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The effects of microbial biopesticides used for mosquito control on autotrophic microorganisms such as nanophytoplankton are equivocal. We examined impacts of mosquito biopesticides and mosquito larvae on primary producers in two independent experiments. In the first experiment, we examined the effects of a commonly used microbial biopesticide formulation (VectoMax® CG) on a unicellular microalga, Selenastrum capricornatum Printz, under axenic laboratory conditions. The biopesticide treatments included two concentrations (0.008 and 0.016 g liter-1 ) of VectoMax® CG and two controls (one untreated and another with autoclaved 0.016 g VectoMax® CG liter-1 ) in replicated axenic experimental microcosms. Spectrophotometric analysis of chlorophyll a (proxy for algal biomass) and direct enumeration of algal cells following the treatments revealed no significant effects of the microbial biopesticide on algal population growth during the four-week study. In the second experiment, we tested the effects of different densities of Culex larvae on the population of S. capricornatum. Effects of mosquito larvae feeding on S. capricornatum were significant with a curvilinear relationship between larval density and algal abundance in the water column. Together, these studies demonstrated a lack of direct cytological/toxicological effects of Bacillus-based microbial pesticides on freshwater primary production and support the hypothesis that the reduction in algal primary production previously reported when Bti products were applied to aquatic environments was likely independent of the Bacillus-based larvicidal toxins. Instead, it was likely mediated by microbial interactions in the water column and the trophic cascade effects that resulted from the removal of larval mosquitoes. These studies suggest that mosquito larvae independent of pesticide application can influence primary production. Our method of evaluating biopesticides against small photoautotrophs can be very useful for studying the unintended effects on autotrophic microorganisms of other pesticides, including herbicides and pesticides applied to aquatic environments.

Gravid mosquito collections were compared among several large-volume (infusion volume ≥35 liters) gravid trap designs and the small-volume (infusion volume  =  6 liters) Centers for Disease Control and Prevention (CDC) gravid trap used routinely by vector control districts for vector and pathogen surveillance. The numbers of gravid Culex quinquefasciatus, Cx. tarsalis, and Cx. stigmatosoma collected by large gravid traps were greater than by the CDC gravid trap during nearly all overnight trials. Large-volume gravid traps collected on average 6.6-fold more adult female Culex mosquitoes compared to small-volume CDC gravid traps across 3 seasons during the 3 years of the studies. The differences in gravid mosquito collections between large-versus small-volume gravid traps were greatest during spring, when 8- to 56-fold more Culex individuals were collected using large-volume gravid traps. The proportion of gravid females in collections did not differ appreciably among the more effective trap designs tested. Important determinants of gravid trap performance were infusion container size and type as well as infusion volume, which determined the distance between the suction trap and the infusion surface. Of lesser importance for gravid trap performance were the number of suction traps, method of suction trap mounting, and infusion concentration. Fermentation of infusions between 1 and 4 wk weakly affected total mosquito collections, with Cx. stigmatosoma collections moderately enhanced by comparatively young and organically enriched infusions. A suction trap mounted above 100 liters of organic infusion housed in a 121-liter black plastic container collected the most gravid mosquitoes over the greatest range of experimental conditions, and a 35-liter infusion with side-mounted suction traps was a promising lesser-volume alternative design.

Like many insects, mosquitoes, rely on endosymbionts to grow and develop. These can be acquired from the environment. We used next generation 454 pyrosequencing to discern the whole-body microbiome of the mosquito species Culex quinquefasciatus in various larval stadia and following exposure to common pharmaceutical and personal care products (PPCPs) found in wastewater. PPCP treatments included environmentally-relevant concentrations; 1) a combination of common antibiotics, 2) a combination of mammalian hormones, 3) a mixture of the antibiotic and hormone treatments plus acetaminophen and caffeine and, 4) an untreated control. Within control groups, the predominant families of bacterial symbionts change with each larval instar despite consistent diets and rearing conditions. This trend was also seen in hormone treatments but not in the antibiotic or the mixture treatments. Richness and evenness were reduced in both antibiotic and mixture treatments, suggesting that antibiotics remove certain bacteria or inhibit them from increasing to proportions seen in the control treatment. Interestingly, the mixture treatments had greater richness and evenness compared to antibiotic alone treatments, possibly due to the other contaminants facilitating growth of different bacteria. These findings illuminate the complexity of the microbiome of C. quinquefasciatus and may have implications for more effective control strategies.

The increasing demand for fresh water has forced many countries to use reclaimed wastewater for agricultural purposes. This water contains pharmaceuticals and personal care products (PPCPs) that remain biologically active following passage through wastewater treatment plants. Run-off from farms and contaminated water from treatment facilities exposes aquatic ecosystems to PPCPs. This study examined the effects of PPCPs on a lower trophic organism. Culex quinquefasciatus larvae were reared in water contaminated with environmentally relevant concentrations of common PPCPs. Acetaminophen alone and a mixture of contaminants were found to increase developmental time of larvae. Susceptibility to Bti increased in larvae exposed to antibiotics, acetaminophen, or a mixture of PPCPs. Antibiotics, hormones, and the mixture altered the mosquito bacterial microbiome. Overall, the results indicate that at environmentally relevant concentrations, PPCPs in reclaimed water can have biologically important effects on an ecologically and medically important lower trophic level insect.

BackgroundThe native microflora associated with mosquitoes have important roles in mosquito development and vector competence. Sequencing of bacterial V3 region from 16S rRNA genes across the developmental stages of Culex mosquitoes (early and late larval instars, pupae and adults) was used to test the hypothesis that bacteria found in the larval stage of Culex are transstadially transmitted to the adult stage, and to compare the microbiomes of field-collected versus laboratory-reared mosquitoes.ResultsBeta diversity analysis revealed that bacterial community structure differed among three life stages (larvae, pupae and adults) of Culex tarsalis. Although only ~2% of the total number of bacterial OTUs were found in all stages, sequences from these OTUs accounted for nearly 82% of the total bacterial sequences recovered from all stages. Thorsellia (Gammaproteobacteria) was the most abundant bacterial taxon found across all developmental stages of field-collected Culex mosquitoes, but was rare in mosquitoes from laboratory-reared colonies. The proportion of Thorsellia sequences in the microbiomes of mosquito life stages varied ontogenetically with the greatest proportions recovered from the pupae of C. tarsalis and the lowest from newly emerged adults. The microbiome of field-collected late instar larvae was not influenced significantly by differences in the microbiota of the habitat due to habitat age or biopesticide treatments. The microbiome diversity was the greatest in the early instar larvae and the lowest in laboratory-reared mosquitoes.ConclusionsBacterial communities in early instar C. tarsalis larvae were significantly more diverse when compared to late instar larvae, pupae and newly emerged adults. Some of the bacterial OTUs found in the early instar larvae were also found across developmental stages. Thorsellia dominated the bacterial communities in field-collected immature stages but occurred at much lower relative abundance in adults. Differences in microbiota observed in larval habitats did not influence bacterial community profiles of late instar larvae or adults. However, bacterial communities in laboratory-reared C. tarsalis larvae differed significantly from the field. Determining the role of Thorsellia in mosquitoes and its distribution across different species of mosquitoes warrants further investigation.

Arsenic contamination of global water supplies has come to the forefront in policy decisions in recent decades. However, the effects of arsenic on lower trophic levels of insects inhabiting contaminated ecosystems are not well understood. One approach to document both acute and sublethal effects of toxicants like arsenic is to assay them in combination with microbial pathogens to evaluate shifts in survival curves of the test organisms. Larvae of Culex quinquefasciatus and Culex tarsalis were reared in water containing 0 or 1 000 μg/L of arsenate or arsenite. Fourth instars were then exposed to a range of doses of Bacillus thuringiensis subsp. israelensis (Bti) or Lysinibacillus sphaericus (Ls), with shifts in lethal concentrations determined. Arsenic accumulation in 4th instars was also quantified, and a relative growth index (RGI) calculated for the treatments and compared to controls. Larvae of both species accumulated between 4 447 ± 169 ng As/g and 6 983 ± 367 ng As/g, though RGI values indicated accumulation did not affect growth and development. In all cases, the LC50 's and LC90 's of Cx. quinquefasciatus exposed jointly with arsenic and Bti/Ls were higher than Cx. tarsalis. Cx. tarsalis reared in arsenite showed a significant reduction in their Bti LC90 values compared to the control, indicating a sublethal effect of Bti. When exposed jointly with Ls, arsenite was more toxic than arsenate in Cx. tarsalis. Overall, these results indicate tolerance of these Culex species to arsenic exposures, and why this may occur is discussed.

The impact of emergent macrophyte species and crepuscular sprinkler disturbance on mosquito abundance over a 2-year period was measured in wetland mesocosms. Mosquito oviposition and abundance of immature mosquitoes and aquatic invertebrates were monitored in monotypic plots of small-stature (height of mature stands 2 m) California bulrush (Schoenoplectus californicus) without or with daily sprinkler showers to deter mosquito egg laying. Relative to wetlands without operational sprinklers, oviposition by culicine mosquitoes was reduced by > 99% and immature mosquito abundance was reduced by > 90% by crepuscular sprinkler applications. Mosquito abundance or distribution in wetlands did not differ between the two bulrush species subjected to the sprinkler treatment. Alkali bulrush wetlands without daily sprinkler treatments contained more egg rafts but significantly fewer mosquito larvae than did California bulrush wetlands. Predaceous damselfly naiads were 3-5 times more abundant in alkali bulrush than in California bulrush. Stem density, rate of spread, and autumnal mortality of alkali bulrush were higher than for California bulrush. Replacement of large emergent macrophytes by smaller species may enhance the efficacy of integrated mosquito management programs to reduce mosquito-transmitted disease cycles associated with multipurpose constructed wetlands used worldwide for water reclamation and habitat restoration.

The movement of energy and nutrients from aquatic to terrestrial ecosystems can be substantial, and emergent aquatic insects can serve as biovectors not only for nutrients, but also for contaminants present in the aquatic environment. The terrestrial predators Tenodera aridifolia sinensis (Mantodea: Mantidae) and Tidarren haemorrhoidale (Araneae: Theridiidae) and the aquatic predator Buenoa scimitra (Hemiptera: Notonectidae) were chosen to evaluate the efficacy of arsenic transfer between aquatic and terrestrial environments. Culex tarsalis larvae were reared in either control water or water containing 1000 µg l(-1) arsenic. Adults that emerged from the control and arsenic treatments were fed to the terrestrial predators, and fourth instar larvae were fed to the aquatic predator reared in control or arsenic contaminated water. Tenodera a. sinensis fed arsenic-treated Cx. tarsalis accumulated 658±130 ng g(-1) of arsenic. There was no significant difference between control and arsenic-fed T. haemorrhoidale (range 142-290 ng g(-1)). Buenoa scimitra accumulated 5120±406 ng g(-1) of arsenic when exposed to arsenic-fed Cx. tarsalis and reared in water containing 1000 µg l(-1) arsenic. There was no significant difference between controls or arsenic-fed B. scimitra that were not exposed to water-borne arsenic, indicating that for this species environmental exposure was more important in accumulation than strictly dietary arsenic. These results indicate that transfer to terrestrial predators may play an important role in arsenic cycling, which would be particularly true during periods of mass emergence of potential insect biovectors. Trophic transfer within the aquatic environment may still occur with secondary predation, or in predators with different feeding strategies.

Microbes are important for mosquito nutrition, growth, reproduction and control. In this study, we examined bacterial communities associated with larval mosquitoes and their habitats. Specifically, we characterized bacterial communities associated with late larval instars of the western encephalitis mosquito (Culextarsalis), the submerged portions of two emergent macrophytes (California bulrush, Schoenoplectuscalifornicus and alkali bulrush, Schoenoplectusmaritimus), and the associated water columns to investigate potential differential use of resources by mosquitoes in different wetland habitats. Using next-generation sequence data from 16S rRNA gene hypervariable regions, the alpha diversity of mosquito gut microbial communities did not differ between pond mesocosms containing distinct monotypic plants. Proteobacteria, dominated by the genus Thorsellia (Enterobacteriaceae), was the most abundant phylum recovered from C. tarsalis larvae. Approximately 49% of bacterial OTUs found in larval mosquitoes were identical to OTUs recovered from the water column and submerged portions of the two bulrushes. Plant and water samples were similar to one another, both being dominated by Actinobacteria, Bacteroidetes, Cyanobacteria, Proteobacteria and Verrucomicrobia phyla. Overall, the bacterial communities within C. tarsalis larvae were conserved and did not change across sampling dates and between two distinct plant habitats. Although Thorsellia spp. dominated mosquito gut communities, overlap of mosquito gut, plant and water-column OTUs likely reveal the effects of larval feeding. Future research will investigate the role of the key indicator groups of bacteria across the different developmental stages of this mosquito species.

Wetlands are valuable habitats that provide important social, economic, and ecological services such as flood control, water quality improvement, carbon sequestration, pollutant removal, and primary/secondary production export to terrestrial and aquatic food chains. There is disagreement about the need for mosquito control in wetlands and about the techniques utilized for mosquito abatement and their impacts upon wetlands ecosystems. Mosquito control in wetlands is a complex issue influenced by numerous factors, including many hard to quantify elements such as human perceptions, cultural predispositions, and political climate. In spite of considerable progress during the last decades, habitat protection and environmentally sound habitat management still remain inextricably tied to politics and economics. Furthermore, the connections are often complex, and occur at several levels, ranging from local businesses and politicians, to national governments and multinational institutions. Education is the key to lasting wetlands conservation. Integrated mosquito abatement strategies incorporate many approaches and practicable options, as described herein, and need to be well-defined, effective, and ecologically and economically sound for the wetland type and for the mosquito species of concern. The approach will certainly differ in response to disease outbreaks caused by mosquito-vectored pathogens versus quality of life issues caused by nuisance-biting mosquitoes. In this contribution, we provide an overview of the ecological setting and context for mosquito control in wetlands, present pertinent information on wetlands mosquitoes, review the mosquito abatement options available for current wetlands managers and mosquito control professionals, and outline some necessary considerations when devising mosquito control strategies. Although the emphasis is on North American wetlands, most of the material is applicable to wetlands everywhere.

Combining Bacillus sphaericus with the Cyt1Ab1 or Cyt2Ba1 cytotoxins from Bacillus thuringiensis can overcome a mosquito's resistance to B. sphaericus alone. This combination suppressed resistance of Culex quinquefasciatus to B. sphaericus from greater than 17,000- to 2-fold.

The Cyt1A protein from Bacillus thuringiensis increases the toxicity of Bacillus sphaericus toward Aedes aegypti more than 3,000-fold. This is important because even though B. sphaericus is toxic to Culex and Anopheles mosquitoes, it has little effect on A. aegypti.

Strains of the mosquito Culex quinquefasciatus with high levels of resistance to single and multiple toxins of Bacillus thuringiensis subsp. israelensis display low levels of cross-resistance to B. thuringiensis subsp. jegathesan. Moreover, the strains display moderate to high levels of cross-resistance to Cry11B, one of the toxins in B. thuringiensis subsp. jegathesan. However, the cross-resistance to Cry11B can be suppressed in two of the strains by combining Cry11B with Cyt1A.

The Cry19A polypeptide from Bacillus thuringiensis subsp. jegathesan may be effective against Culex quinguefasciatus mosquitoes that are resistant to B. thuringiensis subsp. israelensis toxins. There are no field reports of such resistance as of April, 2001, but laboratory studies show that resistance can develop.

The abilities of two mosquitocidal toxins (Mtx) of Bacillus sphaericus were analyzed. Results concluded that both the Mtx were useful in recombinant bacterial insecticides as a resistance management tool because of their toxicity and synergistic interactions with other mosquitocidal toxins.

Various experiments were reported in which separate Culex quinquefasciatus populations were selected for 20 generations to recombinant strains of Bacillus thuringiensis that produced either Cyt1Aa, Cry11Aa, or a 1:3 mixture of these strains. The results indicated that Cyt1Aa is the principal factor responsible for delaying the evolution and expression of resistance to mosquitocidal Cry proteins.