Evaluation of a new tablet formulation based on Bacillus thuringiensis sorovar. israelensis for larvicidal control of Aedes aegypti

The effect of sunlight on the efficacy and persistence of an experimental tablet formulation based on Bacillus thuringiensis sorovar. israelensis (C4P1) was evaluated against Aedes aegypti larvae under simulated field conditions. The initial mortality ranged from 93 to 100%, and the residual activity (> or = 70% mortality) recorded in containers exposed to sunlight or shade were, respectively, 13-35 days and 40-54 days. The results suggest that C4P1 can provide long-term larvicidal effect and operational advantages.     

Evaluation of an experimental product based on Bacillus thuringiensis sorovar. israelensis against Aedes aegypti larvae (Diptera:Culicidae)

The larvicidal activity of an experimental formulation of Bacillus thuringiensis israelensis (Bti) against Aedes aegypti larvae was evaluated under laboratory and simulated field conditions (SFC). Samples of technical powder (TP) were assayed to establish the LC₅₀ and the potency of the product. The larvicidal activity of the TP and the tablet (T) were evaluated under SFC to assess the efficacy and the residual activity, measured against Ae. aegypti larvae. Either a T or 250 mg of TP were added to 50 L of water in plastic containers. Containers were exposed to sunlight or kept in the shade. Results showed a LC₅₀ of 0.26 mg/L and a potency of 750 ITU/mg. In spite of differences in the toxicity amongst TP and T samples, all of them killed 98–100% of the larvae and the mortality remained high for six months, in the shade. The replacement of 20% or 60% of the water volume did not affect the activity of the product. Seasonal differences influenced the persistence of the product in containers exposed to sunlight. Both formulations showed an excellent performance, especially when kept in the shade. The Bti tablet evaluated in this study is potentially very useful in programs to control dengue vectors.     

The naturally derived insecticide spinosad is highly toxic to Aedes and Anopheles mosquito larvae

Spinosad is a naturally derived biorational insecticide with an environmentally favourable toxicity profile, so we investigated its potency against mosquito larvae (Diptera: Culicidae). By laboratory bioassays of a suspension concentrate formulation of spinosad (Tracer), the 24 h lethal concentration (LC50) against Aedes aegypti (L.) third and fourth instars was estimated at 0.025 p.p.m. following logit regression. The concentration-mortality response of third- and fourth-instar Anopheles albimanus Weidemann did not conform to a logit model. The LC50 value of spinosad in Anopheles albimanus was 0.024 p.p.m. by quadratic linear regression. A field trial in southern Mexico demonstrated that spinosad 1 p.p.m. compared with the standard temephos (Abate) 1% granules 100 g/m3 water prevented Ae. aegypti breeding in plastic containers of water for 8 weeks; at 10 p.p.m. spinosad prevented breeding for > 22 weeks. In another field trial, spinosad at 5 p.p.m. and temephos both completely eliminated reproduction of Ae. aegypti for 13 weeks. In contrast, the bacterial insecticide Bacillus thuringiensis var. israelensis (Bti, Vectobac) AS) performed poorly with just 2 weeks of complete inhibition of Ae. aegypti breeding. Spinosad also effectively prevented breeding of Culex mosquitoes and chironomids in both trials to a degree similar to that of temephos. We conclude that spinosad merits evaluation as a replacement for organophosphate or Bti treatment of domestic water tanks in Mesoamerica. We also predict that spinosad is likely to be an effective larvicide for treatment of mosquito breeding sites.     

Combination of Mesocyclops thermocyclopoides and Bacillus thuringiensis var. israelensis: a better approach for the control of Aedes aegypti larvae in water containers.

The efficacy of a local Thai-strain of the copepod, Mesocyclops thermocyclopoides and the larvicide, Bacillus thuringiensis var. israelensis (Bti), used jointly and singly, was studied against Aedes aegypti in water containers. In a laboratory test, copepods alone produced mortality of 98-100% in 1st instar larvae of Ae. aegypti at copepod:larvae ratios ranging from 1:1 to 1:4. In an outdoor field simulated experiment that ran for 16 wk, after a single inoculation, the treatment of copepods and Bti combined yielded the better, more sustainable results than the agents used individually. Numbers of mosquito larvae per sample in the combined treatment were zero during the first 8 wk; larval numbers then increased but were maintained at a very low level for the next 4 wk after which the larval numbers increased moderately but still remained below numbers in the control. Bti alone kept the larvae at the zero level for the first 4 wk after which their numbers increased slightly and were at low levels up to 12 wk. Copepods alone maintained larval numbers at a low level as compared with those of the control. During the course of the experiment the larval numbers in the control were greater than 20 per sample. Statistically significant differences were noted among treatment means (F = 23.083, df = 3/60, P<0.01) over the total period of the study. The number of copepods in the joint treatment was significantly higher than in the copepod alone treatment for the first 8 wk (t = -4.97, df = 14, P<0.01). The density of copepods, however, for the whole 16-wk period was not significantly different in these two treatments (t = -1.51, df = 30, P>0.1).     

Effect of sunlight on enumeration of indicator bacteria under field conditions.

The effect of sunlight on the enumeration of fecal coliform (FC) and fecal streptococcal (FS) bacteria when water samples are collected in containers and brought back to the laboratory for analysis or when the water samples are filtered through membranes on site was determined. FC and FS in raw sewage stored in clear glass or translucent polyethylene containers were resistant to the effects of sunlight. However, under the same conditions of storage and exposure to sunlight, 90% of FC and FS in sewage diluted 1:100 in seawater were inactivated within 13 to 32 min. When sewage was similarly diluted in stream water and exposed to sunlight, 90% of FC were inactivated after 28 to 38 min, whereas 90% of FS were not inactivated even after a 2-h exposure to sunlight. Other experiments showed that 90 to 99% of FC and FS retained on membranes were inactivated when these membranes were exposed to sunlight for 10 to 15 min. FS were inherently more resistant to sunlight inactivation than were FC. Finally, evidence was obtained to show that sunlight initially stresses the bacteria but eventually causes cell death.     

Enduring toxicity of transgenic Anabaena PCC 7120 expressing mosquito larvicidal genes from Bacillus thuringiensis ssp. israelensis

Persistence of biological control agents against mosquito larvae was tested under simulated field conditions. Mosquito larvicidal activity of transgenic Anabaena PCC 7120 expressing cry4Aa, cry11Aa and p20 from Bacillus thuringiensis ssp. israelensis was greater than B. thuringiensis ssp. israelensis primary powder (fun 89C06D) or wettable powder (WP) (Bactimos products) when either mixed with silt or exposed to sunlight outdoors. Reduction of Bactimos primary powder toxicity was at least 10-fold higher than Anabaena's after mixing with silt. In outdoors experiments, Bactimos WP remained toxic (over 30% mortality of 3rd instar Aedes aegypti larvae) for 2-4 days only, while transgenic Anabaena's toxicity endured 8-21 days.     

Variation in Aedes aegypti (Diptera: Culicidae) container productivity in a slum and a suburban district of Rio de Janeiro during dry and wet seasons

Seasonal variation in container productivity and infestation levels by Aedes aegypti were evaluated in two areas with distinct levels of urbanization degrees in Rio de Janeiro, a slum and a suburban neighborhood. The four most productive containers can generate up to 90% of total pupae. Large and open-mouthed containers, such as water tanks and metal drums, located outdoors were the most productive in both areas, with up to 47.49% of total Ae. aegypti pupae collected in the shaded sites in the suburban area. Water-tanks were identified as key containers in both areas during both the dry and rainy seasons. Container productivity varied according to seasons and urbanization degree. However, the mean number of pupae per house was higher in the suburban area, but not varied between seasons within each area (P > 0.05). High infestation indexes were observed for both localities, with a house index of 20.5-21.14 in the suburban and of 9.56-11.22 in the urban area. This report gives potential support to a more focused and cost-effective Ae. aegypti control in Rio de Janeiro.     

Comparison of the insecticide susceptibilities of laboratory strains of Aedes aegypti and Aedes albopictus

A susceptible strain of Aedes albopictus derived from the Gainesville strain (Florida, USA) was established in our laboratory. The larvicidal efficacies of the neurotoxic insecticides temephos, permethrin and the pure cis and trans-permethrin isomers and the microbial insecticide Bacillus thuringiensis israelensis (Bti) against Ae. albopictus were estimated and compared to a susceptible strain of Aedes aegypti. The larvicidal effect of insect growth regulator pyriproxyfen was also evaluated in both mosquito strains. The median lethal concentration/median emergency inhibition values for Ae. aegypti and Ae. albopictus, respectively, were: temephos, 3.058 and 6.632 ppb, permethrin, 3.143 and 4.933 ppb, cis-permethrin, 4.457 and 10.068 ppb, trans-permethrin, 1.510 and 3.883 ppb, Bti, 0.655 and 0.880 ppb and pyriproxyfen, 0.00774 and 0.01642 ppb. Ae. albopictus was more tolerant than Ae. aegypti to all six larvicides evaluated. The order of susceptibility for Ae. aegypti was pyriproxyfen > Bti > trans-permethrin > temephos > permethrin > cis-permethrin and for Ae. albopictus was pyriproxyfen > Bti > trans-permethrin > permethrin > temephos > cis-permethrin. Because both species can be found together in common urban, suburban and rural breeding sites, the results of this work provide baseline data on the susceptibility of Ae. albopictus to insecticides commonly used for controlling Ae. aegypti in the field.     

Evaluation of Mesocyclops annulatus (Copepoda: Cyclopoidea) as a control agent of Aedes aegypti (Diptera: Culicidae) in Argentina

We evaluated the potential of Mesocyclops annulatus as a control agent of Aedes aegypti in La Plata city (Argentina). Mosquito larval survivorship due to predation by these copepods was estimated at weekly intervals during the oviposition period of A. aegypti. Mean weekly A. aegypti larval survivorship in cylindrical plastic containers (12 cm height and 11 cm diameter) with copepods was significantly lower than in control containers. Furthermore, weekly larval survival was negatively correlated with M. annulatus adult density, and approximately 23 adult copepods/container would be a threshold density over which the weekly mosquito larval survivorship approached zero. The copepods were able to persist in all containers during approximately 100 days (in three of them until the end of the experiment: 155 days) without the resource represented by A. aegypti larvae. The predation and persistence observed suggest that M. annulatus is a potential control agent to be considered in biological control programs.     

Effects of elevated CO2 and vascular plants on evapotranspiration in bog vegetation

We determined evapotranspiration in three experiments designed to study the effects of elevated CO₂ and increased N deposition on ombrotrophic bog vegetation. Two experiments used peat monoliths with intact bog vegetation in containers, with one experiment outdoors and the other in a greenhouse. A third experiment involved monocultures and mixtures of Sphagnum magellanicum and Eriophorum angustifolium in containers in the same greenhouse. To determine water use of the bog vegetation in July–August for each experiment and each year we measured water inputs and outputs from the containers. We studied the effects of elevated CO₂ and N supply on evapotranspiration in relation to vascular plant biomass and exposure of the moss surface (measured as height of the moss surface relative to the container edge). Elevated CO₂ reduced water use of the bog vegetation in all three experiments, but the CO₂ effect on evapotranspiration interacted with vascular plant biomass and exposure of the moss surface. Evapotranspiration in the outdoor experiment was largely determined by evaporation from the Sphagnum moss surface (as affected by exposure to wind) and less so by vascular plant transpiration. Nevertheless, elevated CO₂ significantly reduced evapotranspiration by 9–10% in the outdoor experiment. Vascular plants reduced evapotranspiration in the outdoor experiment, but increased water use in the greenhouse experiments. The relation between vascular plant abundance and evapotranspiration appears to depend on wind conditions; suggesting that vascular plants reduce water losses mainly by reducing wind speed at the moss surface. Sphagnum growth is very sensitive to changes in water level; low water availability can have deleterious effects. As a consequence, reduced evapotranspiration in summer, whether caused by elevated CO₂ or by small increases in vascular plant cover, is expected to favour Sphagnum growth in ombrotrophic bog vegetation.     

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.     

INCREASE IN LARVAL GUT PROTEOLYTIC ACTIVITIES AND Bti RESISTANCE IN THE DENGUE FEVER MOSQUITO

The bioinsecticide Bacillus thuringiensis var. israelensis (Bti) is increasingly used worldwide for mosquito control. Although no established resistance to Bti has been described in the field so far, a resistant Aedes aegypti strain (LiTOX strain) was selected in the laboratory using field‐collected leaf litter containing Bti toxins. This selected strain exhibits a moderate resistance level to Bti, but a high resistance level to individual Cry toxins. As Bti contains four different toxins, generalist resistance mechanisms affecting mosquito tolerance to different toxins were expected in the resistant strain. In the present work, we show that the resistant strain exhibits an increase of various gut proteolytic activities including trypsins, leucine‐aminopeptidases, and carboxypeptidase A activities. These elevated proteolytic activities resulted in a faster activation of Cry4Aa protoxins while Cry4Ba or Cry11Aa were not affected. These results suggest that changes in proteolytic activities may contribute to Bti resistance in mosquitoes together with other mechanisms.     

Serum concentrations of melatonin in prepubertal or postpubertal gilts exposed to artificial lighting or sunlight

To determine if the type of environmental lighting or reproductive status influences secretory patterns of serum melatonin, gilts were exposed to artificial light or full sunlight during the summer months. In Experiment 1, eight prepubertal and eight postpubertal gilts (Hampshire x Yorkshire x Duroc) were exposed to light intensity of 700 lux in an environmentally controlled room from 0730 to 1900 h daily. An additional eight prepubertal and eight postpubertal gilts were reared outdoors in an open modified-front gestation building and fed on a concrete apron outdoors where light intensity approached 50,000 lux in full sunlight. After 2 mo of acclimating to these environmental conditions, blood samples were drawn from each gilt at 2-h intervals from 1000 to 0200 h. Serum concentrations of melatonin were assayed utilizing Guildhay antisera. The experiment was repeated during the same months of the following year utilizing different gilts (Experiment 2). During both replications, neither light intensity nor reproductive status affected the secretory patterns of melatonin during the sampling period (P >0.05). In both prepubertal and postpubertal gilts, serum concentrations of melatonin were not reduced (P >0.05) by exposure to direct sunlight. Since baseline concentrations of serum melatonin were not reduced by sunlight during the day, the incidence of nocturnal rises of melatonin was not increased (P >0.05) in either prepubertal or postpubertal gilts.     

Construction and characterization of a recombinant Bacillus thuringiensis subsp. israelensis strain that produces Cry11B

The mosquitocidal bacterium Bacillus thuringiensis subsp. israelensis (Bti) produces four major endotoxin proteins, Cry4A, Cry4B, Cry11A, and Cyt1A, and has toxicity in the range of many synthetic chemical insecticides. Cry11B, which occurs naturally in B. thuringiensis subsp. jegathesan, is a close relative of Cry11A, but is approximately 10-fold as toxic to Culex quinquefasciatus. To determine whether the addition of Cry11B to Bti would improve its toxicity, we produced this protein in Bti. High levels of Cry11B synthesis were obtained by expression of the cry11B gene under the control of cyt1A promoters and the STAB-SD sequence. This construct was cloned into the shuttle vector pHT3101, yielding the derivative plasmid pPFT11Bs, which was then transformed by electroporation into acrystalliferous (4Q7) and crystalliferous (IPS-82) strains of Bti. Synthesis of Cry11B in Bti 4Q7 produced crystals approximately 50% larger than those produced with its natural promoters without STAB-SD. However, less Cry11B was produced per unit culture medium with this construct than with the wild-type construct, apparently because the latter construct produced more cells per unit medium. Nevertheless, the Bti IPS-82 strain that produced Cry11B with pPFT11Bs was twice as toxic as the parental IPS-82 strain (LC(50) = 1.4 ng/ml versus 3.3 ng/ml, respectively) to fourth instars of C. quinquefasciatus. Against fourth instars of Aedes aegypti, no statistically significant difference between parental Bti IPS-82 (LC(50) = 4.7 ng/ml) and the Bti IPS-82 recombinant producing Cry11B (LC(50) = 3.5 ng/ml) was found in toxicity.     

Sequential release and residual activity of temephos applied as sand granules to water-storage jars for the control of Aedes aegypti larvae (Diptera: Culicidae).

Two long-term experiments were carried out on the release profile and efficacy of temephos 1% GR (sand granules) against Aedes aegypti larvae in water-storage containers. In the first experiment, the efficacy of temephos 1% GR enclosed and tied in a muslin cloth and placed in water at the bottom of 200 L earthen water-storage jars was studied by exposing the packets for four to nine wk in one set ofjars and then transferring them sequentially to new sets ofjars four times successively. Temephos released slowly from the granules, the magnitude of release being adequate in the initial period of two to three wk after treatment. Following this period, the efficacy of the granules increased substantially where 92-100% inhibition of emergence even at the lowest dosage of 1 g/100 L (0.05 mg/L AI) was obtained for about another five mo or longer. On removal of the packets from a given set of jars, the released residues remaining in the jars and water lasted a maximum of one to six wk post-removal depending on the magnitude of prior release into the jars. This experiment provided clear evidence that temephos is released slowly over a long period of time in water-storage jars. In the second experiment, we compared the efficacy of temephos 1% GR at 1 and 10 g (0.05 and 0.5 mg/L AI) per 200-L water in jars painted and unpainted on the inside. The efficacy in the painted jars, although high, was consistently lower than that in the unpainted jars, where 99-100% control of larvae was achieved at both rates for a minimum of five mo after treatment. On the basis of this experimental evidence, it is desirable to study the efficacy of lower dosages of temephos than those currently used in Ae. aegypti control programs. The use of controlled release formulations or sachets that are retrievable during cleaning and washing will be more practical and desirable. Both of these interventions will make the program more cost effective.     

In vitro assay for testing the toxicity of Bacillus thuringiensis subsp. israelensis delta endotoxin using blood agarose plates

An in vitro assay system that used erythrocytes was developed to assess the toxicity of delta endotoxin of Bacillus thuringiensis subsp. israelensis (Bti), a mosquito larvicidal agent. This endotoxin was activated at high alkaline pH without causing alkaline injury to the erythrocytes. The assay is carried out on blood agarose plates, prepared in MOPS-buffered saline (0·01 mol 1^-1), pH 7·0. The threshold dose for toxicity of Bti HD-567 was 8 ng protein and that of Bti H-14 and IPS-82 standard were 6 ng and 4 ng protein respectively. The assay is rapid, sensitive, quantitative and applicable to different Bti formulations to assess the toxicity of the products. Only the activated toxin is detected by this method and has good correlation with insect bioassay. The percentage toxicity dose of different Bti preparations agrees well with percentages of LC₅₀ values when the second instar larvae of Aedes aegypti were tested.     

Optimization of medium composition for the production of mosquitocidal toxins from Bacillus thuringiensis subsp. israelensis

Optimization of chicken feather (CF) based culture medium for the production of Bacillus thuringiensis subsp. israelensis (Bti) biomass in combination with the agro industrial by-product (coconut cake, CC) and manganese chloride (MnCl2) has been evaluated. The biomass yield of Bti spore/crystal toxin was highest (12.06 g/L) from the test medium (CF+CC+MnCl2) compared to the reference medium (Luria Bertani, LB). Toxicity assay with Bti produced from the test medium against mosquito vectors (Culex quinquefasciatus, Anopheles stephensi and Aedes aegypti) was also satisfactory and results were comparable with bacteria produced from LB. The results suggest that Bti can be produced to the maximum extent possible as a potential mosquitocidal activity as suggested by the test medium (CF+CC+MnCl2).     

Novel fermentation media for production of Bacillus thuringiensis subsp. israelensis

The production of Bacillus thuringiensis subsp. israelensis (deBarjac) (Bti) as a biopesticide is not cost-effective using existing fermentation technology. In this study, we explored the use of several less expensive alternative culture media (potato, common sugar, and Bengal gram) for the growth and production of Bti. Growth was obtained in all tested media and was comparable to that obtained in conventional medium (Luria-Bertani). Toxicity assays showed that the toxin produced from the novel growth media were effective in killing larvae of Culex quinquefasciatus, Anopheles stephensi, and Aedes aegypti and toxicity was comparable to that produced from Luria-Bertani medium. These observations suggest that potato can be used as a cheap source of culture medium for the production of Bti toxin in mosquito control programs.