We describe and compare 2 qualitative serologic techniques for detecting West Nile virus (WNV)-specific antibodies in mosquito blood meals. The techniques are the biotin microsphere immunoassay (b-MIA) and the inhibition platform of the VectorTest™ WNV antigen assay (VecTest-inhibition). To demonstrate the ability of these tests to detect WNV-neutralizing antibodies, we experimentally exposed feeding mosquitoes to blood containing 5 concentrations of 6B6C-1, a flavivirus-neutralizing monoclonal antibody. Antibody concentrations were quantified using the 90% plaque-reduction neutralization test (PRNT₉₀). After 24 h of blood-meal digestion at 22.5°C, the threshold PRNT₉₀ titer of detection was ≤18 for b-MIA and ≤50 for VecTest-inhibition. Both tests reliably detected antibodies in 3 of 3 blood meals that had been digested for up to 30 h, or were about 25% digested. The b-MIA was also applied to mosquitoes that had engorged on avian blood in Arizona following a WNV epidemic in 2010. There was no significant difference in the WNV antibody prevalence determined by b-MIA (52% of 71 avian blood meals) compared to the WNV-neutralizing antibody prevalence in birds determined by direct sampling (49% of 234 birds). VecTest-inhibition requires fewer resources and may be used in the field without a laboratory, but consumes the entire blood meal and relies on subjective interpretation of results. The b-MIA requires a laboratory and sophisticated equipment and reagents. Results for b-MIA are analyzed objectively and can be applied to mosquito blood meals with greater confidence than the VecTest-inhibition method and thus can contribute substantially to research and surveillance programs that would benefit from the detection of specific WNV antibodies in mosquito blood meals.

In the Culex pipiens complex, a common mechanism of insecticide resistance is amplification of esterase genes leading to overproduction of detoxifying esterase enzymes. A number of electrophoretic esterase alleles have been identified, and in field populations individuals with the same esterase electromorph can exhibit a wide range of esterase enzyme activities. We isolated and characterized esterase activity variants associated with the esterase B1 electromorph from a field strain. A mating scheme was used to isolate chromosomes with esterase genes from the strain into 45 families. Twenty-six of the families received esterase genes from the field strain that conferred elevated esterase activity. Mean esterase activities in these families ranged from 43 to 695 nmoles α-naphthyl acetate hydrolyzed/min/mg protein. Variance components indicated that genetic variance (i.e., genetic differences among families) accounted for 77% of the total variation in esterase activity. A comparison of mean esterase activities indicated that there were at least 11 different esterase activity variants contributing to the observed genetic variation in esterase activity among the 26 families. The relevance of these results to understanding the dynamics of amplified esterase genes in populations is discussed.

Since its introduction and establishment in Italy during the early 1990s, the Asian tiger mosquito, Aedes albopictus, has spread over large parts of Italy and other Mediterranean countries. Aedes albopictus is both a nuisance and a competent vector for various arthropod-borne pathogens. Although efficient traps for Ae. albopictus exist and are used for population monitoring, their use as a control tool has not yet been studied. We evaluated Biogents BG-Sentinel mosquito traps, used with the BG Lure, as control tools in northern Italy. The trial was performed as a controlled experiment in which 3 intervention sites, equipped with 7 or 8 BG-Sentinel traps each, were matched with 3 comparable control sites. Trap density ranged from 1 trap per 150 m² to 1 per 350 m². Mosquito populations were monitored at both the intervention and control sites with weekly human landing collections (HLC) and ovitraps. Between 64% and 87% fewer Ae. albopictus individuals were collected by HLC at the intervention sites with the BG-Sentinel mosquito traps, as compared to the untreated control sites. These results indicate that the sustained use and proper placement of efficient mosquito traps can significantly reduce Ae. albopictus biting pressure.

The BG-Sentinel® (BGS) trap and oviposition cups (OCs) have both proven effective in the surveillance of Aedes species. This study aimed to determine which of the 2 traps could best characterize the relative population sizes of Aedes albopictus and Aedes aegypti in an urban section of Jacksonville, FL. Until 1986, Ae. aegypti was considered the dominant container-breeding species in urban northeastern Florida. Since the introduction of Ae. albopictus, Ae. aegypti has become almost completely extirpated. In 2011, a resurgence of Ae. aegypti was detected in the urban areas of Jacksonville; thus this study initially set out to determine the extent of Ae. aegypti reintroduction to the area. We determined that the BGS captured a greater number of adult Ae. aegypti than Ae. albopictus, while OCs did not monitor significantly different numbers of either species, even in areas where the BGS traps suggested a predominance of one species over the other. Both traps were effective at detecting Aedes spp.; however, the BGS proved more diverse by detecting over 20 other species as well. Our results show that in order to accurately determine vectorborne disease threats and the impact of control operations on these 2 species, multiple trapping techniques should be utilized when studying Ae. aegypti and Ae. albopictus population dynamics.

The aim of our study was to assess the potential of Macrocyclops albidus as a biological control agent against the 1st and 2nd instars of Culex pipiens and Aedes albopictus. Under laboratory conditions of prey saturation (50 1st instars/copepod), an average of 58.98% of Cx. pipiens and 54.99% of Ae. albopictus larvae were killed by 1 copepod in 24 h. Trials run in big drums containing 200 liters of water showed that the M. albidus population, inoculated in April, efficiently controlled the mosquito population for the entire season. The predator was particularly effective against Ae. albopictus, as only 2 larvae of this species were found in the treated drums, compared to 814 larvae in untreated control drums throughout the study period. No difference was observed in the control efficacy between the 2 initial densities of copepods used. The reduction in Ae. albopictus density in the drums with 100 and 500 M. albidus with respect to the control drums was 99.90 ± 0.35% and 100.0 ± 0.0%, respectively. For Cx. pipiens, the reduction in density was 88.69 ± 13.51% and 84.65% in drums inoculated with 100 and 500 copepods, respectively. Macrocyclops albidus populations survived through the winter and continued to keep the mosquito population under control during the 2008 season. The M. albidus population developed very well both in drums placed in sunny and shaded areas and proved to be tolerant to both high (summer) and low (winter) temperatures. Trials performed on M. albidus survival in catch basins showed that after a few weeks, the copepod population dramatically decreased and subsequently disappeared. The main problem for copepod survival in catch basins seemed to be the low oxygen tension and accumulation of toxic substances, rather than copepods being flushed out in heavy rainfall episodes. During the period when copepods were present, they maintained the mosquito population under control; their partial disappearance from the catch basins, however, would require more recolonization intervention to maintain mosquito control during the season.

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%.

We describe 2 new mosquito bioassays for use with insecticide-treated netting or other textiles. The 1st is a cylinder bioassay in which a mosquito is forced to contact treated material regardless of where it lands within the bioassay construct. The 2nd is a repellency/irritancy and biting-inhibition bioassay (RIBB) in which human arms and breath are used as attractants. Mosquitoes have the choice to pass through holes cut in untreated or treated netting to move from a center release chamber into side chambers to reach arms and potentially bite. Trials were conducted with pyrethroid-susceptible (New Orleans), moderately resistant (Hunucmá), and highly resistant (Vergel) strains of Aedes aegypti. Tests with netting treated with different pyrethroids demonstrated the utility of the cylinder bioassay to quantify knockdown and mortality following exposure to treated netting, and of the RIBB to quantify spatial repellency/contact irritancy of the treated netting and biting inhibition after females land on and then pass through holes in the treated netting. Both tested brands of pyrethroid-treated mosquitocidal netting (DuraNet® and NetProtect®) were effective against New Orleans but ineffective against Vergel strains. Mortality in the cylinder bioassay was 100% for New Orleans for all tested brands of treated netting, but only 10–14% for Vergel. Rates of passage through treated netting to reach a human arm in the RIBB were 10–15% for New Orleans versus 24–37% for Vergel. The reduction in biting after passage through treated netting, compared with untreated netting in the same trial replicates, was 12–39% for New Orleans versus ≤9% for Vergel.

The attraction of female Aedes aegypti to single compounds and binary compositions containing L-lactic acid and an additional saturated compound from a set of ketones, sulfides, and chloroalkanes was studied using a triple-cage dual-port olfactometer. These chemical classes were studied because of their structural relation to acetone, dimethyl disulfide, and dichloromethane, which have all been reported to synergize attraction to L-lactic acid. Human odors, carbon dioxide, and the binary mixture of L-lactic acid and CO₂ served as controls for comparison of attraction responses produced by the binary mixtures. All tested mixtures that contained chloroalkanes attracted mosquitoes at synergistic levels, as did L-lactic acid and CO₂. Synergism was less frequent in mixtures of L-lactic acid with sulfides and ketones; in the case of ketones, synergistic attraction was observed only for L-lactic acid combined with acetone or butanone. Suppression or inhibition of attraction response was observed for combinations that contained ketones of C₇-C₁₂ molecular chain length (optimum in the C₈-C₁₀ range). This inhibition effect is similar to that observed previously for specific ranges of carboxylic acids, aldehydes, and alcohols.

The objective of this study was to determine the larvicidal activity of an active compound isolated from Cercis chinensis heartwood and its structurally related analogs against 4th-stage Aedes aegypti, Culex pipiens pallens, and Ae. togoi. The larvicidal compound of C. chinensis was isolated with the use of various chromatographic techniques and identified as analogs of 1,4-naphthalenedione. Based on the median lethal concentration (LC₅₀) values of commercially procured analogs against Ae. aegypti larvae, the most toxic analog was 2-bromo-1,4-naphthalenedione (1.19 µg/ml); followed by 5-hydroxy-1,4-naphthalenedione (1.72 µg/ml); 2-methyl-1,4-naphthalenedione (9.12 µg/ml); 2-hydroxy-1,4-naphthalenedione (10.76 µg/ml); and 2-methoxy-1,4-naphthalenedione (12.50 µg/ml). Similar results were observed against Cx. p. pallens and Ae. togoi larvae with 1,4-naphthalenedione analogs. These results also showed that 1,4-naphthalenedione analogs were less toxic than the organophosphate pirimiphos-methyl. Nonetheless, naturally occurring C. chinensis–derived materials and 1,4-naphthalenedione analogs have potential for development as mosquito larvicidal agents.

Accumulations of dormant eggs in container habitats allow Aedes aegypti populations to survive harsh environmental conditions and may frustrate control interventions directed at larval and adult life stages. While sodium hypochlorite solutions (NaOCl) have long been recognized as ovicides for use against dengue vectors, the susceptibility of eggs to spray applications has not been robustly evaluated on substrate materials representative of the most frequently utilized artificial container habitats. Experiments were performed under controlled and natural conditions by applying dilutions of household bleach (52.5 ppt NaOCl) as a spray to eggs on plastic, rubber, and concrete surfaces, with and without a smectite clay thickener. Laboratory assays identified the minimum NaOCl concentrations required to eliminate eggs on plastic (10 ppt), rubber (20 ppt) and concrete (20 ppt) surfaces. Addition of smectite clay reduced the minimum effective concentration to 10 ppt NaOCl for all 3 substrates. A minimum exposure period of 24 h was required to completely eliminate egg viability on concrete surfaces, even at the highest NaOCl concentration (52.5 ppt). Field experiments verified that spray application of a 1∶3 dilution of household bleach mixed with smectite clay can reduce egg hatching by ≥ 99% in shaded and sun-exposed plastic containers. Similarly, 4∶1 dilution of household bleach (with or without smectite clay) eliminated ≥ 98% of eggs from concrete surfaces in outdoor, water-filled drums. In this study, we propose a practical, effective and safe strategy for using household bleach to eliminate Ae. aegypti eggs in a range of artificial container habitats.

The effectiveness and penetration of a novel, truck-mounted mist sprayer (3WC-30-4P provided by American LongRay) was evaluated with bifenthrin in a large, park-like setting with historic floodwater and woodland mosquito populations. Efficacy evaluations were determined through adult population collections and excised leaf bioassays. Trapping results showed a mean reduction of 77% in mosquito populations in the treated area for 5 sampling events up to 4 wk posttreatment. Leaf bioassays revealed an average mortality of 80% at 2.7 m and 51% at 5.5 m against laboratory-reared Aedes aegypti for 5 posttreatment samples. Leaves collected from the treated areas caused higher mortality at distances closer to the sprayer, though the distance and coverage of bifenthrin application was effective up to 5 m.

A series of statewide surveys were conducted in Oklahoma in the summers between 1991 and 2004 to identify the distribution of Aedes albopictus. Adult mosquitoes were identified in 63 counties, bringing the currently known distribution of Ae. albopictus in the state to 69 of 77 counties. The widespread presence of Ae. albopictus in Oklahoma has important current and future public and veterinary health implications for surveillance and control efforts.

Specimens of a mosquito new to the continental USA, Aedes pertinax, were retrospectively identified from 2 collections made in 2011 in Indian River County, FL. Routine mosquito surveillance in subsequent years yielded more than 700 specimens appearing in 100 collections. The distribution of this mosquito in Florida and the United States is currently unknown, and recognition of the adult female is likely hampered by morphological similarities to Ae. atlanticus and Ae. tormentor.

We provide an updated checklist of 67 endemic mosquito species known from Ontario, Canada. Nine endemic species are added to the checklist found in Darsie and Ward (2005): Aedes cantator, Ae. churchillensis, Ae. nigripes, Ae. pullatus, Anopheles perplexens, An. crucians, An. smaragdinus, Culex erraticus, and Cx. salinarius. Only 4 specimens of Ae. albopictus have been recorded in Ontario since 2001 despite concerted efforts to find this species; therefore, it is considered an “accidental” species and is excluded from the checklist.

The correct type localities were noted for the following mosquito species: 1) Sabethes fabricii, municipality of Angra dos Reis, state of Rio de Janeiro, Brazil; 2) Sa. soperi 1942, municipality of Nova Iguaçu, state of Rio de Janeiro, Brazil; and 3) Wyeomyia finlayi 1942, Xerém, in the municipality of Duque de Caxias, state of Rio de Janeiro, Brazil.

The typical attractant used in gravid trapping of Culex quinquefasciatus is an aged infusion of organic materials, which can change in attractiveness over time. A standardized chemical attractant dispenser derived from grass infusion, the AtrAedes™ lure, has been produced for the surveillance of the dengue vector Aedes aegypti. A study using this lure in combination with the Centers for Disease Control and Prevention gravid traps was conducted in Tanga, Tanzania. The addition of the lure to traps baited with either grass infusion or tap water did not result in significant increases in trap catch. Grass infusion–baited traps (with and without the AtrAedes lure) collected significantly more Cx. quinquefasciatus than traps baited with AtrAedes tap water, tap water alone, or AtrAedes alone. The catches of the traps baited with AtrAedes tap water, tap water alone, and AtrAedes alone were not significantly different from each other. Although the placement of the lure in the base of the trap may have decreased trap catches, it seems that the AtrAedes is not as effective as grass infusion for collecting Cx. quinquefasciatus in Tanzania.

The novel Mos-Hole trap™ with liquid attractant naphtha™ from Korea was compared with BG Sentinel™ trap and Mosquito Magnet X™ trap for field collection of adult mosquitoes in St. Johns County, northeastern Florida, from May to October 2013. The novel Mos-Hole trap baited with naphtha (liquid attractant) collected similar numbers of mosquitoes, compared with the number of mosquitoes caught by BG Sentinel traps baited with BG Lure™. Both Mos-Hole and BG Sentinel traps collected a significantly greater number of mosquitoes compared with the numbers collected by Mosquito Magnet X traps. In other field evaluations when switching lures, the Mos-Hole traps baited with BG Lure caught more mosquitoes than the BG Sentinel trap baited with liquid naphtha attractant. The results showed that the novel Mos-Hole trap has the potential to be used as an additional effective sampling tool for population surveillance and control of adult mosquitoes.

Although it is widely accepted that black fly larvae employ filter feeding as their primary mode of nutrient intake, other forms of food acquisition, such as predation, may be more prevalent than previously realized. It has been suggested that environments where particulate matter is low, such as high-elevation seasonal streams, may drive predatory behavior in black fly larvae. Relatively little is known about the frequency at which larvae prey on other organisms or if predation may be obligate in some species. In order to supplement the idea that larval black fly predation may be a common method of feeding under certain conditions, a preliminary survey of predation by Prosimulium larvae was conducted in order to assess predation frequency at high-elevation sites (> 3,200 m) in the Colorado Rocky Mountains. Larvae were sampled from alpine and subalpine locations, and their gut content analysis revealed evidence of facultative predation and possible cannibalism. Evidence of predation was observed in all but 1 Prosimulium species collected. Predation frequency was highest in the North Fork Snake River headwater location, a small tributary stream of the Snake River in central Colorado. This survey suggests that further inquiry into predatory behavior of black fly larvae should be conducted to determine the mechanisms, behavior, and ecological impact of this understudied feeding strategy.