Effects of mosquito larval feeding behavior on Bacillus sphaericus efficacy

Three species of mosquito larvae, representing three genera, were exposed from 10 min to 2 hr to the pathogen Bacillus sphaericus. Culex quinquefasciatus rapidly ingested the bacterium with resulting high mortality. Anopheles albimanus ingested it at a much lower rate initially with correspondingly low mortality. At the longest time interval they had accumulated approximately the same number of bacterial cells as C. quinquefasciatus but achieved a lower mortality. Aedes aegypti ingested and accumulated bacterium at the same rate as C. quinquefasciatus but was resistant to all time intervals. Utilizing ¹⁴C-labeled bacteria, we demonstrated that these differences were attributable to larval behavior in the case of A. albimanus but not in the case of A. aegypti.     

A radiotracer method for determining ingestion of Bacillus sphaericus by mosquito larvae

Bacillus sphaericus was labeled with carbon-14 by culturing in a medium containing U-¹⁴C-l-amino acid mixture or d-[U-¹⁴C]glucose, the former giving consistently higher labeling. Less than 5% of the label was lost during the 2-hr experimental period. Groups of mosquito larvae were exposed to labeled bacteria for variable times, washed to remove external radioactivity, and then were either analyzed by combustion and liquid scintillation counting for internal ¹⁴C or held in fresh water for mortality counts. Culex quinquefasciatus larvae rapidly ingested bacteria during the first 30 min followed by a reduced rate of accumulation. the rate and quantity of bacteria ingested was greater with later instars. The method is applicable for the study of environmental and physiological factors influencing the ingestion and susceptibility of different species of mosquito larvae to bacterial pathogens.     

Correlation between population dynamics of mosquito larvae and their habitat qualities

Surveillance of anopheline and culicine larvae was conducted fortnightly to determine population composition and density at seven sites in the area of the Mathura Refinery, Mathura, India during 2005–2006. The correlation between population of the vectors and their habitat quality was established. Temperature (0.978 and 0.85°C), pH (0.99 and 0.95), conductivity (0.98 and 0.98 mho) and calcium ion hardness (0.978 and 0.85 p.p.m.) were positively correlated with the populations of both larval species. In contrast, the correlation coefficients between the parameters total hardness, magnesium ion hardness, biochemical oxygen demand and chemical oxygen demand varied with the larval species. The anopheline larval population was dominant over the culicine population in the surveillance area. The distribution of anopheline mosquito larvae during the study period was found to be constant, and the average density of anopheline and culicine larvae was 84.70 and 15.30%, respectively, across all seven sites. Thus, the study demonstrated spatial and temporal population differences of mosquito larvae with respect to environmental factors, including water quality.     

Effects of a fire-retardant on oviposition habitat selection and larval development of the mosquito Culiseta longiareolata

1. Fire retardants are composed of fertilising salts that are commonly used for fighting wildfires. These chemicals have various effects on individual arthropod species and aquatic communities. 2. This study investigated the effects of four treatments of a prevalent fire retardant [FR CROS 134 (FR), applied at concentrations of 0, 3.8 × 10², 7.6 × 10² and 11.4 × 10² mg litre⁻¹] on oviposition habitat selection (OHS) by Culiseta longiareolata and Culex laticinctus mosquitoes in an outdoor mesocosm. Additionally, larval development of C. longiareolata was examined in response to this fire retardant. 3. The results demonstrate that OHS increased in both species, whereas survivorship (0–86.7%) and time to metamorphosis (5.4–23.1 days) of C. longiareolata were not affected by any of the treatments. However, cyanobacterial biomass increased and heterotrophic bacteria decreased in response to the addition of FR. 4. It was found that FR applications can have unintended consequences which can increase OHS by female mosquitoes. The growing use of fire retardants worldwide, and especially in Israel, justify evidence-based assessment and environmental management of their use. This study is a first critical step in filling the knowledge gap with respect to the impact of FR on aquatic ecosystems.     

Cross-resistance between strains of Bacillus sphaericus but not B. thuringiensis israelensis in colonies of the mosquito Culex quinquefasciatus

Two colonies of Culex quinquefasciatus Say (Diptera: Culicidae) were selected with Bacillus sphaericus strains C3-41 and IAB59 in the laboratory for 13 and 18 generations; they attained 145,000- and 48.3-fold resistance, respectively, in comparison with a susceptible laboratory colony (SLCq) and showed very high levels of cross-resistance (8500- to 145,000-fold) to B. sphaericus strains C3-41, 1593, 2297 and 2362. They were relatively susceptible to B. sphaericus strains LP1-G and 47-6B (only 0.8- to 2.8-fold tolerance), with 24.8- to 48.3-fold cross-resistance to strain IAB59. B. sphaericus-resistant mosquito colonies remained highly susceptible to B. thuringiensis israelensis, suggesting that B.t.i. would be of value in the management of B. sphaericus-resistant Cx. quinquefasciatus colonies. The demonstration of low or no cross-resistance of two selected resistant Cx. quinquefasciatus colonies to IAB59, LP1-G and 47-6B strains of B. sphaericus and the finding of a major 49 kDa protein in these strains suggest that there is likely to be another mosquitocidal factor in the three strains.     

Oviposition habitat selection by the mosquito, Culiseta longiareolata: effects of conspecifics, food and green toad tadpoles

Abstract. 1. Ovipositing females should choose egg-laying sites which maximize the growth and survivorship of their offspring. Accordingly, they should avoid habitats with high densities of competitors and predators and should choose sites with adequate food. We tested experimentally the oviposition response of the mosquito, Culiseta longiareolata Macquart, to extra food and two species which are both potential competitors and predators of its offspring: conspecific larvae and green toad ( Bufo viridis ) tadpoles.
2. We conducted the experiment in outdoor artificial pools where wild C. longiareolata females were presented with choices of all combinations of the presence or absence of: (1) C.longiareolata larvae, (2) B.viridis tadpoles, and (3) extra food (a mixture of ground fish food and mouse chow).
3. Oviposition rates averaged approximately 4 times greater in response to extra food.
4. The presence of the tadpoles, particularly later in the experiment when they were larger, caused a significant reduction (approximately 50%) in oviposition rate.
5. We did not detect a statistically significant effect of conspecific larvae on oviposition habitat selection.
6. Our results indicate that Culiseta females choose habitats for oviposition based on food availability. They also tend to avoid habitats containing Bufo tadpoles, presumably to avoid predation and/or competition.     

Interaction of the Bacillus sphaericus mosquito larvicidal proteins

Genes for 51.4- and 41.9-kDa insecticidal proteins of Bacillus sphaericus were separately cloned and expressed in Escherichia coli. Both proteins were required for toxicity. Approximately equal numbers of cells containing the 51.4- and 41.9-kDa proteins produced the greatest toxicity; excess 41.9-kDa protein did not affect toxicity, whereas excess 51.4-kDa protein reduced activity. Larvae were killed when 41.9-kDa protein was fed up to 24 h after the 51.4-kDa protein, but not when the order of feeding was reversed. Radiolabelled toxins bound in approximately equal amounts to the gastric caecum and posterior midgut of Culex quinquefasciatus larvae. Radiolabelled 51.4-kDa protein was rapidly degraded by ca. 12-13 kDa in the larval gut, while 41.9-kDa protein was degraded by 1-2 kDa. Nonreduced toxin extracted from B. sphaericus produced a band on SDS-PAGE of ca. 68-74 kDa that contained both 51.4- and 41.9-kDa proteins based on sequence analysis, and a band of ca. 51 kDa that contained primarily 41.9-kDa protein. Escherichia coli containing 51.4-kDa protein enhanced toxicity of the latter eluted SDS-PAGE band. These proteins may associate very strongly, and trace amounts of 51.4-kDa protein in preparations of 41.9-kDa protein from B. sphaericus may be responsible for the previously reported toxicity of the latter.     

Comparison of adult mosquito community structure on various habitats

The community structure of adult mosquitoes was compared from New Jersey light trap collections in six different types of habitats in Citrus County, Florida, USA. From October 1998 to December 2000, mosquitoes were collected three times a week from the following habitats (swamps, swamps and freshwater marshes, pine fiat-woods, pine fiat-woods and scrub, salt marshes, and salt marshes and mangroves). Mosquito density was highest in the swamps and freshwater marshes habitat, with an average of 95.65 specimens per trap.Density was lowest in the flatwoods and scrub habitat, with an average of 14.38 specimens per trap. Species dominance differed among habitats. Salt marshes produced the greatest aggregation index, while pine flatwoods produced the lowest. Conversely, diversity analysis showed that pine flatwoods had the greatest diversity, while salt marshes the lowest diversity. Similarity indices indicated that the adult mosquito communities from pine flatwoods and pine flatwoods and scrub were very similar (0.8583). The adult mosquito community of salt marshes was different from that of swamps and freshwater marshes (the similar index was 0.0217).     

Carbohydrate metabolism in the mosquito pathogen Bacillus sphaericus 2362

Bacillus sphaericus 2362 is pathogenic for mosquito larvae and is being considered for large-scale production as a larvicide. The inability of the bacteria to metabolize carbohydrates requires that they be grown on proteinaceous media. This bacterium was found to be unable to transport glucose or sucrose into the cell, and it lacked glucokinase and hexokinase activity. In addition, it lacked phosphoglucose isomerase, phosphofructokinase, and glucose 6-phosphate dehydrogenase, which are early enzymes of the Embden-Myerhof-Parnas and hexose monophosphate pathways. The presence of other enzymes in these pathways was indicated by assay, by the metabolism of glycerol to acetate, and by growth on acetate and gluconate as sole carbon sources. Critical enzymes of the Entner-Doudoroff pathway were also shown to be absent.     

Habitat stability and the larval mosquito community in treeholes and other containers on a temperate Island

The temporal fluctuation of water levels and the presence of mosquito larvae were investigated for four types of small container habitats (treeholes, bamboo stumps, cemetery stone vases, and cemetery stone vessels) on Kabeshima Island in Kyushu, southwestern Japan. The probability that containers held water was positively correlated with the quantity of the preceding rainfall and with the depth and volume of the containers. It was estimated that dehydration occurred more regularly in autumn and winter than in summer. The probability that mosquito larvae were present in each type of container was positively correlated with habitat stability in terms of the probability of the existence of standing water and the coefficient of variation of the water level. Twelve species of mosquito larvae, including two rare predators, were found. Species composition differed between the different types of container. Although 4 to 10 species used each type of container, the median number of species per container was two for treeholes and bamboo stumps, and one for the others. The dominant species wasTripteroides bambusa in treeholes and bamboo stumps,Aedes albopictus in stone vases, andA. japonicus in stone vessels. The larval mosquito community, which lacked major predators, possessed the following features that may facilitate the coexistence of many species: (1) niche segregation amongst species in terms of their selection of container types; (2) an aggregated distribution of the individual species among containers of the same type; (3) high intraspecific mean crowding (and hence probably intense intraspecific competition) in the dominant species in each type of container; (4) independent species associations within the same type of container; and (5) low interspecific mean crowding (and hence probablynot intense interspecific competition) between species in the same type of container.     

Strong habitat selection and the development of population structure in a mosquito

Summary Remarkably simple and regular patterns of genic variation for two electrophoretically assayed loci are presented against a background of strong habitat selection, population isolation, and population structuring in the pitcher-plant mosquitoWyeomyia smithii. The genic variation assessed thus far comes from 29 local populations, all but one of which lie within the glaciated region of North America. Although the conclusions drawn from these patterns are still quite tentative, it appears that the fact that the same higher frequency electromorphs occurred everywhere for both loci may be due more to selection than drift. Drift or selection, acting alone or in concert may be responsible for the almost complete absence of electromorphs of intermediate frequency. A weak association was found between the two-locus genic variation and the well-studied diapause-development time variation in this species. It is suggested that a Wrightian interaction system and fluctuating-stabilizing selection might account for maintenance of the genic level polymorphism. The observations reported are circumstantially in accord with Wright's shifting balance process of evolution. Future studies in the older parts of the species range will put these hypotheses to a strong test.     

Carbohydrate metabolism in the mosquito pathogen Bacillus sphaericus 2362.

Bacillus sphaericus 2362 is pathogenic for mosquito larvae and is being considered for large-scale production as a larvicide. The inability of the bacteria to metabolize carbohydrates requires that they be grown on proteinaceous media. This bacterium was found to be unable to transport glucose or sucrose into the cell, and it lacked glucokinase and hexokinase activity. In addition, it lacked phosphoglucose isomerase, phosphofructokinase, and glucose 6-phosphate dehydrogenase, which are early enzymes of the Embden-Myerhof-Parnas and hexose monophosphate pathways. The presence of other enzymes in these pathways was indicated by assay, by the metabolism of glycerol to acetate, and by growth on acetate and gluconate as sole carbon sources. Critical enzymes of the Entner-Doudoroff pathway were also shown to be absent.     

Ultrastructural effects of the Bacillus sphaericus mosquito larvicidal toxin on cultured mosquito cells

Crude Bacillus sphaericus extracts and purified toxin derived from these extracts caused very rapid changes in cultured Culex quinquefasciatus cells, including dilation of mitochondrial cristae, endoplasmic reticula, and Golgi secretory vesicles, and condensation of the mitochondria. The cell membrane gradually lost integrity as intoxication progressed. These observations are compared to the ultrastructure of the pathology due to Bacillus thuringiensis in cultured cells and larvae, and are discussed in relation to binding and internalization of the toxin.     

Comparison of the cellular fatty acid composition of a bacterium isolated from a human and alleged to be Bacillus sphaericus with that of Bacillus sphaericus isolated from a mosquito larvicide.

The cellular fatty acid (CFA) composition of the cytoplasmic membrane of a bacillus isolated from a human lung and deposited in the National Collection of Type Cultures as Bacillus sphaericus NCTC 11025 was determined by gas-liquid chromatography. The CFA composition of B. sphaericus 2362, isolated from a microbial larvicide, and those of B. sphaericus reference strains obtained from public collections were also determined. Samples were grouped by hierarchical cluster analysis based on the unpaired-group method using arithmetic averages. Samples that linked at a Euclidean distance of < or = 2.0 U were considered to belong to the same strain. NCTC 11025 and the type strain of B. sphaericus, ATCC 14577, were mixed; all other isolates were monotypic. The predominant fatty acid in NCTC 11025 was 12-methyltetradecanoic acid, while the predominant fatty acid in the remaining isolates was 13-methyltetradecanoic acid. NCTC 11025 linked to the other isolates at a Euclidean distance of 83.8 U, and we concluded that it belongs to a different species that we could not identify. We could distinguish among six DNA homology groups of B. sphaericus by using fatty acids. Within DNA homology group IIA, strain 2362 could be distinguished from other strains belonging to serotype H5a, 5b. We concluded that CFA analysis is a useful technique to determine if future human isolates identified as B. sphaericus in fact belong to other species of bacteria or whether the isolates originated from commercial products.