William E. Walton

Effects of vegetation management in constructed wetland treatment cells on water quality and mosquito production

The impact of three vegetation management strategies on wetland treatment function and mosquito production was assessed in eight free water surface wetland test cells in southern California during 1998–1999. The effectiveness of the strategies to limit bulrush Schoenoplectus californicus culm density within the cells was also investigated. Removing accumulated emergent biomass and physically limiting the area in which vegetation could reestablish, significantly improved the ammonia–nitrogen removal efficiency of the wetland cells, which received an ammoniadominated municipal wastewater effluent (average loading rate = 9.88 kg/ha per day NH4-N). We determined that interspersing open water with emergent vegetation is critical for maintaining the wetland’s treatment capability, particularly for systems high in NH4-N. Burning aboveground plant parts and thinning rhizomes only temporarily curtailed vegetation proliferation in shallow zones, whereas creating hummocks surrounded by deeper water successfully restricted the emergent vegetation to the shallower hummock areas. Since the hummock configuration kept open water areas interspersed throughout the stands of emergent vegetation, the strategy was also effective in reducing mosquito production. Decreasing vegetation biomass reduced mosquito refuge areas while increasing mosquito predator habitat. Therefore, the combined goals of water quality improvement and mosquito management were achieved by managing the spatial pattern of emergent vegetation to mimic an early successional growth stage, i.e. actively growing plants interspersed with open water.

Cyt1A of Bacillus thuringiensis Delays Evolution of Resistance to Cry11A in the Mosquito Culex quinquefasciatus

ABSTRACT Insecticides based on Bacillus thuringiensis subsp. israelensis have been used for mosquito and blackfly control for more than 20 years, yet no resistance to this bacterium has been reported. Moreover, in contrast to B. thuringiensis subspecies toxic to coleopteran or lepidopteran larvae, only low levels of resistance to B. thuringiensis subsp. israelensis have been obtained in laboratory experiments where mosquito larvae were placed under heavy selection pressure for more than 30 generations. Selection of Culex quinquefasciatus with mutants of B. thuringiensis subsp. israelensis that contained different combinations of its Cry proteins and Cyt1Aa suggested that the latter protein delayed resistance. This hypothesis, however, has not been tested experimentally. Here we report experiments in which separate C. quinquefasciatus populations were selected for 20 generations to recombinant strains of B. thuringiensis that produced either Cyt1Aa, Cry11Aa, or a 1:3 mixture of these strains. At the end of selection, the resistance ratio was 1,237 in the Cry11Aa-selected population and 242 in the Cyt1Aa-selected population. The resistance ratio, however, was only 8 in the population selected with the 1:3 ratio of Cyt1Aa and Cry11Aa strains. When the resistant mosquito strain developed by selection to the Cyt1Aa-Cry11Aa combination was assayed against Cry11Aa after 48 generations, resistance to this protein was 9.3-fold. This indicates that in the presence of Cyt1Aa, resistance to Cry11Aa evolved, but at a much lower rate than when Cyt1Aa was absent. These results indicate that Cyt1Aa is the principal factor responsible for delaying the evolution and expression of resistance to mosquitocidal Cry proteins.

Cyt1A from Bacillus thuringiensis Synergizes Activity of Bacillus sphaericus against Aedes aegypti (Diptera: Culicidae)

ABSTRACT Bacillus sphaericus is a mosquitocidal bacterium recently developed as a commercial larvicide that is used worldwide to control pestiferous and vector mosquitoes. Whereas B. sphaericus is highly active against larvae of Culexand Anopheles mosquitoes, it is virtually nontoxic toAedes aegypti, an important vector species. In the present study, we evaluated the capacity of the cytolytic protein Cyt1A fromBacillus thuringiensis subsp. israelensis to enhance the toxicity of B. sphaericus toward A. aegypti. Various combinations of these two materials were evaluated, and all were highly toxic. A ratio of 10:1 of B. sphaericus to Cyt1A was 3,600-fold more toxic to A. aegypti than B. sphaericus alone. Statistical analysis showed this high activity was due to synergism between the Cyt1A toxin and B. sphaericus. These results suggest that Cyt1A could be useful in expanding the host range of B. sphaericus.

Cyt1A from Bacillus thuringiensis Restores Toxicity of Bacillus sphaericus Against Resistant Culex quinquefasciatus (Diptera: Culicidae)

Abstract The 2362 strain of Bacillus sphaericus, which produces a binary toxin highly active against Culex mosquitoes, has been developed recently as a commercial larvicide. It is being used currently in operational mosquito control programs in several countries including Brazil, France, India, and the United States. Laboratory studies have shown that mosquitoes can develop resistance to B. sphaericus, and low levels of resistance have already been reported in field populations in Brazil, France, and India. To develop tools for resistance management, the Cyt1A protein of Bacillus thuringiensis subsp. israelensis De Barjac was evaluated for its ability to suppress resistance to B. sphaericus in a highly resistant population of Culex quinquefasciatus Say. A combination of B. sphaericus 2362 in a 10:1 ratio with a strain of B. thuringiensis subsp. israelensis that only produces Cyt1A reduced resistance by >30,000-fold. Resistance was suppressed completely when B. sphaericus was combined with purified Cyt1A crystals in a 10:1 ratio. Synergism was observed between the Cyt1A toxin and B. sphaericus against the resistant mosquito population and accounted for the marked reduction in resistance. However, no synergism was observed between the toxins against a nonresistant mosquito population. These results indicate that Cyt1A could be useful for managing resistance to B. sphaericus 2362 in Culex populations, and also provide additional evidence that Cyt1A may synergize toxicity by enhancing the binding to and insertion of toxins into the mosquito microvillar membrane.

Synergy between Toxins of Bacillus thuringiensis subsp. israelensis and Bacillus sphaericus

Abstract Synergistic interactions among the multiple endotoxins of Bacillus thuringiensis subsp. israelensis de Barjac play an important role in its high toxicity to mosquito larvae and the absence of insecticide resistance in populations treated with this bacterium. A lack of toxin complexity and synergism are the apparent causes of resistance to Bacillus sphaericus Neide in particular Culex field populations. To identify endotoxin combinations of the two Bacillus species that might improve insecticidal activity and manage mosquito resistance to B. sphaericus, we tested their toxins alone and in combination. Most combinations of B. sphaericus and B. t. subsp. israelensis toxins were synergistic and enhanced toxicity relative to B. sphaericus, particularly against Culex quinquefasciatus Say larvae resistant to B. sphaericus and Aedes aegypti (L.), a species poorly susceptible to B. sphaericus. Toxicity also improved against susceptible Cx. quinquefasciatus. For example, when the Cyt1Aa toxin from B. t. subsp. israelensis was added to Bin and Cry toxins, or when native B. t. subsp. israelensis was combined with B. sphaericus, synergism values as high as 883-fold were observed and combinations were 4–59,000-fold more active than B. sphaericus. These data, and previous studies using cytolytic toxins, validate proposed strategies for improving bacterial larvicides by combining B. sphaericus with B. t. subsp. israelensis or by engineering recombinant bacteria that express endotoxins from both strains. These combinations increase both endotoxin complexity and synergistic interactions and thereby enhance activity and help avoid insecticide resistance.

LARVIVOROUS FISH INCLUDING GAMBUSIA

Mosquitofish [Gambusia affinis (Baird and Girard) and G. holbrooki Girard] have been used as a biological control agent for mosquitoes for more than 100 years and, when used under appropriate conditions, remain one of the best candidates for biological control programs against mosquitoes. Mosquitofish are prolific, hardy, omnivores that are capable of rapidly producing large populations following introduction into most aquatic habitats. During the 20 century, Gambusia was introduced to more than 60 countries on nearly every continent and the 2 mosquitofish species remain the preferred larvivorous fishes (85–90% of studies) for biological control programs focused on mosquitoes (Gerberich and Laird 1985). The literature on mosquitofish is vast and numerous studies on native fishes as replacements for the mosquitofish in biological control programs have been carried out since Meisch’s (1985) and Bay’s (1985) reviews of larvivorous fishes previously used in mosquito control programs. Recent bibliographies (Haas and Pal 1984, Gerberich and Laird 1985, Ahmed et al. 1988, Legner 1995), as well as a recent publication focusing on mosquitofish culture and use in mosquito control programs (Swanson et al. 1996), illustrate the great interest and integral role that larvivorous fish fulfill in modern mosquito control programs. Ahmed et al. (1988) compiled a bibliography of 204 references related to indigenous fishes for mosquito control and 96 references for fishes for aquatic weed control. Gerberich and Laird’s (1985) review of the literature on larvivorous fishes found that more than 253 fish species have been considered for the biocontrol of mosquitoes. The great interest in Gambusia as a control agent for mosquitoes is tempered by the concerns of icthyologists and ecologists for the potential negative aspects of mosquitofish on non-target organisms and natural ecosystems (Arthington and Lloyd 1989, Meffe and Snelson 1989, Rupp 1996, Gratz et al. 1996). Regardless whether a particular fish species is used in biological control programs or stocked for recreational fisheries, adding non-native fish to ecosystems can have dramatic consequences on the fauna of some aquatic habitats (Goodman 1991, Adams et al. 2003), especially in habitats lacking fish (Wellborn et al. 1996, Hamer et al. 2002). A greater awareness of the ecological consequences of biomanipulation is needed (Rupp 1996) including both the direct effects of predation on non-target taxa and indirect trophic effects such as facilitation of other non-native organisms (Stachowicz 2001, Adams et al. 2003). This chapter provides an overview and highlights some of the important advances in our knowledge of mosquitofish and other larvivorous fishes since Meisch’s (1985) and Bay’s (1985) reviews, respectively. The reader is referred to Swanson et al. (1996) for thorough discussion of mosquitofish biology, culture systems and use of Gambusia spp. as a biological control agent for mosquitoes, and to Gerberich and Laird (1985), Ahmed et al. (1988), Legner (1995) and Hurst (2004) for bibliographies of larvivorous fishes used for mosquito control.

Mtx Toxins Synergize Bacillus sphaericus and Cry11Aa against Susceptible and Insecticide-Resistant Culex quinquefasciatus Larvae

ABSTRACT Two mosquitocidal toxins (Mtx) of Bacillus sphaericus, which are produced during vegetative growth, were investigated for their potential to increase toxicity and reduce the expression of insecticide resistance through their interactions with other mosquitocidal proteins. Mtx-1 and Mtx-2 were fused with glutathione S-transferase and produced in Escherichia coli, after which lyophilized powders of these fusions were assayed against Culex quinquefasciatus larvae. Both Mtx proteins showed a high level of activity against susceptible C. quinquefasciatus mosquitoes, with 50% lethal concentrations (LC50) of Mtx-1 and Mtx-2 of 0.246 and 4.13 μg/ml, respectively. The LC50s were 0.406 to 0.430 μg/ml when Mtx-1 or Mtx-2 was mixed with B. sphaericus, and synergy improved activity and reduced resistance levels. When the proteins were combined with a recombinant Bacillus thuringiensis strain that produces Cry11Aa, the mixtures were highly active against Cry11A-resistant larvae and resistance was also reduced. The mixture of two Mtx toxins and B. sphaericus was 10 times more active against susceptible mosquitoes than B. sphaericus alone, demonstrating the influence of relatively low concentrations of these toxins. These results show that, similar to Cyt toxins from B. thuringiensis subsp. israelensis, Mtx toxins can increase the toxicity of other mosquitocidal proteins and may be useful for both increasing the activity of commercial bacterial larvicides and managing potential resistance to these substances among mosquito populations.

Cyt1Ab1 and Cyt2Ba1 from Bacillus thuringiensis subsp. medellin and B. thuringiensis subsp. israelensis Synergize Bacillus sphaericus against Aedes aegypti and Resistant Culex quinquefasciatus (Diptera: Culicidae)

ABSTRACT The interaction of two cytolytic toxins, Cyt1Ab fromBacillus thuringiensis subsp. medellinand Cyt2Ba from Bacillus thuringiensis subsp.israelensis, with Bacillus sphaericus was evaluated against susceptible and resistant Culex quinquefasciatus and the nonsensitive species Aedes aegypti. Mixtures of B. sphaericus with either cytolytic toxin were synergistic, and B. sphaericusresistance in C. quinquefasciatus was suppressed from >17,000- to 2-fold with a 3:1 mixture of B. sphaericusand Cyt1Ab. This trait may prove useful for combating insecticide resistance and for improving the activity of microbial insecticides.

Evolution of resistance to the Bacillus sphaericus Bin toxin is phenotypically masked by combination with the mosquitocidal proteins of Bacillus thuringiensis subspecies israelensis

Two insecticidal bacteria are used as larvicides to control larvae of nuisance and vector mosquitoes in many countries, Bacillus thuringiensis ssp. israelensis and B. sphaericus. Field studies show both are effective, but serious resistance, as high as 50 000-fold, has evolved where B. sphaericus is used against Culex mosquitoes. To improve efficacy and deal with even greater potential problems of resistance, we previously developed several recombinant larvicidal bacteria that combine the best mosquitocidal proteins of these bacteria. In the present study, we report laboratory selection studies using our best recombinant strain against larvae of Culex quinquefasciatus. This recombinant, Bti/BsBin, is a strain of B. thuringiensis ssp. israelensis engineered to produce a large amount of the B. sphaericus binary (Bin) toxin, which makes it more than 10-fold as mosquitocidal as the its parental strains. Here we show that larvae exposed to Bti/BsBin failed to develop significant resistance after 30 successive generations of heavy selection pressure. The highest level of resistance obtained at the LC(95) level was 5.2-fold, but declined to less than two-fold at the 35th generation. Testing the selected populations against B. sphaericus alone showed resistance to Bin evolved, but was masked by combination with B. thuringiensis ssp. israelensis. These results suggest that recombinant bacterial strains have improved mosquito and vector management properties compared with the wild-type strains used in current commercial formulations, and should prove useful in controlling important human diseases such as malaria and filariasis on a long-term basis, even when used intensively under field conditions.

Bacterial Communities Associated with Culex Mosquito Larvae and Two Emergent Aquatic Plants of Bioremediation Importance

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 ( Culex tarsalis ), the submerged portions of two emergent macrophytes (California bulrush, Schoenoplectus californicus and alkali bulrush, Schoenoplectus maritimus ), 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.