Isolation and characterization of microsatellite loci in the mosquito Anopheles stephensi Liston (Diptera: Culicidae)

The mosquito Anopheles stephensi is an important malaria vector in India, Pakistan, Iran and Afghanistan. Differences in egg morphology and chromosomal characters have been described between urban and rural forms of this mosquito but the population genetic structure remains unclear. In India this species is mainly urban, rural populations are largely zoophilic and not thought to transmit malaria. In eastern Afghanistan and the Punjab and Northwest Frontier Province, Pakistan, it is the major malaria vector. We have developed primers for 16 microsatellite loci to assist in defining the population structure and epidemiological importance of this mosquito.     

Gene flow in malaria vector Anopheles stephensi (Diptera: Culicidae) across the Aravalli Hills in North-west India

The population structure of An. stephensi in North-west India was studied to assess the impact of the Aravalli Hills, as a barrier to gene flow using microsatellite markers. Large and significant genetic differentiation was found along the sides of, as well as across, the Aravalli Hills as the mean F_ST and R_ST on west vs. east of the Aravalli Hills were 0.213, 0.112 and 0.179, 0.056, respectively. Similarly, across the hills, mean values of F_ST and R_ST were 0.100 and 0.094, respectively. Genetic diversity on both sides did not vary significantly. The F_ST values were more sensitive than R_ST values, indicating that genetic drift might have caused genetic differentiation between populations. A positive correlation (r = 0.0149 and 0.157, respective to F_ST and R_ST) was found between genetic differentiations and geographic distances irrespective of the hills. Low level of gene flow was found along both sides (Nm = 0.92 and 0.14; west vs. east of Aravalli Hills, respectively) as compared to across the Aravalli Hills (Nm = 2.25). It was found that the Aravalli Hills are not working as an effective barrier to gene flow for An. Stephensi, maybe because of the low average height and discontinuous hills, however, the distance is playing a major role for differentiation between populations due to active mode of dispersal of An. stephensi mosquitoes which have a short flight range. All this information should help draw the strategies for genetic control of mosquitoes using transgenic mosquitoes.     

Oviposition deterrent, ovicidal and gravid mortality effects of ethanolic extract of Andrographis paniculata Nees against the malarial vector Anopheles stephensi Liston (Diptera: Culicidae)

Oviposition is an important phenomenon of mosquitoes and has recently become the focus in the concept of integrated vector control management. In the present study, we evaluated oviposition deterrent, ovicidal and mortality effects of ethanolic extract of Andrographis paniculata Nees against gravid and oviposited females of Anopheles stephensi Liston. Water treated with the ethanolic extract had a high deterrent activity in ovipositing females: oviposition activity index values for the test species were –0.28, –0.45, –0.49 and –0.59 for extract concentrations of 29, 35, 41 and 46 p.p.m., respectively. High degrees of mortality were observed with various concentrations of extract: 1.12 (control) to 11.70 for gravid females, and 0.65 (control) to 10.25 for oviposited females. The highest mortality in both gravid and oviposited females was observed soon after they came in contact with oviposition medium treated with the extract, and this was found to be significant at doses higher than 35 p.p.m., suggesting possible contact toxicity of the extract. The extract caused moderate ovicidal activity against various age groups of A. stephensi, but it inflicted delayed effects such as high larval, pupal and adult mortality. The age of the eggs and the duration of the extract treatment influenced the ovicidal activity observed. It is clear that ethanolic extract of A. paniculata Nees can affect the oviposition cycle of A. stephensi Liston, thereby suppressing the vector population and adversely influencing transmission of the disease pathogen.     

Efficacy of Melia azedarach L. extract on the malarial vector Anopheles stephensi Liston (Diptera: Culicidae)

Methanolic extracts of leaves and seeds from the chinaberry tree, Melia azedarach L. (Meliaceae) was tested against mature and immature mosquito vector Anopheles stephensi Liston (Diptera) under laboratory condition. The extract showed strong larvicidal, pupicidal, adulticidal, antiovipositional activity, repellency and biting deterency. The M. azedarach seed and leaf extracts were used to determine their effect on A. stephensi adults and their corresponding oviposition and consequent adult emergence in comparison with the control. The seed extracts showed high bioactivity at all doses, while the leaf extracts proved to be active, only in the higher dose. Results obtained from the laboratory experiment showed that the seed extracts suppressed the pupal and adult activity of A. stephensi even at low dose. In general, first and second instar larvae were more susceptible to both leaves and seed extracts. Clear dose-response relationships were established with the highest dose of 2% plant extract evoking 96% mortality. Entire development of A. stephensi was inhibited by M. azedarach treatment. Less expensive (less than 0.50 US dollars per 1 kg seed), naturally accruing bio-pesticide could be an alternative for chemical pesticides.     

Effects of Dysoxylum malabaricum Bedd. (Meliaceae) extract on the malarial vector Anopheles stephensi Liston (Diptera: Culicidae)

In recent years, use of environmentally friendly and biodegradable natural insecticides of plant origin have received renewed attention as agents for disease vector control. Methanol extracts of leaves from the Indian white cedar Dysoxylum malabaricum Bedd. (Meliaceae) were tested against mature and immature Anopheles stephensi Liston (Diptera) mosquitoes under laboratory conditions. The extract showed strong larvicidal, pupicidal, adulticidal, and antiovipositional activity. The maximum leaf extract concentration tested in this study was 4%, which produced pronounced effects. In general, first and second instars were more susceptible to leaf extract than older insects. Clear dose-response relationships were established, with the highest dose of 4% plant extract causing 97% mortality of first instars.     

Larvicidal and insect growth regulator effect of α-amyrin acetate from Catharanthus Roseus Linn against the malaria vector Anopheles Stephensi Liston (Diptera: Culicidae)

Vector control is a serious concern in developing countries. Over the past two decades, phytochemicals have received progressively more attention as insecticide alternatives, and they have recently become the focus in the concept of integrated vector control. α-Amyrin acetate, the n-hexane fraction of acetone extract from the leaves of Catharanthus roseus , was evaluated for its larvicidal, pupicidal and fecundity effects as well as insect growth regulator activity against the malaria vector Anopheles stephensi Liston. The highest concentration of 1 p.p.m. produced 100% mortality in first to second instars and 94% mortality in third and fourth instars. In addition, the duration of larval instars and the total developmental time were prolonged, while female longevity and fecundity were markedly decreased. The suppression of pupation and adult emergence was probably due to its action similar to juvenile hormone analogs in combination with growth regulator activity and toxicity, which reduced the overall performance of the malaria vector An. stephensi .     

Changes in the circadian flight activity of the mosquito Anopheles stephensi associated with insemination, blood-feeding, oviposition and nocturnal light intensity

Abstract. The circadian activity patterns of Anopheles stephensi Liston were examined before and after mating and throughout the gonotrophic cycle. In LD 12:12h, males and virgin females are active at dusk. After insemination, females are active at dusk and also for short bursts during much of the night. After blood-feeding, inseminated females are inactive for two nights. On becoming gravid, these females become highly active at dusk and, to a lesser extent, are also active later in the night. In contrast, blood-feeding and egg maturation have minimal effects on the activity pattern of virgin females, who continue to fly only during dusk periods, i.e. virgins continue with the activity pattern which would most likely lead to mating encounters. After oviposition, parous females resume the activity pattern characteristic of inseminated nulliparous females. In a light regime which simulates moonlit nights and normal days, inseminated females are more active overall and flight-burst durations are much longer than in LD 12:12h.     

斯氏按蚊中Toll受体参与抵抗微生物感染和调控肠道菌群稳态

【目的】Toll信号通路是昆虫天然免疫系统的重要组分,其中Toll受体在激活昆虫病原菌侵染免疫应答方面发挥了关键作用。本研究旨在探究斯氏按蚊Anopheles stephensi Toll受体基因在抵抗微生物侵染和维持肠道菌群稳态过程中的功能。【方法】根据冈比亚按蚊Anopheles gambiae Toll受体家族的蛋白氨基酸序列,通过序列同源比对鉴定斯氏按蚊中相应的Toll受体基因;运用荧光定量PCR检测Toll受体基因在未感染病原菌的斯氏按蚊脂肪体中的相对表达量,以及在真菌球孢白僵菌Beauveria bassiana和革兰氏阴性细菌胡萝卜软腐欧文氏菌Erwinia carotovora subsp. carotovora侵染斯氏按蚊过程中的表达变化;最后,在斯氏按蚊雌成蚊胸部显微注射AsToll1A和AsToll5A的双链RNA进行RNA干扰后,检测RNAi处理的斯氏按蚊受真菌侵染后的存活率、肠道细菌含量变化以及抗菌肽基因表达变化。【结果】在斯氏按蚊中共鉴定到8个Toll受体基因,即AsToll1A, AsToll5A, AsToll6, AsToll7, AsToll8, AsToll9, AsToll10和AsToll11。通过荧光定量PCR检测发现,未感染病原菌的斯氏按蚊雌成蚊脂肪体中AsToll5A表达量最高,AsToll1A表达量次之,其余Toll受体基因表达量极低。在球孢白僵菌和胡萝卜软腐欧文氏菌侵染过程中,与对照(注射PBS)比较,AsToll1A和AsToll5A在斯氏按蚊中的表达量显著升高,其余Toll受体基因表达变化不显著或降低。RNA干扰结果表明,AsToll1A或AsToll5A的表达受到抑制后,斯氏按蚊对球孢白僵菌的抵抗能力显著降低,肠道细菌总量与对照(dsGFP)比较显著增多。而且,抑制AsToll1A后抗菌肽基因DEF1和GAM1的表达受到显著抑制;抑制AsToll5A后仅有GAM1表达量下调。【结论】斯氏按蚊Toll受体在结构和功能上具有高度的保守性,其中AsToll1A和AsToll5A能响应病原真菌和革兰氏阴性细菌侵染并且影响肠道菌稳态。     

斯氏按蚊肽聚糖识别蛋白基因PGRP-LC1的克隆及功能分析

天生免疫系统是昆虫抵御外界病原入侵的主要方式。目前研究发现, Imd信号通路与按蚊感染柏氏疟原虫Plasmodium berghei的强度密切相关, 而PGRP-LC1是Imd信号通路最上游的受体之一。为了研究斯氏按蚊Anopheles stephensi肽聚糖识别蛋白PGRP-LC1, 采用RT-PCR并结合RACE技术克隆斯氏按蚊PGRP-LC1基因, 通过序列比较分析, 得到两条cDNA序列, 其开放阅读框分别为1 365 bp和1 290 bp, 3′非编码区为320 bp, 5′非编码区为240 bp。将两条cDNA分别命名为AsPGRP-LC1a（GenBank注册号 GU214232）和AsPGRP-LC1b（GenBank注册号GU214233）。AsPGRP-LC1a编码454个氨基酸, 分子量约为49.07 kDa;AsPGRP-LC1b编码429个氨基酸, 分子量约为46.3 kDa。AsPGRP-LC1b比AsPGRP-LC1a少一个长度为75 bp的外显子, 该外显子在冈比亚按蚊Anopheles gambiae PGRP-LC1基因的某些可变剪切形式中也有发现。分别将两个斯氏按蚊PGRP-LC1基因在冈比亚按蚊细胞系L3-5和斯氏按蚊细胞系MSQ43中过量表达, 通过双荧光素酶检测系统检测抗菌肽的表达情况, 结果显示克隆得到的PGRP-LC1基因在两种细胞系中均能够启动Imd信号通路, 为进一步研究斯氏按蚊的Imd信号通路提供了依据。     

Effect of the synergist, piperonyl butoxide, on the development of deltamethrin resistance in yellow fever mosquito, Aedes aegypti L. (Diptera: Culicidae)

The larvae and adults of Aedes aegypti were tested for the potential to develop resistance to the synthetic pyrethroid, deltamethrin, alone or a combination of deltamethrin with the synergist, piperonyl butoxide (PBO). Although continuous larval selection for 40 generations resulted in 703-fold resistance, the resistance ratio in the adults was only 1.3. Similarly, adult selections with deltamethrin showed a resistance ratio of less than four after 40 generations, indicating differential response to deltamethrin selection in the two developmental stages of the insect. When the susceptible larvae were subjected to selection pressure of deltamethrin and PBO in the ratio of 1:5 for 20 generations, the speed of selection for deltamethrin resistance slowed down by 60%. The F24 larvae obtained from the strain selected with deltamethrin alone were further subjected to selection pressure with synergized deltamethrin, which resulted in 89% reversal in deltamethrin resistance in just one generation. However, long-term selection with the insecticide-synergist combination returned resistance close to original levels in 15 generations. The data indicate the involvement of cytochrome P450-dependent detoxification as the primary mechanism of development of resistance to deltamethrin in the larvae. Implications of the results on the management of larval and adult stages of Ae. aegypti are discussed.