Genetic sexing of Anopheles stephensi with the larval morphological mutant Bl

The development of two genetic sexing systems for Anopheles stephensi based on the visible mutant black larvae (Bl) are reported. Two Y-linked translocations, T(Y-3)20 and T(Y-3)24, induced by 5 Krads X-ray irradiation were found to have breakpoints almost completely linked to Bl, showing recombination frequencies of less than 0.05% and 0.9% respectively. These strains can be maintained as stable inbreeding populations in which males are easily selected at the late 3rd or 4th larval instar by their half-black appearance, which is distinct from the full black phenotype of the females.A third Y-linked translocation, T(Y-3)9, in which the breakpoint showed only 0.7% recombination with an adult morphological mutant, short palpi (sp) was also isolated. Linkage between the breakpoint of 5 Y-linked translocations and the DDT resistance gene locus (DDT) was established providing incentive for further studies. Only two translocations showing poor linkage between the breakpoint and the dieldrin resistance gene locus (Dl) were identified. Linkage data and cytology indicated that each of the Y-chromosone 3 translocations studied involved the 3R arm, and not 3L where Dl is located.     

Satellite DNA of Anopheles stephensi liston (Diptera: Culicidae)

Four satellite DNAs in the Anopheles stephensi genome have been defined on the basis of their banding properties in Hoechst 33258-CsCl density gradients. Two of these satellites, satellites I and II, are visible on neutral CsCl density gradients as a light density peak forming approximately 15% of total cellular DNA. Hoechst-CsCl density gradient profiles of DNA extracted from polytene tissues indicates that these satellites are underreplicated in larval salivary gland cells and adult female Malpighian tubules and possibly also in ovarian nurse cells. The chromosomal location of satellite I on mitotic and polytene chromosomes has been determined by in situ hybridisation. Sequences complementary to satellite I are present in approximately equal amounts on a heterochromatic arm of the X and Y chromosomes and are also present, in smaller amounts, at the centromere of chromosome 3. A quantitative analysis of the in situ hybridisation experiments indicates that sequences complementary to satellite I at these two sites differ in their replicative behaviour during polytenisation: heterosomal satellite I sequences are under-replicated relative to chromosome 3 sequences in polytene larval salivary gland and ovarian nurse cell nuclei.     

Dramatic developmental changes in larval knockdown response enhance genetic sexing based on DDT resistance in Anopheles stephensi (Diptera: Culicidae)

Genetic sexing systems based on a conditional lethal require good discrimination between the different phenotypes. DDT resistance in the early instars of Anopheles stephensi Liston is not a good candidate when based on mortality, but this study shows that the knockdown response gives exceptional discrimination between heterozygous resistant and homozygous susceptible individuals. One- and two-day-old larvae of the DlDDT strain showed high (417-fold) resistance to knockdown by DDT, but very low resistance to mortality (3.3-fold). This changes with the onset of the third instar, so that by the fourth instar, mortality resistance is high (108-fold) and knockdown resistance is low (6.5-fold). Susceptibility to DDT decreases from first to fourth instar in the susceptible strain by 443-fold for knockdown and 15-fold for mortality and in the resistant strain by 8.5-fold for knockdown and 491-fold for mortality. The DDT knockdown response in young larvae was successfully used to identify two Y-autosome translocations linked to the resistance gene, DDT. T(Y-3)69 and T(Y-3)72 gave recombination values between the translocation breakpoint and the DDT locus of 4.1 and 10.1 crossover units, respectively. T(Y-3)69 proved to be an adequate genetic sexing system for laboratory studies.     

Allozyme variation in Anopheles stephensi Liston from Pakistan (Diptera: Culicidae)

Allozyme variability was analyzed at 16 loci in 11 lines of Anopheles stephensi Liston from Pakistan. Six lines were considered as samples from natural populations. For these lines the mean number of alleles was 1.31-1.63, the degree of polymorphism was 0.188-0.375, the observed heterozygosity was 0.065-0.086, and the genetic distance ranged from 0.001 to 0.016. No population-specific alleles were found. Interbreeding was considerable (mean Fit = 0.183). Differences in allele frequencies were due considerable (mean Fit = 0.183). Differences in allele frequencies were due primarily to local inbreeding (Fis greater than Fst at most loci). The Lahore line, reared for more than 20 generations, had more homozygotes than the other lines. A line refractory to Plasmodium falciparum and a genetic sexing line exhibited decreased allozyme variability. The latter line showed reduced staining intensity at 10 loci. Linkage studies are recommended for the following loci with rare alleles: Acp, Gapdh, Icd-1, Icd-2, Mpi, and Pgd.     

Midgut antibodies reduce the reproductive capacity of Anopheles stephensi (Diptera: Culicidae)

Rabbits immunized with polypeptides of midgut of glucose fed A. stephensi resulted in high titer of antibodies (10(4)-10(6)) as detected by ELISA. Effect of antisera on fecundity, hatchability and engorgement was investigated. Fecundity was reduced drastically (62.4%). Eight polypeptides were recognized by the antisera raised against midgut tissues viz., 92, 85, 55, 52, 45, 38, 29 and 13 kDa. Cross reactivity of these antibodies with different tissues of A. stephensi as well as different species of Anopheles was also analyzed. The results indicated that anti-mosquito midgut antibodies had the potential to disrupt the reproductive physiology of mosquitoes in view of the present study, there is a need for further investigation with target antigens.     

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.     

Escherichia coli expressing a green fluorescent protein (GFP) in Anopheles stephensi: a preliminary model for paratransgenesis

Issues, like emerging insecticide resistance in Anopheles mosquitoes, have led to a breakdown in many vector control programs. In this study, a recombinant Escherichia coli with plasmid expressing a green fluorescent protein (E.coli-GFP) was used as a paratransgenesis model to determine: the possibility of E. coli-GFP trans-stadial transmission. The effect of the water microflora, of bacteria-impregnated sugar solutions, and of blood-feeding on E. coli-GFP colonization and localization within An. stephensi tissues, were studied. The results demonstrated the persistence of E. coli-GFP during molting and metamorphosis events and its trans-stadial transmission. Also the efficacy of bacteria-impregnated sugar solutions for transferring the bacteria to the adult mosquito’s midgut was shown. A blood meal dramatically increased the number of bacteria within 24–48 h post feeding. In addition to fluorescence microscope evaluation, GFP gene PCR amplification showed the presence of the bacteria in the midgut of larvae, pupae, and adults up to 13 days after eclosion. Massive colonization of bacteria was observed in the larvae and in the adult mosquito’s malpighian tubules which may play a role in retaining bacteria in adult mosquitos. The results of this study showed that E. coli could be used as a laboratory model in paratransgenesis studies for the evaluation of various effector molecules as anti-parasite agents for malaria and filariasis.     

Nosema algerae n. sp. (Cnidospora, Microsporida) a Pathogen in a Laboratory Colony of Anopheles stephensi Liston (Diptera, Culicidae)

SYNOPSIS. A new microsporidan, Nosema algerae n. sp., was found in a laboratory colony of Anopheles stephensi. The microsporidan infects a variety of tissues of both larvae and adults and is highly pathogenic for its host. The structure and life cycle of the microsporidan under the light and electron microscope is described and its relationship to other Nosema species infecting mosquitoes is discussed.     

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.     

A photomap of the salivary gland chromosomes of Anopheles stephensi liston (Culicidae: Diptera)

A photomap of the banding pattern of the salivary gland chromosomes of Anopheles stephensi Liston, which is first of its kind, has been prepared. The salivary chromosome complement consists of five arms, the shortest of which represents the telocentric X-chromosome, and the remaining four the autosomal arms. A comparison has been made of the banding pattern of this species with other species of the subgenus Cellia.     

Homologous banding patterns in the polytene chromosomes from the larval salivary glands and ovarian nurse cells of Anopheles stephensi liston (Culicidae)

A comparison of the banding patterns of two homologous polytene chromosome arms from the larval salivary gland and ovarian nurse cell complement of Anopheles stephensi is presented. The homologous chromosomes from the somatic larval salivary glands and germ-line derived ovarian nurse cells have essentially the same band-interband organisation. An analysis of the ³H-uridine labelling patterns of a small chromosome segment from the two tissues indicates that germ-line polytene chromosomes are not radically different from somatic polytene chromosomes in their patterns of gene expression.     

Anopheles stephensi in Africa: vector control opportunities for cobreeding An. stephensi and Aedes arbovirus vectors

Anopheles stephensi is an urban malaria vector native in some Asian countries and recently emerged in Africa as an invasive vector competent in transmitting Plasmodium falciparum and P. vivax. The coexistence of An. stephensi and Aedes arboviral vectors offers an optimal opportunity for successful integrated vector management with limited resources.     

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。     

Characterization of MboI repeat DNA sequence of Anopheles stephensi.

MboI repeat fragment of mosquito Anopheles stephensi has been isolated by molecular cloning. The restriction map and entire nucleotide sequence of the 433bp insert has been determined. Hybridization of this repeat DNA with restriction enzyme digest of mosquito DNA does not show an interspersed pattern but suggests that this repeat may be tandemly repeated at one major site and a few minor sites in the genome of Anopheles stephensi. The hybridization pattern also indicates that this repeat family comprises of many similar but non-identical sequences. An open reading frame encoding 66 amino acids with an initiation and two tandem termination codons has been identified. This putative 66 amino acid polypeptide sequence has significant homology to a small region of RNA tumour viral envelope protein.     

Heat shock response during development of the malaria vector Anopheles stephensi (Culicidae: Diptera)

The pattern of synthesis of heat shock proteins (HSP) and thermotolerance to elevated temperatures during the development of the malaria vector Anopheles stephensi normally reared at 28 +/- 2 degrees C was studied using SDS-PAGE. In total twelve heat shock proteins (i.e. 31, 33, 38, 43, 44, 51, 57, 62, 69, 71, 113 and 121 kD were induced by heat shock during various stages of development. Eight polypeptides (HSP during one or other of the instars) appeared during normal development of the adult, which showed very little response towards heat shock. Only two polypeptides (57 and 69 kD) were induced while the 22.5 kD protein disappeared during adult life. The HSP 62 and 71 kD induced during the larval stages showed a sharp decline in quantity in male and female adults upon heat shock. Three HSP (31, 43 and 44 kD) were induced in pupae due to heat shock. The synthesis of HSP in A. stephensi was correlated with the various morphological and physiological events occurring during development.