive effect on Simulium vittatum. While warming had little effect on the growth of overwintering cohorts of S. vittatum, warming led to an additional cohort during the summer months and increased its abundance, biomass, and production. Overall, family‐level production was enhanced by warming, despite variation in species‐level responses. Our study illustrates that the effects of climate warming are likely to differ even among closely related species. Moreover, our study highlights the need for further investigation into the uneven effects of warming on individual species and how those variable effects influence food web dynamics and ecosystem function.

Evaluation of Larval Medium in the Controlled Current Toxicity Test.

The Controlled Current Toxicity Test (CCTT) is a protocol used by the University of Georgia Black Fly Rearing and Bioassay Laboratory to evaluate the efficacy of larvicides based on Bacillus thuringiensis subsp. israelensis (Bti) against Simulium vittatum larvae. A standard CCTT provides a larval medium with suspended organic nutrients, a sustained current, and a clean surface for black fly larval attachment in each exposure vessel. In an effort to streamline the CCTT and eliminate a potential source of variability, 3 types of larval medium were evaluated: deionized water, distilled water, and moderately hard water, which were compared to the currently used protocol. A statistical significance in mean lethal concentration (LC50) rates was observed between the CCTT (deionized water with suspended organic nutrients) medium and the other 3 experimental media. The presence of suspended organic nutrients in the CCTT significantly improved the efficacy of the Bti-based larvicide. The interaction of the suspended organic nutrient particles and the Bti-based particles in the larvicide appears to produce a mixture that is more efficiently captured and ingested by the black fly larvae than the larvicide formulation particles alone.

Evaluation of Partially Submerged Sticky Traps On Lake Spillways For Adult Black Fly (Diptera: Simuliidae) Surveillance and Arbovirus Detection

Sentinel surveillance systems demonstrate an improved ability to supplement monitoring data and anticipate arbovirus outbreaks (i.e., sentinel avian species). Management complications can arise during unpredictable or unseasonal disease detections, especially in rural areas where resident distribution is patchy. Using spillways near residential lake communities as static surveillance locations, we tested a novel partially submerged sticky trapping technique and screened wild populations of adult female black flies (Diptera: Simuliidae) for West Nile virus (WNV) and eastern equine encephalitis virus (EEEV). Trap site selection criteria considered the density of immature black fly colonization on spillway surfaces and the number of positive detections of arboviral targets in nearby Culex mosquito populations. On average (±standard error), sticky traps captured 134 (±33) adult black flies over a 24-h period, with 1 trap capturing as many as 735 individuals. Although we detected positive cases of WNV from 20 Culex mosquito trapping sites within 16 km (approx. flight radius) of the selected lake spillways, mixed pools of adult female Simulium vittatum complex and Simulium decorum were all negative for both arboviruses. This study yielded an application for partially submerged sticky traps to collect adult female black flies. Its potential uses for monitoring the infection rates of more well-documented Simulium parasites are discussed.

BFPI: A Minimal Prothrombinase Inhibitor from the Saliva of Simulium Vittatum

Background: The black fly, Simulium Vittatum, has an anticoagulant protein in its saliva that allows it to feed on mammalian blood (black fly protease inhibitor; BFPI). Remarkably, BFPI is similar to the human anticoagulant tissue factor pathway inhibitor alpha (TFPIα). TFPIα contains three Kunitz-type protease inhibitor domains (K1, K2, K3), which inhibit factor VIIa (FVIIa) and factor Xa (FXa) and bind the co-factor protein S (PS), respectively; BFPI contains a single Kunitz domain that inhibits FXa. In addition, TFPIα and BFPI contain homologous basic regions (BRs) near their C-termini (252LIKTKRKRKK261 in human TFPIα, LIKTRKRKPKK in BFPI). The TFPIα BR binds a regulatory acidic region (AR) in factor Va (FVa). The AR is present in forms of FVa released by collagen-activated platelets and generated through limited proteolysis by FXa (FVaXa), and is removed by thrombin (FVaIIa). We hypothesized that BFPI, through its Kunitz domain and basic C-terminus, inhibits early forms of the prothrombinase complex, but does not possess the other inhibitory functions of TFPIα: (1) K1-dependent inhibition of the tissue factor (TF)-FVIIa complex; and (2) PS/K3-dependent FXa inhibition.Results: Recombinant BFPI inhibited FXa in an amidolytic activity assay, and PS did not promote this inhibition. BFPI did not inhibit TF-FVIIa-mediated FX activation. As described with TFPIα, FV promoted FXa inhibition by BFPI but FVaIIa did not, suggesting that the BFPI BR is capable of binding the FVa AR. In a purified protein assay, BFPI inhibited prothrombinase assembled with FVaXa (IC50=4.9nM), but not FVaIIa. Similarly, 5nM BFPI increased the lag time for FXa-initiated plasma thrombin generation by 10.4±1.5%.We next used BFPI as a backbone to evaluate a reported human mutation in the TFPIα BR, K254E. Every mammalian, avian, or reptilian TFPIα sequence available contains either a Lys or Arg residue at this position, suggesting that this residue is functionally important. In purified protein assays, BFPI-K254E inhibited FXa amidolytic activity identically to BFPI, but FV did not promote this inhibition, suggesting that BFPI-K254E has a specific defect in its interaction with FV. Consistent with this, BFPI-K254E was a weaker inhibitor of prothrombinase assembled with FVaXa (IC50 = 15.8nM) and FXa-initiated plasma thrombin generation.The results obtained with BFPI-K254E were confirmed using peptides and full-length TFPIα proteins. First, a peptide mimicking the wild type TFPIα BR (LIKTKRKRKK) inhibited prothrombinase assembled with FVaXa (IC50 = 1.0 µM), while the substituted peptide (LIETKRKRKK) was substantially weaker (20% inhibition observed with 340 µM peptide). Second, full-length TFPIα-K254E was a weaker inhibitor of prothrombinase containing FXa-activated FVa (IC50 = 14.8 nM, vs. 1.8 nM for TFPIα) and had greatly reduced anticoagulant activity in plasma-based thrombin generation assays.Conclusions: In summary, the anticoagulant effect of BFPI is mediated through inhibition of early forms of prothrombinase, independent of TF-FVIIa inhibition or PS-dependent FXa inhibition. The natural mutation TFPIα K254E disrupts prothrombinase inhibition, despite the presence of six other conserved basic residues, and is thus procoagulant in human plasma. The absolute conservation of the TFPIα BR, and its usurpation to allow feeding by black flies, point to formation of the initial prothrombinase complex as a key regulatory step in blood coagulation. DisclosuresMast:Novo Nordisk: Research Funding.

A prospective study on insect bite hypersensitivity in horses exported from Iceland into Switzerland

BackgroundInsect bite hypersensitivity (IBH) is an IgE-mediated dermatitis caused by bites of Culicoides spp., which occurs frequently in horses imported from Iceland to continental Europe. IBH does not occur in Iceland because Culicoides species that bite horses are not present. However, Simulium vittatum (S. vittatum) are found in Iceland. In Europe, blood basophils from IBH-affected horses release significantly more sulfidoleukotrienes (sLT) than those from healthy controls after in vitro stimulation with Culicoides nubeculosus (C. nubeculosus) and S. vittatum. Aims of the study were: (I) using the sLT release assay, to test if horses living in Iceland were sensitized to S. vittatum and (II) to determine in a longitudinal study in horses imported from Iceland to Switzerland whether the sLT release assay would allow to predict which horses would develop IBH.ResultsHorses in Iceland, even when living in high S. vittatum areas, were usually not sensitized to S. vittatum or C. nubeculosus. Incidence of IBH in the 145 horses from the longitudinal study was 51% and mean time until IBH developed was 2.5 ± 1 year. Before import and after the first summer following import, there were no significant differences in sLT release between the endpoint healthy (H) and IBH groups. After the 2nd summer, when the number of clinically affected horses increased in the endpoint IBH group, a significantly higher sLT release after stimulation with C. nubeculosus but not with S. vittatum was observed. After the 3rd and 4th summer, the endpoint IBH group had a significantly higher sLT release with C. nubeculosus and S. vittatum than the endpoint H group. Some of the horses that remained healthy became transiently positive in the sLT release assay upon stimulation of their peripheral blood leucocytes with C. nubeculosus.ConclusionsHorses in Iceland are not sensitized to S. vittatum. In horses that develop IBH, sensitization to S. vittatum is secondary to sensitization to C. nubeculosus and probably a result of an immunological cross-reactivity. A sLT release assay cannot be used to predict which horses will develop IBH. A transient positive reaction in the sLT release assay observed in horses that remained healthy suggests that immunoregulatory mechanisms may control an initial sensitization of the healthy horses.

A cryptic species of Onchocerca (Nematoda: Onchocercidae) in blackflies (Simulium spp.) from southern California, USA

BackgroundEntomological surveillance for pathogens based on molecular screening of putative arthropod vectors such as blackflies (Diptera: Simuliidae) is becoming increasingly important. Surveillance provides a means to understand host and geographical patterns of underestimated biodiversity among North American species of Onchocerca and a pathway to identify and track expanding emergence of the zoonotic Onchocerca lupi. Herein, we have screened two blackfly species, Simulium tescorum and Simulium vittatum (s.l.), from Los Angeles County, southern California, USA for DNA of filarioid nematodes to better understand species richness and limits within the genus Onchocerca.MethodsA total of 1056 and 378 female blackflies was collected using CO2-baited mosquito traps from March to November of 2015 and 2016, respectively. All blackflies during 2015 were individually processed for DNA extraction and PCR targeting of the cytochrome c oxidase subunit 1 (cox1) of the mitochondrial DNA (mtDNA). Specimens of S. tescorum collected in 2016 were processed individually with heads and bodies extracted separately, whereas those of S. vittatum (s.l.) were processed in pooled samples with heads and bodies extracted separately. A subset of filarioid-positive samples from 2015 and all samples from 2016 were screened using a PCR targeting the NADH dehydrogenase subunit 5 (nad5) gene (mtDNA).ResultsIn 2015, 356 S. tescorum (33.7%) and 683 S. vittatum (s.l.) (64.7%) were collected, and an additional 17 specimens were not assessed morphologically. In 2016, a total of 378 blackflies was collected. Of these, 43 (11.6%) were S. tescorum and 327 (88.4%) were S. vittatum (s.l.), and an additional 8 specimens were not assessed morphologically. In 2015, Onchocerca sequences were detected in 4.8% (n = 17) of S. tescorum samples, and only one S. vittatum (0.15%). In 2016, only a single S. vittatum pool was positive for the same cryptic Onchocerca species. In phylogenetic comparisons based on nad5, the Onchocerca sequences from California formed a clade with those isolates in white-tailed deer from upstate New York, suggesting these belong to a single widespread cryptic species.ConclusionsAn uncharacterized species of Onchocerca associated with cervid hosts was found in blackflies from southern California. Sequence data demonstrated it is likely conspecific with an unnamed species of Onchocerca previously found in white-tailed deer from upstate New York. Current data support recognition of a broad geographical distribution across North America for an apparently cryptic species of Onchocerca that is discrete from O. cervipedis, considered to be a typical filarioid among cervids. Our data suggest that this cryptic species of Onchocerca may infect subspecies of white-tailed deer (Odocoileus virginianus), and mule and black-tailed deer (Odocoileus hemionus) at temporal latitudes. The blackflies Simulium tescorum and S. vittatum (s.l.) (presumably, S. tribulatum) are putative vectors. Discovery of a cryptic complex indicates that species diversity and putative associations for definitive hosts and vectors of Onchocerca species in North America must be reassessed.

Trophic Strategies Influence Metal Bioaccumulation in Detritus-Based, Aquatic Food Webs.

Metal accumulation in aquatic food webs is mediated by physiochemical parameters of the environment and organismal traits. Trophic strategies influence an organisms' exposure to metal pollution, but links between trophic ecology and exposure to divalent metals are relatively understudied. While organically bound metals are typically considered unavailable for uptake, organisms directly consuming dissolved organic carbon (DOC) and bacteria-via the microbial loop-must also be consuming organically bound metals. Hence, we predicted animals feeding within the microbial loop would accumulate metals through their diet. To test this prediction, we exploited dietary differences between two organisms, Simulium vittatum, a filter-feeding black fly and Hyalella azteca, a shredding detritivore. We exposed both species to three treatments of DOC (labile, recalcitrant, and noadditional DOC) that were crossed with exposure to variable copper (Cu) concentrations (2-14 μg L-1) in laboratory microcosms. As predicted, H. azteca experienced a buffering effect by DOC. However, this pattern was not apparent for S. vittatum. Our results highlight the importance of considering trophic strategies when examining the impacts of metal pollution on aquatic communities, and demonstrate the potential for the microbial loop to facilitate metal uptake in freshwater food webs.

Identification of semiochemicals attractive to Simulium vittatum (IS-7).

Many blackfly species (Diptera: Simuliidae) are economically important insect pests, both as nuisance biters and as vectors of pathogens of medical and veterinary relevance. Among the important blackfly pest species in North America is Simulium vittatum Zetterstedt sensu lato. The objective of this study was to identify compounds excreted by mammalian hosts that are attractive to host-seeking S. vittatum females. The attractiveness of putative compounds to colonized S. vittatum was tested through electrophysiological (electroantennography; n = 58 compounds) and behavioural (Y-tube assays; n = 7 compounds in three concentrations) bioassays. Five compounds were significantly attractive to host-seeking S. vittatum females: 1-octen-3-ol; 2-heptanone; acetophenone; 1-octanol, and naphthalene. These candidate compounds might be useful as attractants in traps that could be developed for use in alternative or complementary management tactics in programmes to suppress nuisance blackfly populations, or for the collection of samples in which to study the transmission ecology of pathogens transmitted by blackflies of the S. vittatum complex.

Pathogenesis of Vesicular Stomatitis New Jersey Virus Infection in Deer Mice ( Peromyscus maniculatus) Transmitted by Black Flies ( Simulium vittatum).

The natural transmission of vesicular stomatitis New Jersey virus (VSNJV), an arthropod-borne virus, is not completely understood. Rodents may have a role as reservoir or amplifying hosts. In this study, juvenile and nestling deer mice ( Peromyscus maniculatus) were exposed to VSNJV-infected black fly ( Simulium vittatum) bites followed by a second exposure to naive black flies on the nestling mice. Severe neurological signs were observed in some juvenile mice by 6 to 8 days postinoculation (DPI); viremia was not detected in 25 juvenile deer mice following exposure to VSNJV-infected fly bites. Both juvenile and nestling mice had lesions and viral antigen in the central nervous system (CNS); in juveniles, their distribution suggested that the sensory pathway was the most likely route to the CNS. In contrast, a hematogenous route was probably involved in nestling mice, since all of these mice developed viremia and had widespread antigen distribution in the CNS and other tissues on 2 DPI. VSNJV was recovered from naive flies that fed on viremic nestling mice. This is the first report of viremia in a potential natural host following infection with VSNJV via insect bite and conversely of an insect becoming infected with VSNJV by feeding on a viremic host. These results, along with histopathology and immunohistochemistry, show that nestling mice have widespread dissemination of VSNJV following VSNJV-infected black fly bite and are a potential reservoir or amplifying host for VSNJV.

Feeding behavior of Simulium vittatum larvae in response to various suspended materials: implications for control using biological larvicides

AbstractBiologically based larvicides used to suppress nuisance populations of black flies (Diptera: Simuliidae) require that the larvae ingest the larvicide for mortality to occur. Materials present in the habitat that alter larval feeding behavior may reduce the effectiveness of the larvicide. This study was conducted to determine the effects that commonly occurring materials in larval habitats have on Simulium vittatum Zetterstedt feeding behavior. Larvae were exposed to 50 p.p.m. of kaolinite clay and 4 p.p.m. of purified diatom frustules. Larvae were also exposed to 10 000 cells or colonies per ml of viable green algae, Chlorella vulgaris Pratt and Scenedesmus quadricauda (Turp) Bréb, and the diatom Cyclotella meneghiniana Kützing to determine the effects that these materials have on larval feeding behavior. Individual larvae were recorded using a digital camcorder to determine the average number of times the larvae extended and retracted their cephalic fans per min (flick rate) in each medium. Following exposure to the selected material, neon red particles (NRP) (DayGlo) were added to the larval medium for 20 min. A spectrophotometric analysis was conducted to determine the average quantity of NRP consumed by larvae per min in each medium. The flick rate and quantity of NRP consumed by larvae was not significantly affected by clay material or viable Chlorella cells. When Scenedesmus colonies or Cyclotella cells were present in the larval medium, larval flick rate and NRP ingestion was significantly reduced. The presence of diatom frustules in the medium resulted in the most severe reduction in larval flick rate and NRP ingestion. The results suggest that larval flick rate can change depending on environmental conditions, and that suppression programs using biologically based larvicides may benefit from investigating the potential correlation between algal counts and treatment efficacy.

Drift and activity responses of black flies (Simulium vittatum) in the field: influences of tactile and injury-released stimuli from simulated predation

Prey must manage threat from many types of predators; therefore, selection should favor sensory mechanisms that allow the refinement of defensive behavior. To assess responses to tactile and chemical stimuli related to predation, we observed drift and activity of larval black flies (Simulium vittatum) to simulated predator contact intended to imitate benthic and drift predators as well as a combination of tactile and injury-released stimuli. In the field, larvae responded to tactile stimuli applied to the head with a higher frequency of curling and posterior abdominal segments with a higher frequency of drifting. Additionally, chemical cues from injured conspecifics followed by tactile stimuli applied to the head resulted in a higher frequency of drifting than to either cue independently and this effect was more pronounced at night. The results of our study indicate that larval black flies may utilize multiple cues to determine their antipredator and predator avoidance strategies.

Identification of communal oviposition pheromones from the black fly Simulium vittatum.

The suite of pheromones that promote communal oviposition by Simulium vittatum, a North American black fly species, was identified and characterized using gas chromatography-mass spectrometry, electrophysiological, and behavioral bioassays. Behavioral assays demonstrated that communal oviposition was induced by egg-derived compounds that were active at short range and whose effect was enhanced through direct contact. Three compounds (cis-9-tetradecen-1-ol, 1-pentadecene, and 1-tridecene) were identified in a non-polar solvent extract of freshly deposited S. vittatum eggs that were capable of inducing the oviposition response. Electroantennography demonstrated that two of these three compounds (1-pentadecene and 1-tridecene) actively stimulated antennal neurons. Identification of the oviposition pheromones of this family may be helpful in developing control measures for nuisance black flies and for medically-important species such as Simulium damnosum sensu lato.

Mortality Patterns of Simulium vittatum Larvae (Diptera: Simuliidae) Following Exposure to Insecticidal Proteins Produced by Bacillus thuringiensis var. israelensis.

Products containing insecticidal crystalline proteins (ICPs) produced by Bacillus thuringiensis var. israelensis (Bti ICPs) are used to suppress vector and nuisance populations of black flies. The efficacy of an application of these products is often determined by a posttreatment evaluation of larval mortality. Larvae are typically removed from the substrate at some point in time after application of the product and mortality is determined. The time necessary for the effects of Bti ICPs to cause morality in exposed larvae can vary, and there is little consensus on how long operators should wait before evaluating larval mortality. This study was conducted to provide more information to larvicide applicators when performing posttreatment evaluations. Simulium vittatum larvae were exposed to Bti ICPs under controlled conditions and the mortality was monitored over time. Larvae exposed to operational concentrations of ICPs exhibited maximum mortality, approximately 87%, after 4h. Exposure of larvae to 1/3 of that concentration resulted in similar mortality; however, the maximum mortality was not reached until 8h postexposure. Additional experiments revealed that maximum mortality and time to maximum mortality can also be affected by components in the larval medium. Larval mortality was compared between larvae exposed to Bti ICPs in moderately hard water, medium containing 50 parts per million (ppm) of kaolinite, and medium containing 50ppm of cellulose. The clay material had no significant effect on larval mortality or time to achieve maximum mortality. When cellulose was present in the medium, the time to maximum mortality was increased 50% and overall mortality was reduced by more than 40%.

Larval black fly (Simulium vittatum) responses to chemical stimuli from predators and injured conspecifics under field conditions

We examined chemically-mediated antipredator behaviour in larval Simulium vittatum Zetterstedt 1838 by exposing individuals to one of five stimuli during daytime (1500 hr) and night-time (2300 hr) trials: distilled water, injured earthworm (Lumbricus sp.), injured conspecific (S. vittatum), predator (Corydalus sp.), and a combination of injured conspecific and predator. Larval responses were not homogenous with respect to treatment during daytime and night-time trials and the adjusted residuals show that a higher frequency of larvae curled in response to damaged conspecifics and the combination cue, and drifted when exposed to the combination cue. Additionally, the curling response was significantly longer when larvae were exposed to the three cues associated with predation. Lastly, larvae were more likely to drift at night in response to the combination cue suggesting that larvae responded differentially to chemical stimuli associated with predation and exhibited diel variability.

Major histocompatibility complex and other allergy-related candidate genes associated with insect bite hypersensitivity in Icelandic horses

Insect bite hypersensitivity (IBH) is an allergic dermatitis of horses caused by bites of insects. IBH is a multifactorial disease with contribution of genetic and environmental factors. Candidate gene association analysis of IBH was performed in a group of 89 Icelandic horses all born in Iceland and imported to Europe. Horses were classified in IBH-affected and non-affected based on clinical signs and history of recurrent dermatitis, and on the results of an in vitro sulfidoleukotriene (sLT)-release assay with Culicoides nubeculosus and Simulium vittatum extract. Different genetic markers were tested for association with IBH by the Fisher's exact test. The effect of the major histocompatibility complex (MHC) gene region was studied by genotyping five microsatellites spanning the MHC region (COR112, COR113, COR114, UM011 and UMN-JH34-2), and exon 2 polymorphisms of the class II Eqca-DRA gene. Associations with Eqca-DRA and COR113 were identified (p < 0.05). In addition, a panel of 20 single nucleotide polymorphisms (SNPs) in 17 candidate allergy-related genes was tested. During the initial screen, no marker from the panel was significantly (p < 0.05) associated with IBH. Five SNPs associated with IBH at p < 0.10 were therefore used for analysis of combined genotypes. Out of them, SNPs located in the genes coding for the CD14 receptor (CD14), interleukin 23 receptor (IL23R), thymic stromal lymphopoietin (TSLP) and transforming growth factor beta 3 (TGFB3) molecules were associated with IBH as parts of complex genotypes. These results are supported by similar associations and by expression data from different horse populations and from human studies.

A spectrophotometric technique for measuring particle ingestion by black fly larvae

AbstractA spectrophotometric technique was developed to provide insight into the feeding behavior of Simulium vittatum Zetterstedt (Diptera: Simuliidae) larvae. Larvae were exposed to water insoluble Neon Red particles (NRP) (DayGlo®) in a controlled current. The insoluble particles were available for capture by the cephalic fans of the larvae and subsequent ingestion. The length of gut occupied by the particles after a given exposure time was determined by visual inspection and measured with the aid of a dissecting microscope. Larvae were then homogenized in acetone to solubilize the particles. After filtration, the quantity of pigmented particles in the alimentary tract of the larvae was determined using spectrophotometric analysis. The quantity of particles per unit length of the alimentary tract was calculated. Experiments were conducted to determine the ideal concentration of NRP for obtaining an accurate measure of ingestion without interfering with normal larval feeding behavior. Larval mortality following ingestion of insecticidal proteins produced by Bacillus thuringiensis ssp. israelensis was used as an indirect measure of feeding behavior for these experiments. A concentration of 15 p.p.m. of NRP in the larval medium was the highest concentration used that did not interfere with larval mortality following exposure to the insecticidal proteins. Additional experiments demonstrated that components of the experimental matrix did not interfere with NRP absorbance. The final experiment revealed that the consumption of NRP and insecticidal proteins by larvae was influenced by clay and cellulose in the larval medium.

Simukunin from the Salivary Glands of the Black Fly Simulium vittatum Inhibits Enzymes That Regulate Clotting and Inflammatory Responses

BackgroundBlack flies (Diptera: Simuliidae) feed on blood, and are important vectors of Onchocerca volvulus, the etiolytic agent of River Blindness. Blood feeding depends on pharmacological properties of saliva, including anticoagulation, but the molecules responsible for this activity have not been well characterized.Methodology/Principal FindingsTwo Kunitz family proteins, SV-66 and SV-170, were identified in the sialome of the black fly Simulium vittatum. As Kunitz proteins are inhibitors of serine proteases, we hypothesized that SV-66 and/or −170 were involved in the anticoagulant activity of black fly saliva. Our results indicated that recombinant (r) SV-66 but not rSV-170 inhibited plasma coagulation. Mutational analysis suggested that SV-66 is a canonical BPTI-like inhibitor. Functional assays indicated that rSV66 reduced the activity of ten serine proteases, including several involved in mammalian coagulation. rSV-66 most strongly inhibited the activity of Factor Xa, elastase, and cathepsin G, exhibited lesser inhibitory activity against Factor IXa, Factor XIa, and plasmin, and exhibited no activity against Factor XIIa and thrombin. Surface plasmon resonance studies indicated that rSV-66 bound with highest affinity to elastase (KD = 0.4 nM) and to the active site of FXa (KD = 3.07 nM). We propose the name “Simukunin” for this novel protein.ConclusionsWe conclude that Simukunin preferentially inhibits Factor Xa. The inhibition of elastase and cathepsin G further suggests this protein may modulate inflammation, which could potentially affect pathogen transmission.