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.

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 4 h. Exposure of larvae to 1/3 of that concentration resulted in similar mortality; however, the maximum mortality was not reached until 8 h 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 50 ppm 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%.

[Economic losses during an outbreak of Simulium (Wilhelmia) species (Diptera: Simuliidae) in the Cappadocia region of Turkey]. / Türkiye'de Kapadokya Bölgesinde Simulium (Wilhelmia) Türlerinin (Diptera: Simuliidae) Istilasinda Olusan Ekonomik Kayiplar.

OBJECTIVE: The aim of this study was to calculate the economic losses during an outbreak of Simulium (Wilhelmia) spp. in the Cappadocia Region of Turkey. METHODS: The economic costs associated with a 2006-2007 outbreak of Simulium (Wilhelmia) spp. in the Cappadocia region of Turkey were calculated by summing losses to the livestock (dairy) industry and tourism (hotels), plus ongoing control expenditures. RESULTS: More than 2.000.000 domestic and foreign tourists, 60.000 animals, and the local population were disturbed by the flies. Tourism was the most affected sector from the Simulium outbreak. CONCLUSION: The calculated cost of the outbreak according to 2013 prices was 10.626.966 TL (US$ 5.45 million).

Host predilection and transmissibility of vesicular stomatitis New Jersey virus strains in domestic cattle (Bos taurus) and swine (Sus scrofa).

BACKGROUND: Epidemiologic data collected during epidemics in the western United States combined with limited experimental studies involving swine and cattle suggest that host predilection of epidemic vesicular stomatitis New Jersey virus (VSNJV) strains results in variations in clinical response, extent and duration of virus shedding and transmissibility following infection in different hosts. Laboratory challenge of livestock with heterologous VSNJV strains to investigate potential viral predilections for these hosts has not been thoroughly investigated. In separate trials, homologous VSNJV strains (NJ82COB and NJ82AZB), and heterologous strains (NJ06WYE and NJOSF [Ossabaw Island, sand fly]) were inoculated into cattle via infected black fly bite. NJ82AZB and NJ06WYE were similarly inoculated into swine. RESULTS: Clinical scores among viruses infecting cattle were significantly different and indicated that infection with a homologous virus resulted in more severe clinical presentation and greater extent and duration of viral shedding. No differences in clinical severity or extent and duration of viral shedding were detected in swine. CONCLUSIONS: Differences in clinical presentation and extent and duration of viral shedding may have direct impacts on viral spread during epidemics. Viral transmission via animal-to-animal contact and insect vectored transmission are likely to occur at higher rates when affected animals are presenting severe clinical signs and shedding high concentrations of virus. More virulent viral strains resulting in more severe disease in livestock hosts are expected to spread more rapidly and greater distances during epidemics than those causing mild or inapparent signs.

The lack of effect of low temperature and high turbidity on operational Bacillus thuringiensis subsp. israelensis activity against larval black flies (Diptera: Simuliidae).

Black fly suppression programs are conducted across a wide range of environmental conditions, targeting a variety of pest species with diverse life histories. Operational applications of Vectobac 12AS (Bacillus thuringiensis subsp. israelensis) were conducted during times characterized by water temperature and turbidity extremes. Applications were conducted in the Yellow River in central Wisconsin targeting Simulium annulus and S. johannseni when water temperatures were 1-2 degrees C. Applications were conducted in the Green River in western North Carolina targeting the S. jenningsi group after a rain event, when portions of the treatment zone experienced turbidities of 276 nephelometric turbidity units. Excellent larvicidal activity was observed in both programs, with 97% mortality or greater being observed at distances over 5 km downstream of a treatment site. Mortality data for larval black flies in 2 operational suppression programs conducted in 2011 demonstrated a negligible effect of near-freezing water temperatures and exceptionally high turbidity on Bti activity.

Chironomus calligraphus (Diptera: Chironomidae), a new pest species in Georgia.

Chironomid midges are ubiquitous and ecologically important aquatic insects. However, some species can become pests when they occur in extremely high numbers, particularly those that colonize man-made habitats. Chironomus calligraphus is a Neotropical, pan-American species that has recently been found in the Nearctic region. This paper represents the 1st reported occurrence of C. calligraphus in Georgia. Extensive larval populations were found in the leaf sheaths and root masses of cattails and in the firm sandy substrates of a wastewater lake at an industrial site in coastal Georgia. Chironomus calligraphus was causing a significant economic impact at this site.

Comparison of morning and evening larvicide applications on black fly (Diptera: Simuliidae) mortality.

The downstream movement, or drift, of larval black flies as well as many other aquatic insects has been well documented. This phenomenon has most often been associated with the dusk-to-overnight time frame. Operationally, larvicide applications targeting black flies are typically initiated early in the day and can extend until near sunset. To determine if larvicide applications conducted late in the day would be affected by the drift behavior of larval black flies, 6 larvicide applications were conducted. Three applications were conducted at midmorning and 3 during the evening. Larvicidal applications of Bacillus thuringiensis subsp. israelensis insecticidal proteins targeting Simulium appalachiense demonstrated no difference in larval mortality between morning and evening applications. These findings indicate that the larvae responded in a similar manner to the larvicide during the late morning to early afternoon and evening to night. The drift behavior of larval black flies does not appear to be an impediment to black fly suppression activities.

Field evaluation of bactimos pt against chironomid midge (Diptera: Chironomidae) larvae in residential ponds.

We evaluated Bactimos PT (Bacillus thuringiensis subsp. israelensis [Bti]) against larval populations of Glyptotendipes paripes at 30 kg/ha in man-made lakes on Hilton Head Island, SC. Three treatments were "whole-pond" treatments, while the 4th consisted of treating a "band" along the edge of a pond where significant larval populations had been observed. Larval populations were reduced by an average of 95% at day 7, 70% at day 14, and 50% at day 21 posttreatment in the whole-pond treatments. Initial larval suppression with the band treatment was similar to the whole-pond treatments, indicating that suppression activities can be targeted to specific areas of a larval habitat.

The effect of seston on mortality of Simulium vittatum (Diptera: Simuliidae) from insecticidal proteins produced by Bacillus thuringiensis subsp. israelensis.

Water was collected from a site on the Susquehanna River in eastern Pennsylvania, where less-than-optimal black fly larval mortality had been occasionally observed after treatment with Bacillus thuringiensis subsp. israelensis de Barjac insecticidal crystalline proteins (Bti ICPs). A series of experiments was conducted with Simulium vittatum Zetterstedt larvae to determine the water related factors responsible for the impaired response to Bti ICPs (Vectobac 12S, strain AM 65-52). Seston in the water impaired the effectiveness of the ICPs, whereas the dissolved substances had no impact on larval mortality. Individual components of the seston then were exposed to the larvae followed by exposure to Bti ICPs. Exposure of larvae to selected minerals and nutritive organic material before ICP exposure resulted in no significant decrease in mortality. Exposure of larvae to silicon dioxide, cellulose, viable diatoms, and purified diatom frustules before Bti ICP exposure resulted in significant reductions in mortality. Exposure of larvae to purified diatom frustules from Cyclotella meneghiniana Kützing resulted in the most severe impairment of mortality after Bti ICP exposure. It is postulated that frustule-induced impairment of feeding behavior is responsible for the impairment of larval mortality.

Influence of selected antibiotics on the response of black fly (Simulium vittatum) larvae to insecticidal proteins produced by Bacillus thuringiensis subsp. israelensis.

A controlled current toxicity test (CCTT) was developed to evaluate the response of black fly (Simulium vittatum) larvae to insecticidal proteins following exposure to various antibiotics. The bacterium, Bacillus thuringiensis subsp. israelensis (Bti), produces proteins that are toxic to Nemotoceran Diptera, such as black flies and mosquitoes, when ingested. These insecticidal crystalline proteins (ICPs) are highly efficacious in controlling black flies; however, speculation has arisen regarding the potential for antibiotic contamination of waterways to mitigate the toxicity of these proteins. A series of experiments was conducted with the CCTT in which black fly larvae were exposed to enrofloxacin, tylosin, sulfamethoxazole, and trimethoprim followed by exposure to Bti ICPs. These antibiotics were selected based on their use in agricultural and documented anthropogenic contamination of rivers. Anthropogenic concentrations of a mixture of these four antibiotics did not affect the response of the larvae to Bti ICPs. Subsequent experiments were conducted with antibiotic concentrations 10,000 to 80,000 times higher than those found in contaminated rivers. Exposure of black fly larvae to high levels of enrofloxacin (0.5 mg/L) had no effect upon the susceptibility to Bti ICPs; however, exposure to high levels of tylosin (8 mg/L) resulted in a significant increase in the susceptibility of the larvae to Bti ICPs at 72 h of exposure, but not at 48 h. Exposure of black fly larvae to high concentrations of a mixture of sulfamethoxazole and trimethoprim resulted in a significant increase in the efficacy of the larvicide after 48 and 72 h of exposure. These results suggest that impairment of the efficacy of Bti ICPs to black fly larvae is not due to antibiotic contamination of the larval environment.

Mechanical transmission of vesicular stomatitis New Jersey virus by Simulium vittatum (Diptera: Simuliidae) to domestic swine (Sus scrofa).

Biting flies have been suggested as mechanical vectors of vesicular stomatitis New Jersey Virus (family Rhabdoviridae, genus Vesiculovirus, VSNJV) in livestock populations during epidemic outbreaks in the western United States. We conducted a proof-of-concept study to determine whether biting flies could mechanically transmit VSNJV to livestock by using a black fly, Simulium vittatum Zetterstedt (Diptera: Simuliidae), domestic swine, Sus scrofa L., model. Black flies mechanically transmitted VSNJV to a naive host after interrupted feeding on a vesicular lesion on a previously infected host. Transmission resulted in clinical disease in the naïve host. This is the first demonstration of mechanical transmission of VSNJV to livestock by insects.

Mitigating the effects of the green alga Scenedesmus quadricauda on the efficacy of Bacillus thuringiensis var. israelensis against larval black flies.

The green alga Scenedesmus quadricauda has been shown to decrease the efficacy of Bacillus thuringiensis var. israelensis (Bti) to black fly larvae in laboratory experiments. In this study, 2 approaches to mitigating the effects of S. quadricauda were tested, increasing the dose of Bti exposed to the larval black flies and increasing the length of the exposure period. Results showed that increasing the dose as well as increasing the exposure time were effective methods for mitigating the effects of S. quadricauda on Bti efficacy. Assessment of larval mortality at different exposure periods to Bti indicate that the manifestation of toxicity or acquisition of toxic doses of the Bti formulation takes place approximately 2-3 times slower when S. quadricauda is present. These results provide information that might be useful in developing a plan of action to combat this complex situation in field operations.

Ochlerotatus japonicus japonicus (Theobald) in Georgia and North Carolina.

Although the 1st published record of Ochlerotatus japonicus japonicus in Georgia (Rabun County) occurred in 2004, we report here a 2002 collection and identification in Fulton County, Georgia (metro Atlanta). The finding of Oc. j. japonicus in Fulton County represents the most southern record of this species in the United States to date. Also, subsequent collections in North Carolina and 4 additional counties in northeast Georgia are reported.

Effects of algae on the efficacy of Bacillus thuringiensis var. israelensis against larval black flies.

Personnel from several black fly control programs have reported that the efficacy of Bacillus thuringiensis var. israelesis (Bti) is reduced during periods when algal concentrations are high in the waterways. Although the reduction in Bti-induced mortality in black fly larvae is presumed to be related to the presence of algae, no scientific data support this theory. In this study, 4 genera of algae (Microcytis, Scenedesmus, Dictrosphaerium, and Chlorella) commonly detected in Pennsylvania rivers where Bti-induced mortality in black fly larvae has been reduced were assessed to determine their respective effects on Bti-induced mortality by using an orbital shaker bioassay with laboratory-reared black fly larvae (Simulium vittatum cytospecies IS-7). A significant reduction in Bti-induced mortality was observed when Scenedesmus was present in the flasks at concentrations > or = 16,000 cells/ml. The Bti-induced mortality of larvae was not significantly reduced when Chlorella, Dictyosphaerium, or Microcytis was present in the flasks, even at concentrations > or = 250,000 cells/ml. These results indicate that the presence of certain types of algae can reduce the mortality of black flies exposed to Bti. Although not clearly defined, the mechanisms involved may be related to algal morphology due to overall size and structures associated with certain types of algae, and possible interference with feeding.

Biological transmission of vesicular stomatitis virus (New Jersey serotype) by Simulium vittatum (Diptera: Simuliidae) to domestic swine (Sus scrofa).

The role of hematophagous arthropods in vesicular stomatitis virus (New Jersey serotype; VSV-NJ) transmission during epizootics has remained unclear for decades in part because it has never been shown that clinical or subclinical disease in a livestock host results from the bite of an infected insect. In this study, we investigated the ability of VSV-NJ-infected black flies (Simulium vittatum Zetterstedt) to transmit the virus to domestic swine, Sus scrofa L. Experimental evidence presented here clearly demonstrates that VSV-NJ was transmitted from black flies to the swine. Transmission was confirmed by seroconversion or by the presence of clinical vesicular stomatitis followed by seroconversion. Our results represent the first report of clinical vesicular stomatitis in a livestock host after virus transmission by an insect.

Black fly involvement in the epidemic transmission of vesicular stomatitis New Jersey virus (Rhabdoviridae: Vesiculovirus).

The transmission routes of Vesicular stomatitis New Jersey virus (VSNJV), a causative agent of vesicular stomatitis, an Office International des Epizooties List-A disease, are not completely understood. Epidemiological and entomological studies conducted during the sporadic epidemics in the western United States have identified potential virus transmission routes involving insect vectors and animal-to-animal contact. In the present study we experimentally tested the previously proposed transmission routes which were primarily based on field observations. Results obtained provide strong evidence for the following: (1) hematophagous insects acquire VSNJV by unconventional routes while blood feeding on livestock, (2) clinical course of VSNJV infection in livestock following transmission by an infected insect is related to insect bite site, (3) infection of livestock via insect bite can result in multiple transmission possibilities, including animal-to-animal contact. Taken together, these data significantly add to our understanding of the transmission routes of a causative agent of one of the oldest known infectious diseases of livestock, for which the details have remained largely unknown despite decades of research.