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.     

Factors Affecting Residual Dosages of Two Formulations of Bacillus thuringiensis subsp. israelensis Tested in the Same Stream During a 3-Year Experiment

Although many field trials have been conducted using Bacillus thuringiensis subsp. israelensis (Bti)-based formulations, most have been in rivers with different biotic and abiotic conditions thus rendering the evaluation of their performance very difficult. Recently, results of a threeyear experiment using a new field procedure brought new insight into the behavior and the performance (carry) of two liquid formulations of Bti, Teknar HP-D and Vectobac 1200L, tested in the same lotic environment and under similar abiotic and biotic conditions. Factors such as discharge, water temperature and the hyporheic zone were identified as elements affecting the downstream loss of activity of both products. However, to better understand the reduction of black fly mortality along a stream (measured by using gutters), data of residual dosages of both products (measured by laboratory assay with mosquito larvae) were compared with reduction of black fly mortality. Bti toxic activity was monitored from water samples taken at different distances downstream from an application point, and from probes driven into the hyporheic zone, to study the effects of abiotic factors on the loss of the toxic crystals. Results showed that the loss of dosage was exponential for both products but more crystals were recovered from Vectobac 1200L along the stream than from Teknar HP-D. However, the latter was more efficient, i.e. less toxins were needed to kill 50% of black fly larvae both in temperate (16°C) and warmer (19.5-22°C) water. Also, a rise in water temperature had a greater effect in the kill induced by Vectobac 1200L compared to Teknar HP-D. For the same residual dosages present at the stations, longer carries of toxin activity (higher mortalities) were obtained in warmer water. Finally, the hyporheic zone was identified as a major source of loss of activity of Bti products. Large stream discharges decreased the effect of the hyporheic zone and that was reflected in longer carry of the products.     

Storage Stability of Two Liquid Formulations of Bacillus thuringiensis subsp. israelensis and Effect of Freezing Over Time

Over the last two decades, many tests have been performed in the field to investigate the behaviour (persistence, carry, loss of activity, etc.) of different Bacillus thuringiensis subsp. israelensis ( Bti ) formulations. Depending on the experimental protocols, a single container of a formulation could be used more than once over time and field samples containing Bti may have to be frozen to preserve them for bioassays to be performed later. Thus, it is necessary to know how long a formulation could keep its level of efficacy and also the effects of time on frozen samples. Our results showed that the efficacy of two commercial liquid formulations of Bti (Teknar HP-D and Vectobac 1200L) when tested against Aedes triseriatus behaved differently over time when kept at room temperature. Teknar HP-D remained stable for the first two years and its LC 50 increased by 20% the third year. For Vectobac 1200L, although its larvicidal activity was better than that of Teknar HP-D every year, there was an increase in LC 50 by 22% the second year and by another 20% the third year for a total loss of activity of 46% over the three-year study. The efficacy of suspensions made with both formulations was greatly affected by freezing and the loss of efficacy increased over time. About half of the efficacy of Teknar HP-D was lost after one week of freezing and stayed at that level for three months, while with Vectobac 1200L, no significant effect of freezing was seen after one month, when compared to fresh material. However, both products showed similar efficacy after three or six months of freezing. Overall, the LC 50 s of both products had increased by a factor of about 2.5 after six months of freezing.     

苏云金杆菌以色列亚种cryIVB基因在E.coli中的高表达

韭菜迟眼蕈蚊（Ｂｒａｄｙｓｉａｏｄｏｒｉｐｈａｇａ）幼虫以取食根部危害韭菜的生长．为了研究有效的生防制剂,采用ＤＮＡ聚合酶链式反应方法,从苏云金杆菌以色列亚种（Ｂｔｉ）得到３．５ｋｂ的开放阅读框架（ＯＲＦ）,其包括Ｂｔｉ杀虫蛋白基因ｃｒｙＩＶＢ的结构基因和部分上游启动区．将结构基因亚克隆到高表达载体ｐＱＥ５２中,构建了高表达质粒ｐＱＥ５２Ｂ．在Ｅ．ｃｏｌｉＳＧ１３００９（ｐＲＥＰ４）中,经ＩＰＴＧ诱导,１３０ｋＤ的蛋白表达量约占细胞总蛋白的７．９％,其对韭菜迟眼蕈蚊三龄幼虫具有较强的毒杀作用．为进一步利用ｃｒｙＩＶＢ基因,构建生防工程菌株打下了基础     

Genome scan in the mosquito Aedes rusticus: population structure and detection of positive selection after insecticide treatment

Identification of genes involved in local adaptation is particularly challenging for species functioning as a network of interconnected populations undergoing frequent extinctions–recolonizations, because populations are submitted to contrasted evolutionary pressures. Using amplified fragment length polymorphism markers, population genetic structure of the mosquito Aedes rusticus was analysed in five geographical areas of the French Rhône‐Alpes region. We included a number of sites that were treated with the bio‐insecticide Bacillus thuringiensis israelensis (Bti) for more than 15 years. Analysis of molecular variance revealed that most of the genetic variability was found within populations (96%), with no significant variation among geographical areas, although variation among populations within areas (4%) was significant. The global genetic differentiation index F_ST was low (0.0366 ± 0.167). However, pairwise F_ST values were significant and no isolation‐by‐distance at the regional level was observed, suggesting a metapopulation structure in this species. Bti‐treatment had no effect on genetic structure and on within‐population genetic diversity. Potential signatures of positive selection associated with Bti‐treatment were detected for five loci, even though toxicological bioassays performed on field‐collected larvae showed no significant difference in mortality between Bti‐treated and nontreated sites. The difficulty of detecting moderate resistance in field‐collected larvae together with possible differential persistence of toxins in the environment may explain our inability to detect a toxicological response to Bti in treated sites. The evidence for positive selection occurring at several genomic regions suggests a first step towards Bti resistance in natural mosquito populations treated with this bio‐insecticide. Furthermore, this signal was detectable using genomic tools before any toxicological evidence for resistance could be identified.     

供水系统摇蚊污染的微生物控制实验研究

为了研究微生物防治对水厂摇蚊污染的控制效果,测定了一种苏云金杆菌以色列亚种(Bacillus thuringiensis subsp.israelensis,Bti)制剂对摇蚊的LC50,并利用其对某水厂沉淀池孳生的摇蚊进行了现场控制试验。结果表明,Bti粉剂对摇蚊的LC50为0.318mg/L,使沉淀池中摇蚊幼虫在24h得到了有效控制,成虫在36h后得以完全控制。该制剂对摇蚊的控制时效达10周,且控制成本是采用液氯费用的1/3,采用微生物手段能使供水系统的摇蚊污染得到高效、经济的治理。     

Bacterial larvicides for vector control: mode of action of toxins and implications for resistance

Vector control can be an effective strategy to interrupt disease transmission and biolarvicides based on the entomopathogenic bacteria Bacillus sphaericus, and Bacillus thuringiensis serovar israelensis (Bti) have been successfully used to control species of public health relevance from the genera Aedes, Culex, Anopheles and Simulium. The most important feature of these agents is their ability to produce insecticidal proteins with selective action on the larval midgut. These protoxins are produced as crystals that, once ingested by larvae, are processed into active toxins, interact with receptors in the midgut epithelium and trigger cytopathological effects leading to larval death. B. sphaericus and Bti toxins share the initial steps of the mode of action; however, they interact with different midgut molecules. B. sphaericus presents a single larvicidal factor, the binary (Bin) toxin, whose action relies on the binding to one class of midgut receptors, while Bti crystals contain four protoxins (Cry4Aa, Cry4Ba, Cry11Aa and Cyt1Aa), which display interactions with multiple midgut receptors. The mode of action of B. sphaericus displays a greater potential for resistance selection, compared to Bti, and, to date, there is no record of insect resistance to the latter, contrarily to B. sphaericus. The set of mosquitocidal toxins and their interaction with midgut target sites are described in this review, as well as the implications for the potential to select resistance amongst exposed populations. These biolarvicides have specific mode of action that rely on unique interactions and make them the most selective agents to control Diptera insects actually available.     

Evaluating large‐scale effects of Bacillus thuringiensis var. israelensis on non‐biting midges (Chironomidae) in a eutrophic urban lake

1. There is continued interest in controlling non‐biting midges (chironomids) in some freshwaters because of the potential nuisance caused by emergent adults. One option is to apply Bacillus thuringiensis var. israelensis (Bti), primarily used against mosquitoes and blackflies, to control benthic chironomid larvae. Chironomids are also at risk of collateral effects where Bti is used on other targets, and such instances may increase if climate change alters the incidence of dipteran‐borne diseases. However, most research on Bti effects on chironomids is available from mesocosms or ponds and might not scale‐up to larger waterbodies. 2. We present results on the effects of Bti on larval chironomids from eight experimental treatments, over 3 years, on a newly created eutrophic, urban lake of 200 ha, Cardiff Bay. 3. The first two experimental years provided limited evidence of Bti effects, with chironomid densities reduced by up to 14%. Increased scale of application and altered experimental design in the third year revealed reductions in chironomid larval densities of around 35% following Bti treatment, with suppression lasting several months. 4. These large‐scale Bti experiments – among the largest ever undertaken on chironomids – complement previous smaller‐scale experiments in illustrating how field conditions and application methods influence Bti effects on chironomid densities. Specifically, the work reveals how near‐neutral buoyancy formulations of Bti can reduce chironomid numbers in large lakes exceeding 3 m depth, but only where treatment methods avoid over‐dispersion. We advocate further evaluation to assess whether chironomids can be suppressed over longer periods using whole‐lake application without long‐term ecological implications or excessive cost. We also suggest further consideration be given to non‐buoyant Bti formulations for use in deeper lakes.     

Tolerance to individual and joint effects of arsenic and Bacillus thuringiensis subsp,israelensis or Lysinibacillus sphaericus in Culex mosquitoes

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 000μg/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 4- 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. tarsafis. Cx. tarsafis 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.     

Factors Affecting Black Fly Larval Mortality and Carry of Two Formulations of Bacillus thuringiensis subsp. israelensis Tested in the Same Stream During a 3-Year Experiment

Over the last 20 years, many formulations of Bacillus thuringiensis subsp. israelensis (Bti) have been commercialized for the biological control of black flies in abatement programs. However, products were tested in different streams and under different environmental conditions, thus rendering the evaluation of the performance of liquid formulations of Bti very arbitrary or even impossible. Using a new field procedure, two commercial liquid formulations of Bti, Teknar HP-D and Vectobac 1200L, were for the first time tested within short time intervals in the same stream and under environmental conditions that allowed better comparison of the behavior and the performance (carry) of both products. Field results from tests using the products tested in either the same (replicate) or different environmental (abiotic) conditions were analyzed and compared. Results of the 3-year experiment showed that both water temperature and discharge had an important effect on the performance of both products (Teknar HP-D and Vectobac 1200L). In temperate water temperature (16°C) and similar discharge (70 l s -1 ), Teknar HP-D and Vectobac 1200L did not show a significant difference in their carry50 (distance where 50% of black fly larvae mortality is recorded). However, an increase in water temperature (20- 22°C) or in the discharge (250-350 l s -1 ) enhanced significantly the carry50 of both products with Vectobac 1200L having a longer carry than Teknar HP-D. Other abiotic elements such as the hyporheic zone and adsorption onto periphyton were also identified as factors affecting the behavior and the carry of the products. However, more work should be done on the loss of the residual dosages along the stream to better understand the relation between the dosages and the resulting field mortality that would lead to suggestions on how to improve Bti formulations.