Physiological and biochemical response of Aedes aegypti tolerance to Bacillus thuringiensis

Bacillus thuringiensis subsp. israelensis (Bti) represents the only eco-friendly bio-degradable insecticide for mosquito-borne disease control. Our research aims to identify if mosquito detoxification enzymes play an important role in Bti tolerance mechanisms in the dengue vector, Aedes aegypti. Several enzymes, such as amylase, cytochromes P450, Na⁺/K⁺-ATPase, acetylcholinesterase, protease and glutathione S-transferase (GST) were analysed and level of activity determined in Ae. aegypti larvae after Bti treatment. Bti exposure significantly increased the level of amylase (183.2%) as well as the activity of cytochromes P450 (177.5%), and Na⁺/K⁺-ATPase (142.9%). On the other hand, there was a decrease of 8.6% and 11.4% in acetylcholinesterase and GST activity, and no significant effect in the total level of protease activity. We suggest that the variation in amylase, cytochromes P450, Na⁺/K⁺-ATPase, acetylcholinesterase, protease and GST activity may be associated with the Bti insecticidal mechanism. This study provides the basis of detoxifying enzymes in Bti tolerance.     

Comparative study of insecticide susceptibility and activities of detoxification enzymes in larvae and adults of cotton bollworm,Heliothis armigera

Susceptibility of moths and larvae of cotton bollworm to ten different insecticides by topical application and their effect on enzymes involved in insecticide detoxification were determined. The moths were more susceptible than larvae to the insecticides tested, with the exception of pyrethroids and sulprofos. Combination of several insecticides with the synergists piperonyl butoxide (PB) and S,S,S-tributylphosphorotrithioate (TBPT) showed that lower level of carbamate and organophosphate toxicity in larvae, as compared to moths, was the result of higher detoxification enzyme activities. Studies of the post-treatment fate of ¹⁴C-labeled malathion and ³H-trans-permethrin indicated that both the cuticular penetration, internal accumulation, and excretion of applied toxicants and their metabolites occurred more rapidly in larvae than in moths. The activities in vitro of esterases, glutathione S-transferases, and monooxygenases were determined but there were no correlations with either toxicity of insecticides or synergistic effect for combination of insecticides with PB and TBPT in moths. © 1996 Wiley-Liss, Inc.     

Characterization of a new highly mosquitocidal isolate of Bacillus thuringiensis – An alternative to Bti?

The mosquito is a very important vector involved in the worldwide transmission of disease-causing viruses and parasites. Controlling the mosquito population remains one of the best means for preventing the serious infectious diseases of malaria, yellow fever, dengue, filariasis and so on and there has been an increasing interest in developing biopesticides as a useful substitute to chemical insecticides. As a result, Bacillus thuringiensis subsp. israelensis (Bti) has been extensively used due to its specificity and high toxicity to a variety of mosquito larvae. However it is prudent to seek alternatives to Bti with alternative spectra of mosquitocidal activity or that are able to overcome any resistance that might develop against Bti. The Bt S2160-1 strain was isolated from soil samples collected from Southern China and found to have a comparable mosquitocidal activity to Bti. However there were significant differences in terms of their plasmid profiles, crystal proteins produced and cry gene complement. A PCR-restriction fragment length polymorphism identification system was developed and used in order to identify novel cry-type genes and four such genes (cry30Ea, cry30Ga, cry50Ba and cry54Ba) were identified in Bt S2160-1. In conclusion, Bt S2160-1 has been identified as a potential alternative to Bti, which could be used for the control of mosquito populations in order to reduce the incidence of mosquito-borne diseases.     

Mediation of pyrethroid insecticide toxicity to honey bees (Hymenoptera: Apidae) by cytochrome P450 monooxygenases

Honey bees, Apis mellifera L., often thought to be extremely susceptible to insecticides in general, exhibit considerable variation in tolerance to pyrethroid insecticides. Although some pyrethroids, such as cyfluthrin and lambda-cyhalothrin, are highly toxic to honey bees, the toxicity of tau-fluvalinate is low enough to warrant its use to control parasitic mites inside honey bee colonies. Metabolic insecticide resistance in other insects is mediated by three major groups of detoxifying enzymes: the cytochrome P450 monooxygenases (P450s), the carboxylesterases (COEs), and the glutathione S-transferases (GSTs). To test the role of metabolic detoxification in mediating the relatively low toxicity of tau-fluvalinate compared with more toxic pyrethroid insecticides, we examined the effects of piperonyl butoxide (PBO), S,S,S-tributylphosphorotrithioate (DEF), and diethyl maleate (DEM) on the toxicity of these pyrethroids. The toxicity of the three pyrethroids to bees was greatly synergized by the P450 inhibitor PBO and synergized at low levels by the carboxylesterase inhibitor DEF. Little synergism was observed with DEM. These results suggest that metabolic detoxification, especially that mediated by P450s, contributes significantly to honey bee tolerance of pyrethroid insecticides. The potent synergism between tau-fluvalinate and PBO suggests that P450s are especially important in the detoxification of this pyrethroid and explains the ability of honey bees to tolerate its presence.     

甜菜夜蛾田间种群对甲氨基阿维菌素苯甲酸盐和高效氯氰菊酯的代谢抗性机制

甜菜夜蛾Spodoptera exigua(Hübner)云南晋宁、上海奉贤和江苏六合种群对甲氨基阿维菌素苯甲酸盐抗性为45～437倍,对高效氯氰菊酯抗性为211～555倍,对其它药剂抗性不明显。这3个田间种群3龄幼虫多功能氧化酶、谷胱甘肽S-转移酶和酯酶的活力分别为室内敏感种群的2.7～8.4倍、1.9～8.6倍和1.6～5.7倍。多功能氧化酶抑制剂PBO和酯酶抑制剂DEF对甲氨基阿维菌素苯甲酸盐的增效比为1.2～4.3和1.3～7.7;PBO和DEF对高效氯氰菊酯的增效比为1.8～58和3.6～245;谷胱甘肽S-转移酶抑制剂DEM对这2种药剂均无增效作用。上述结果表明,解毒代谢增强可能是甜菜夜蛾田间种群对甲氨基阿维菌素苯甲酸盐和高效氯氰菊酯的重要抗性机理,与酯酶和多功能氧化酶活性升高有关,与谷胱甘肽S-转移酶活性升高无关。本文的研究结果还表明,对于代谢抗性机理复杂的多抗性田间种群,根据不同解毒酶抑制剂对药剂的增效作用判断不同解毒代谢酶在抗性形成中的作用更加可靠。     

Bioinsecticide and leaf litter combination increases oviposition and reduces adult recruitment to create an effective ovitrap for Culex mosquitoes

Mosquito egg traps, aquatic habitats baited with oviposition attractant and insecticide, are important tools for surveillance and control efforts in integrated vector management programs. The bioinsecticide Bacillus thuringiensis subsp. israelensis (Bti) is increasingly used as an environmentally friendly alternative to chemical insecticides and the combination of Bti with a simple oviposition attractant like leaf litter to create an effective egg trap seems appealing. However, previous research suggests that Bti may itself alter oviposition, and that leaf litter may dramatically reduce Bti toxicity. Here we present results from field experiment designed to link the effects of litter and Bti on mosquito oviposition habitat selection and post‐colonization survival to production of adult mosquitoes. Tripling litter increased Culex spp. oviposition nearly nine‐fold, while Bti had no effect on oviposition. Neither factor altered egg survival, thus larval abundance reflected the effects of litter on oviposition. Both Bti and litter reduced larval survival by ～60%. We found no evidence that increased litter reduced Bti toxicity. Adult production was dependent upon both litter and Bti. In the absence of Bti, effects of litter on oviposition translated into three‐fold more adults. However, in the presence of Bti, initial increases in oviposition were erased by the combined negative effects of Bti and litter on post‐colonization survival. Thus, our study provides field evidence that combined litter and Bti application creates an effective ovitrap. This combined treatment had the highest oviposition and the lowest survival, and thus removed the greatest number of mosquitoes from the landscape.     

Time-of-day specific changes in pesticide detoxification ability of Spodoptera litura (Lepidoptera: Noctuidae)

Circadian clocks govern daily physiological and molecular rhythms, and putative rhythms in the expression of metabolizing xenobiotics have been described in insects. Such rhythms could have important consequences for outcomes of chemical exposures at different times of the day. The proportion of photophase (light) and scotophase (dark) also influence the enzyme activities. Several studies have been done on the mechanism of insecticide resistance in Spodoptera litura exposed to chemical insecticides. This study is aimed at understanding the circadian variations of cypermethrin detoxification mechanisms in S. litura. The toxicity of insecticide, cypermethrin exposed to three different photoperiods in 3rd instar larvae of S. litura has been investigated. Detoxification enzyme profiles of α- and β-esterases, glutathione S-transferase (GST), and cytochrome P450 (Cyt P450) were assessed. The results showed that larvae were more tolerant to cypermethrin treated larvae at 8 h L: 16 h D photoperiod as compared with two other photoperiods tested. We observed significant increases in α- and β-esterases and cyt P450 activities in 4 and 8 h at different photoperiods. GST activity was significantly changed at different photoperiods at different timings. Activities of specific detoxification enzymes fluctuated during the time, and for specific insecticides, the concentration resulting in 50% mortality varied significantly during the different photoperiods. The time of the day when chemical exposure is imposed should be an important consideration in the experimental design, and use of pesticides.     

Sustainable control of mosquito larvae in the field by the combined actions of the biological insecticide Bti and natural competitors

Integrated management of mosquitoes is becoming increasingly important, particularly in relation to avoiding recolonization of ponds after larvicide treatment. We conducted for the first time field experiments that involved exposing natural populations of the mosquito species Culex pipiens to: a) application of the biological insecticide Bacillus thuringiensis israelensis (Bti), b) the introduction of natural competitors (a crustacean community composed mainly of Daphnia spp.), or c) a combined treatment that involved both introduction of a crustacean community and the application of Bti. The treatment that involved only the introduction of crustaceans had no significant effect on mosquito larval populations, while treatment with Bti alone caused only a significant reduction in the abundance of mosquito larvae in the short‐term (within 3–10 days after treatment). In contrast, the combined treatment rapidly reduced the abundance of mosquito larvae, which remained low throughout the entire observation period of 28 days. Growth of the introduced crustacean communities was favored by the immediate reduction in the abundance of mosquito larvae following Bti administration, thus preventing recolonization of ponds by mosquito larvae at the late period (days 14–28 after treatment). Both competition and the temporal order of establishment of different species are hence important mechanisms for efficient and sustainable mosquito control.     

Insect growth regulatory and larvicidal activity of chalcones against Aedes albopictus

Widespread use of chemical insecticides has resulted in development of insect resistance and natural products with biological activities could become an attractive alternative to control insect pests. In order to find more effective insecticides for controlling mosquito, various mosquitocidal compounds are studied. Recently, juvenile hormone antagonists (JHANs) have been found to be to safe and effective insecticides for control of mosquito. In order to identify novel insecticidal compounds with JHAN activity, several chalcones were surveyed on their JHAN activities and larvicidal activities against Aedes albopictus larvae. Among them, 2′‐hydroxychalcone and cardamonin showed high levels of JHAN and mosquito larvicidal activities. These results suggested that chalcones with JHAN activity could be useful for control of mosquito larvae.     

新疆和云南番茄潜叶蛾种群对六种杀虫剂的敏感性及其与解毒酶活性的关系

【目的】番茄潜叶蛾Tuta absoluta 是新入侵我国的对番茄具有毁灭性危害的入侵害虫,目前入侵我国的番茄潜叶蛾种群对杀虫剂的抗性尚无报道。本研究旨在明确新疆和云南番茄潜叶蛾田间种群对6种常用杀虫剂的敏感性及其与解毒酶活性的关系。【方法】采用浸叶法测定6种常用杀虫剂对番茄潜叶蛾新疆和云南种群2龄幼虫的室内毒力。通过对2龄幼虫的生物测定确定3种增效剂［CYP450抑制剂胡椒基丁醚(PBO)、酯酶抑制剂磷酸三苯酯(TPP)和GST抑制剂丁烯二酸二乙酯(DEM)］对氯虫苯甲酰胺的增效作用。采用酶活性分析测定室内敏感种群和田间抗性种群(新疆种群) 2龄幼虫体内解毒酶［细胞色素P450酶(CYP450)、谷胱甘肽S-转移酶(GST)和羧酸酯酶(CarE)］活性,以确定杀虫剂抗性与解毒酶活性的关系。【结果】番茄潜叶蛾云南种群对6种杀虫剂的敏感性由高到低依次为甲维盐、溴虫腈、多杀菌素、茚虫威、氯虫苯甲酰胺和高效氯氰菊酯。新疆种群对6种杀虫剂的敏感性由高到低依次为甲维盐、溴虫腈、氯虫苯甲酰胺、多杀菌素、茚虫威和高效氯氰菊酯。与室内敏感种群相比,云南和新疆种群对氯虫苯甲酰胺的抗性水平最高,抗性倍数分别为212.7和169.3倍。生物测定结果表明,3种增效剂PBO, TPP和DEM均对氯虫苯甲酰胺无明显增效作用。酶活性测定结果表明,番茄潜叶蛾室内敏感种群和田间抗性种群之间2龄幼虫中CYP450, GST和CarE活性无显著差异。【结论】番茄潜叶蛾新疆和云南种群对测试的6种杀虫剂产生不同程度的抗性,对氯虫苯甲酰胺的抗性最高,番茄潜叶蛾对杀虫剂的抗性与解毒酶活性无关。本研究的结果对番茄潜叶蛾的田间防治和杀虫剂抗性治理具有指导意义。     

Factors influencing the toxicity of xenobiotics against larval mosquitoes

In order to examine the factors influencing xenobiotic toxicity against larval mosquitoes, the larvicidal performances of two conventional insecticides (temephos and Bacillus thuringiensis var. israelensis: Bti) and a new potential phyto-insecticide (decomposed leaf litter) were compared under different conditions against three detritivorous larval mosquito types. Bioassays performed under standard conditions indicated differential tolerance levels according to the xenobiotic and the larval type. Bioassays performed under different conditions of xenobiotic dose and geometry of the water column indicated differential effects of those parameters on mortality rates. This allowed us to distinguish the performances of temephos versus those of Bti and leaf litter. These toxicological performances were examined as indicators for analysis of xenobiotic bioavailability for mosquito larvae in environmental water, and also for their comparative interest in field mosquito control.     

Persistence of Toxic Activity and Recycling of Bacillus thuringiensis var. israelensis in Cold Water: Field Experiments Using Diffusion Chambers in a Pond

The persistence of the larvicidal activity of Bacillus thuringiensis var. israelensis (Bti) was tested over a five-month period in a low-temperature aquatic environment. Diffusion chambers filled with a suspension of Bti (100 mg l - 1) and different experimental substrates (pond water, periphyton, sediments and vegetation), but without mosquito larvae, were placed near the bottom of a large pond and removed at various intervals to measure residual toxic activity to mosquito larvae, spore concentration and proteolytic activity. Within the pond water substrate, 50% of the initial toxicity was still present after one month in cold water, while with the periphyton substrate, 30% remained in the liquid fraction after the same period of exposure. Within the vegetation substrate (blue-joint grass, Calamagrostis canadensis), an average of 30% of the initial toxicity was still present in the liquid fraction between day 84 to day 154. Solid fractions of vegetation became toxic very early and remained toxic for five months. At the end (day 154), there was still 54% of the original toxic activity put in the chambers associated with the vegetation samples. In the absence of mosquito larvae, spore recycling was observed in the chambers especially with sediments and vegetation. But spore recycling did not appear to play a major role in the observed persistence, but rather rapid absorption onto vegetation substrates was responsible for the persistence of Bti in a cold climate.     

Effects of mosquito larvae removal with Bacillus thuringiensis israelensis (Bti) on natural protozoan communities

The protein crystals produced by Bacillus thuringiensis israelensis (Bti) are used against the larvae of pestiferous flood-water mosquitoes in ephemeral wetlands. Although mosquito larvae are considered important predators on protozoans and bacteria, it is not known how a distinct reduction of mosquito larvae density in natural wetlands caused by application of Bti may indirectly affect these microbial communities. Here we show, in a large scale experiment in six natural wetlands, that the densities of heterotrophic protozoans was on an average 4.5 times higher in wetland areas treated with Bti than in control areas. In addition, the taxonomic richness of heterotrophic protozoans increased on an average of 60% in areas with Bti application compared to control areas. The increase in protozoan density and richness was fairly consistent among sites of different wetland habitats. We discuss the potential implications of our results for other parts of the ecosystem. Handling editor: K. Martens     

Production of the bioinsecticide Bacillus thuringiensis subsp. israelensis with deltamethrin increases toxicity towards mosquito larvae

Bacillus thuringiensis subsp. israelensis is a bioinsecticide used for larval mosquito control and it represents a safe alternative to chemical insecticides. Despite its environmental safety, it is less efficient and persistent than chemical insecticides. To bypass these limitations, we propose to combine the advantages of chemical and biological insecticides by producing Bti in a medium supplemented with a chemical insecticide (DDT, deltamethrin, permethrin, propoxur or temephos). Among the investigated insecticides, the addition of deltamethrin in the medium induced a higher toxicity (over 6·72‐fold) of the composite deltamethrin‐Bti towards mosquito larvae as compared to Bti alone. This was mainly due to the insertion of deltamethrin into the membranes of Bti spores, as evidenced by a quantification of membrane‐extracted deltamethrin by HPLC. This composite larvicide is a promising tool to decrease the quantity of chemicals dispersed in the environment, to increase the efficacy of Bti and to facilitate its widespread use as a transition between chemical and biological insecticides. Further experiments are required to characterize the mechanisms that underline the incorporation of deltamethrin into Bti to optimize the production and the toxicity of this composite larvicide.

Significance and Impact of the Study

This study is the first report of an increased efficacy of the mosquitocidal bioinsecticide Bacillus thuringiensis subsp. israelensis (Bti) when produced with a chemical insecticide. The results clearly demonstrate that deltamethrin is able to synergize the insecticidal activity of Bti through inclusion into spore membranes, reducing off‐target and nonspecific toxicity occurring when the chemical is used alone as sprays. This new composite chemical–biological insecticide can become an invaluable tool as an intermediate between single chemical usage and the widespread use of Bti, notably in developing countries with limited financial resources for intensive mosquito control campaigns.     

Piperonyl butoxide synergizes the larvicidal activity of Origanum vulgare essential oil and its major constituents against the larvae of Aedes albopictus and Culex pipiens quinquefasciatus

Mosquitoes bite human beings and transmit many diseases, such as malaria, dengue fever, and Zika virus. Vector control of mosquitoes is an effective strategy for reducing the spread of disease. However, extensive use of insecticides (e.g. pyrethroids and organophosphorus) has caused resistance in mosquitoes, which weakens the effectiveness of mosquito control. Phytochemicals have been considered an alternative approach to mosquito control. Essential oil (EO) was obtained from the leaves and flowers of Origanum vulgare, and its synergistic activity with piperonyl butoxide (PBO) was tested against Aedes albopictus and Culex pipiens quinquefasciatus larvae. Thirty-seven compounds were identified, among which carvacrol and thymol were two major constituents (30.73 % and 18.81 %, respectively). O. vulgare EO had a significant toxic effect against fourth-stage larvae of Cx. p. quinquefasciatus and Ae. albopictus, with LC₅₀ values of 17.51 and 75.90 mg/L. Carvacrol and thymol also each appeared to be more effective against Cx. p. quinquefasciatus (LC₅₀ = 19.30 and 11.56 mg/L, respectively) than Ae. albopictus (LC₅₀ = 26.62 and 26.66 mg/L, respectively). PBO interacted synergistically with O. vulgare EO, carvacrol and thymol with 2.60–6.26 times as much of the active compound needed without PBO as against Cx. p. quinquefasciatus and Ae. albopictus larvae. Overall, our results contribute to the development of new natural mosquito insecticides.     

Crustacean biodiversity as an important factor for mosquito larval control

Newly established ponds, which are highly dynamic systems with changing levels of biological interactions among species, are common larval mosquito habitats. We investigated the impact of crustacean abundance and taxa diversity on mosquito oviposition and larval development. The effects of the biological larvicide Bacillus thuringiensis israelensis (Bti) on mosquito larvae were monitored according to fluctuations in crustacean communities. Populations of the mosquito Culex pipiens colonized artificial ponds that contained crustacean communities at different time points of colonization by crustaceans: 1) ‘no colonization’ (no crustaceans), 2) ‘simultaneous colonization’ by crustaceans and mosquitoes, and 3) ‘head‐start colonization’ by crustaceans (preceding colonization by mosquitoes). All types of ponds were treated with three concentrations of Bti (10, 100, or 1,000 µg/liter). Colonization of all ponds by Cx. pipiens (in terms of oviposition, larval abundance, and larval development) decreased significantly with increasing diversity of crustacean taxa. The total abundance of crustaceans had a minor effect on colonization by Cx. pipiens. The presence of crustaceans increased the sensitivity of Cx. pipiens larvae to Bti treatment by a factor of 10 and delayed the time of recolonization. This effect of Bti was relevant in the short term. In the long term, the presence of Cx. pipiens was determined by crustacean biodiversity.