Susceptibility of Aedes aegypti larvae to temephos and Bacillus thuringiensis var israelensis in integrated control

The susceptibility of field collected Aedes aegypti larvae was evaluated in terms of median lethal time (LT50) and final mortality, when treated with temephos, Bacillus thuringiensis var israelensis as well as mixtures of these two agents. Third instar larvae were shown to be more susceptible than early and late fourth instar ones to the entomopathogen. Survival of some individuals when exposed to temephos suggest possible resistance. Temporal synergism in early fourth instar larvae was detected when they were exposed to mixtures of Bti-temephos. The possibility of this integrated treatment is commented on.

Cumulative mortality of Aedes aegypti larvae treated with compounds

OBJECTIVE To evaluate the larvicidal activity of Azadirachta indica, Melaleuca alternifolia, carapa guianensis essential oils and fermented extract of Carica papaya against Aedes aegypti (Linnaeus, 1762) (Diptera: Culicidae). METHODS The larvicide test was performed in triplicate with 300 larvae for each experimental group using the third larval stage, which were exposed for 24h. The groups were: positive control with industrial larvicide (BTI) in concentrations of 0.37 ppm (PC1) and 0.06 ppm (PC2); treated with compounds of essential oils and fermented extract, 50.0% concentration (G1); treated with compounds of essential oils and fermented extract, 25.0% concentration (G2); treated with compounds of essential oils and fermented extract, 12.5% concentration (G3); and negative control group using water (NC1) and using dimethyl (NC2). The larvae were monitored every 60 min using direct visualization. RESULTS No mortality occurred in experimental groups NC1 and NC2 in the 24h exposure period, whereas there was 100% mortality in the PC1 and PC2 groups compared to NC1 and NC2. Mortality rates of 65.0%, 50.0% and 78.0% were observed in the groups G1, G2 and G3 respectively, compared with NC1 and NC2. CONCLUSIONS The association between three essential oils from Azadirachta indica, Melaleuca alternifolia, Carapa guianensis and fermented extract of Carica papaya was efficient at all concentrations. Therefore, it can be used in Aedes aegypti Liverpool third larvae stage control programs.

Residual effect of two Bacillus thuringiensis var. israelensis products assayed against Aedes aegypti (Diptera: Culicidae) in laboratory and outdoors at Rio de Janeiro, Brazil

Resistance of the dengue vector to temephos stimulated its substitution for Bacillus thuringiensis var. israelensis (Bti) since 2001 in Brazil. The persistence of the two Bti formulations employed at that time by the Health Ministry, Vectobac G and Aquabac G, was assayed under laboratory and outdoor conditions. Both formulations were tested at 0.2 g/10 liters of water, the same concentration applied in the field for vector control. The tests were done against Ae. aegypti third instar larvae (Rockefeller strain). In the laboratory, Vectobac G and Aquabac G caused at least 95% mortality until 101 and 45 days after treatment, respectively. In the outdoor assays, test containers of different materials were treated with either formulation and placed in a shaded area. Larvae were introduced each 3-6 days and mortality was recorded 24 and 48 hours later. In the first set of assays, performed in June 2001, mortality levels of 70% or more were attained for 2-5 weeks for both formulations in all containers. The exception was for the iron one that rusted, resulting in low mortality after seven days. In the second set of assays (August 2001), 70% mortality was attained for just 1-2 weeks for all the containers and both formulations.

SUSCEPTIBILIDADE DE Anopheles triannulatus(NEIVA & PINTO, 1922) E ESPÉCIES NÃO ALVO A LARVICIDAS À BASE DE TEMEPHOS E DE Bacillus thuringiensiaH-14

O presente trabalho foi desenvolvido em uma área com ríeco de malária do Projeto Carajás (Carajás, PA). Foi avaliado um larvicida químico amplamente utilizado, o temephos, e um formulado a base da bactéria Bacillus thuringiensis H-14 (Bti), isoladamente ou em conjunto, contra larvae de Anopheles triannulatus; vetor da malária. A susceptibilidade foi expressa em termos do TL50 e da mortandade final. A mistura dos dois larvicidas mostrou sinergismo temporal, larvas de Culex rorotaensis e notonectideos também tiveram sua susceptibilidade avaliada respectivamente ao Bti e ao temephos. Discute-se a relevância dos resultados no manejo integrado de A. triannulatus.

Development of a cyanobacterial biolarvicide

Results of studies on a larvicidal cyanobacterium that expresses a Bti cryIVD gene fusion are reported. Genetically altered Agmenellum quadruplicatum PR-6 is shown to be toxic to larvae of three major genera of disease-bearing mosquitos. Factors affecting expression of Bti genes in cyanobacteria are discussed.

Biological control program against simuliids in the State of São Paulo, Brazil

In Brazil, the use of biological vector-control methods has been largely confined to experimental research, with little or no application of such techniques by public institutions responsible for implementing control programs. The notable exceptions have been the black fly control program carried out by the Health Secretariat in the State of São Paulo. Since the 1980s, São Paulo's "Superintendência de Controle de Endemias" has been conducting studies on the viability of using Bacillus thuringiensis (H-14) for simuliid control, and the results have been so encouraging that the agency has now incorporated this method into its Simuliid Control Program.

Biochemical, immunological and toxicological characteristics of the crystal proteins of Bacillus thuringiensis subsp. medellin

Characterization of the insecticidal and hemolytic activity of solubilized crystal proteins of Bacillus thuringiensis (Bt) subsp. medellin (Btmed) was performed and compared to solubilized crystal proteins of isolates 1884 of B. thuringiensis subsp. israelensis (Bti) and isolate PG-14 of B. thuringiensis subsp. morrisoni (Btm). In general, at acid pH values solubilization of the Bt crystalline parasporal inclusions (CPI) was lower than at alkaline pH. The larvicidal activity demonstrated by the CPI of Btmed indicated that optimal solubilization of CPI takes place at a pH value of 11.3, in Bti at pH values from 5.03 to 11.3 and in Btm at pH values from 9.05 to 11.3. Hemolytic activity against sheep red blood cells was mainly found following extraction at pH 11.3 in all Bt strains tested. Polyacrylamide gel electrophoresis under denaturing conditions revealed that optimal solubilization of the CPI in all Bt strains takes place at the alkaline pH values from 9.05 to 11.3. An enriched preparation of Btmed crystals was obtained, solubilized and crystal proteins were separated on a size exclusion column (Sephacryl S-200). Three main protein peaks were observed on the chromatogram. The first peak had two main proteins that migrate between 90 to 100 kDa. These proteins are apparently not common to other Bt strains isolated to date. The second and third peaks obtained from the size exclusion column yielded polypeptides of 68 and 28-30 kDa, respectively. Each peak independently, showed toxicity against 1st instar Culex quinquefasciatus larvae. Interestingly, combinations of the fractions corresponding to the 68 and 30 kDa protein showed an increased toxicity. These results suggest that the 94 kDa protein is an important component of the Btmed toxins with the highest potency to kill mosquito larvae. When crystal proteins of Bti were probed with antisera raised independently against the three main protein fractions of Btmed, the only crystal protein that showed cross reaction was the 28 kDa protein. These data suggest that Btmed could be an alternative bacterium for mosquito control programs in case mosquito larval resistance emerges to Bti toxic proteins.

Cloning, Expression and Toxicity of a Mosquitocidal Toxin Gene of Bacillus thuringiensis subsp. medellin

Bacillus thuringiensis (Bt) subsp. medellin (Btmed) produces parasporal crystalline inclusions which are toxic to mosquito larvae. It has been shown that the inclusions of this bacterium contain mainly proteins of 94, 68 and 28-30 kDa. EcoRI partially digested total DNA of Btmed was cloned by using the Lambda Zap II cloning kit. Recombinant plaques were screened with a mouse policlonal antibody raised against the 94 kDa crystal protein of Btmed. One of the positive plaques was selected, and by in vivo excision, a recombinant pBluescript SK(-) was obtained. The gene encoding the 94 kDa toxin of Btmed DNA was cloned in a 4.4 kb DNA fragment. Btmed DNA was then subcloned as a EcoRI/EcoRI fragment into the shuttle vector pBU4 producing the recombinant plasmid pBTM3 and used to transform by electroporation Bt subsp. israelensis (Bti) crystal negative strain 4Q2-81. Toxicity to mosquito larvae was estimated by using first instar laboratory reared Aedes aegypti, and Culex quinquefasciatus larvae challenged with whole crystals. Toxicity results indicate that the purified inclusions from the recombinant Bti strain were toxic to all mosquito species tested, although the toxicity was not as high as the one produced by the crystal of the Btmed wild type strain. Poliacrylamide gel electrophoresis indicate that the inclusions produced by the recombinant strain Bti (pBTM3) were mainly composed of the 94 kDa protein of Btmed, as it was determined by Western blot

Evaluation of the impact of Bacillus thuringiensis serovar israelensis and Temephos, used for the control of Simulium (Chirostilbia) pertinax Kollar, 1832 (Diptera, Simuliidae) on the associated entomofauna, Paraty, state of Rio de Janeiro, Brazil

The study was set up to evaluate the impact of two commercial larvicide formulations, Bacillus thuringiensis serovar israelensis base (Bti) at 15 ppm/1 min and temephos at 0.03 ppm of active ingredient, used to control Simulium pertinax populations, on associated non-target entomofauna occupying the same breeding sites. The experiments were carried out on the Pedra Branca and Muricana rivers, on the slopes of Serra do Mar massif, municipality of Paraty, state of Rio de Janeiro, Brazil. Bti was applied to the river Pedra Branca and temephosto theriver Muricana. On both rivers, treatment and control sections were labeled as such, each one with two observation posts: slow moving water and fast water regions respectively. Artificial substrata was used to evaluate the abundance of associated entomofauna. Attached immature stages of arthropods were removed from both of its surfaces fortnightly. Were collected, from the two rivers, 28 477 specimens of the entomofauna associated with S. pertinax. The families Hydropsychidae, Chironomidae, Bactidae, Simuliidae, Blephariceridae and Megapodagrionidae were represented. These was an impact of temephos on the entomofauna associated with S. pertinax only in Simuliidae and Chironomidae, and to Bti only in Simuliidae. However, the reduction in their numbers was not statistically significant.

Histopathological and ultrastructural effects of delta-endotoxins of Bacillus thuringiensis serovar israelensis in the midgut of Simulium pertinax larvae (Diptera, Simuliidae)

The bacterium Bacillus thuringiensis (Bt) produces parasporal crystals containing delta-endotoxins responsible for selective insecticidal activity on larvae. Upon ingestion, these crystals are solubilized in the midgut lumen and converted into active toxins that bind to receptors present on the microvilli causing serious damage to the epithelial columnar cells. We investigated the effect of these endotoxins on larvae of the Simulium pertinax, a common black fly in Brazil, using several concentrations during 4 h of the serovar israelensis strain IPS-82 (LFB-FIOCRUZ 584), serotype H-14 type strain of the Institute Pasteur, Paris. Light and electron microscope observations revealed, by time and endotoxin concentration, increasing damages of the larvae midgut epithelium. The most characteristic effects were midgut columnar cell vacuolization, microvilli damages, epithelium cell contents passing into the midgut lumen and finally the cell death. This article is the first report of the histopathological effects of the Bti endotoxins in the midgut of S. pertinax larvae and the data obtained may contribute to a better understanding of the mode of action of this bacterial strain used as bioinsecticide against black fly larvae.

Developing new approaches for detecting and preventing Aedes aegypti population outbreaks: basis for surveillance, alert and control system

A new approach to dengue vector surveillance based on permanent egg-collection using a modified ovitrap and Bacillus thuringiensis israelensis(Bti) was evaluated in different urban landscapes in Recife, Northeast Brazil. From April 2004 to April 2005, 13 egg-collection cycles of four weeks were carried out. Geo-referenced ovitraps containing grass infusion, Bti and three paddles were placed at fixed sampling stations distributed over five selected sites. Continuous egg-collections yielded more than four million eggs laid into 464 sentinel-ovitraps over one year. The overall positive ovitrap index was 98.5% (over 5,616 trap observations). The egg density index ranged from 100 to 2,500 eggs per trap-cycle, indicating a wide spread and high density of Aedes aegypti (Diptera: Culicidae) breeding populations in all sites. Fluctuations in population density over time were observed, particularly a marked increase from January on, or later, according to site. Massive egg-collection carried out at one of the sites prevented such a population outbreak. At intra-site level, egg counts made it possible to identify spots where the vector population is consistently concentrated over the time, pinpointing areas that should be considered high priority for control activities. The results indicate that these could be promising strategies for detecting and preventing Ae. aegypti population outbreaks.

Impact of water renewal on the residual effect of larvicides in the control of Aedes aegypti

This study was carried out to evaluate the residual effect of three larvicides under laboratory conditions for 100 days in Aedes aegypti. The larval mortality rate was measured without water renewal or with daily water renewal (80%). With temephos, there was 100% mortality in both groups until the 70th day. In the Bacillus thuringiensis israelensis (Bti)-WDG test, there was no difference during the first 20 days. With Bti-G, without water renewal, mortality was sustained above 90% for up to 35 days. The second experiment (with water renewal) reduced the mortality to below 90% after the first 20 days. When renewed water was provided, the residual effect was significantly lower for all larvicides.

Monitoring temporal fluctuations of Culex quinquefasciatus using oviposition traps containing attractant and larvicide in an urban environment in Recife, Brazil

The use of attractants and larvicides in oviposition traps is of practical interest for the surveillance and control of urban mosquitoes. In addition to increasing the safety of the traps, this combination is essential for an attract-and-kill control strategy based on trapping mosquito eggs. The combination of Bacillus thuringiensis var. israelensis (Bti) and grass infusion (GI) vs. GI alone were tested for their ability to attract in paired BR-OVT traps in the backyards of 10 houses in Recife, Brazil, for a period of 45 days. Results show that females prefer to oviposit in traps containing Bti (363 compared with 251 egg rafts over 45 days). Results from a one-year trial on the efficacy of BR-OVT traps loaded with GI and Bti as a sampling tool to monitor temporal fluctuations in the population densities of Culex quinquefasciatus in an urban environment are also reported. From December 2006-January 2007, one trap per home was installed and maintained for 348 consecutive days in 134-151 houses located in three urban blocks. Throughout the one-year field trial a total of 43,151 Culex egg rafts were collected in the traps. The data show that BR-OVT loaded with GI and Bti is sensitive enough to demonstrate continuous reproductive activity of Cux. quinquefasciatus in the study area throughout the year and to monitor temporal fluctuations in population density.

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.

Monitoring resistance to Bacillus thuringiensis subsp. israelensis in the field by performing bioassays with each Cry toxin separately

Bacillus thuringiensis subsp. israelensis (Bti) is increasingly used worldwide for mosquito control and is the only larvicide used in the French Rhône-Alpes region since decades. The artificial selection of mosquitoes with field-persistent Bti collected in breeding sites from this region led to a moderate level of resistance to Bti, but to relatively high levels of resistance to individual Bti Cry toxins. Based on this observation, we developed a bioassay procedure using each Bti Cry toxin separately to detect cryptic Bti-resistance evolving in field mosquito populations. Although no resistance to Bti was detected in none of the three mosquito species tested (Aedes rusticus, Aedes sticticus and Aedes vexans), an increased tolerance to Cry4Aa (3.5-fold) and Cry11Aa toxins (8-fold) was found in one Ae. sticticus population compared to other populations of the same species, suggesting that resistance to Bti may be arising in this population. This study confirms previous works showing a lack of Bti resistance in field mosquito populations treated for decades with this bioinsecticide. It also provides a first panorama of their susceptibility status to individual Bti Cry toxins. In combination with bioassays with Bti, bioassays with separate Cry toxins allow a more sensitive monitoring of Bti-resistance in the field.