Breeding of Anopheles mosquitoes in irrigated areas of South Punjab, Pakistan

As part of investigations on potential linkages between irrigation and malaria transmission, all surface water bodies in and around three villages along an irrigation distributary in South Punjab, Pakistan, were surveyed for anopheline mosquito larvae (Diptera: Culicidae) from April 1999 to March 2000. Samples were characterized according to exposure to sunlight, substratum, presence of vegetation, fauna, inorganic matter and physical water condition (clear/turbid/foul). Also water temperature, dissolved oxygen (DO), electroconductivity (EC) and pH of sites were recorded. A total of 37982 Anopheles larvae of six morphological types were collected from 2992 samples taken from irrigation/agricultural and village/domestic aquatic habitats. Anopheles subpictus Grassi sensu lato was by far the most abundant (74.3%), followed by An. culicifacies Giles s.l. (4.1%), An. stephensi Liston s.l. (2.6%), An. pulcherrimus Theobald (1.8%), An. peditaeniatus Leicester (0.3%) and An. nigerrimus Giles (0.1%). The four most abundant species were significantly associated with waterlogged fields and communal village drinking-water tanks. Habitat characteristics most correlated with occurrence of anophelines were the physical water condition and the absence/presence of fauna, particularly predators. Occurrence and abundance of Anopheles immatures were not significantly correlated with water temperature, DO, EC or pH. Malaria vectors of the Anopheles culicifacies complex occurred at relatively low densities, mainly in irrigated and waterlogged fields. In South Punjab, where rainfall is very low, it should be possible to reduce anopheline breeding through water management, as larvae develop mainly in water bodies that are directly or indirectly related to the extensive canal-irrigation system.     

Pyrethroid-sprayed tents for malaria control

Field trials were undertaken in the North West Frontier Province of Pakistan to determine the effects of pyrethroid-sprayed tents on feeding success, mortality and biting-rates of wild mosquitoes attracted to bait cows confined within the tents. Under natural conditions, endophagic mosquitoes rested only briefly in untreated tents during the night, followed by complete exodus at dawn. In tents sprayed on the interior surface with permethrin 0.5 mg/m² or with deltamethrin 0.03 g/m² the biting rate of Anopheles stephensi was reduced by about 40%; detergency against culicines and other anophelines was much less. Mortality-rates of bloodfed mosquitoes from the treated tents were 75%An.stephensi, 65%An. subpictus but only 10% of culicines. Outer fly-sheets prolonged the effective life of the treatment; bioassays on the sprayed inner-sheets showed that insecticidal efficacy remained high for over a year, whereas on tents without fly-sheets permethrin residual efficacy declined rapidly 20–40 weeks post-treatment. It is concluded that tent-spraying with fast-acting photostable residual pyrethroid insecticide would probably provide effective protection against malaria transmission for the inhabitants of tents in any part of the world where the vector mosquitoes are endophilic and susceptible to pyrethroids.     

Disappearance of malaria vector Anopheles sundaicus from Chilika Lake area of Orissa State in India

Malaria has declined around Chilika Lake (85°20′ E, 19°40′ N) in Orissa State, India, from hyperendemicity in the 1930s to hypoendemicity during recent decades. Six decades ago, 21 spp. of Anopheles mosquitoes (Diptera: Culicidae) were recorded from this area, including the well known Indian malaria vectors An. culicifacies Giles, An. fluviatilis James, An. maculatus Theobald, An. stephensi Liston and An. sundaicus (Rodenwaldt), the last formerly regarded as the main vector locally. Surveys of Chilika area during 1995–96 found 8 spp. of culicine plus 14 spp. of anopheline mosquitoes, the latter comprising An. subpictus Grassi sensu lato, An. hyrcanus (Pallas) s.l., An. vagus Dönitz, An. annularis van der Wulp s.l., An. culicifacies Giles s.l., An. aconitus Dönitz, An. varuna Iyengar, An. barbirostris van der Wulp s.l., An. philippinensis Ludlow, An. ramsayi Covell, An. jeyporiensis James, An. pallidus Theobald, An. tessellatus Theobald and An. karwari James in decreasing order of abundance. Among indoor‐resting female mosquitoes, the anthropophilic index was 4–7% and some species (An. culicifacies, An. subpictus, An. vagus) tended to enter houses for resting after blood‐feeding outside. Females of potentially infective age (three‐parous) were obtained for An. culicifacies (11%) and An. annularis (< 2%), the more abundant established vector in this coastal area, but not for small samples of An. subpictus and An. vagus. Anophelines reported previously but not found in our survey were An. fluviatilis, An. jamesii Theobald, A. maculatus, An. splendidus Koidzumi, An. stephensi, An. theobaldi Giles and the former main vector An. sundaicus.     

Surveillance of vivax malaria vectors and civilian patients for malaria high-risk areas in northern Gyeonggi and Gangwon Provinces near the demilitarized zone,Republic of Korea, 2003–2006

After re-emergence of malaria in 1993, a continued increase in Plasmodium vivax cases was observed from 1993 to 2006 in northern Gyeonggi and Gangwon Provinces adjacent to the demilitarized zone separating North from South Korea. Annual parasite incidence per 1000 people ranged from 0.33 in 2004 to 0.89 in 2006. While malaria case rates declined (22.6%) in 2004, they increased 75.1% in 2005 and 51.7% in 2006 from the previous years. An initial incorrect diagnosis of 46.8% of malaria cases as common cold resulted in a mean delay of 1.3 days for the detection malarial parasites. Of the total cases, 10.2% from December to May were due to latent intrinsic incubation infections acquired the previous malaria season and the rest of the cases from June to November were either latent or short incubation infections. Overall, the peak anopheline population occurred from July to September, resulting in a similar peak in malaria cases. While malaria cases increased during 2005–2006, anopheline populations, based on trap indices, were not significantly different during 4 years of surveillance. To decrease the malaria patient infective period to mosquitoes, public health centers in Paju and Cheorwon in 2006 prescribed chloroquine + primaquine at days 0–3 after initial malaria diagnosis followed by an additional 11 days of primaquine (early primaquine treatment), rather than chloroquine on days 0–3 and primaquine on days 4–17 (delayed primaquine treatment). A reduction in the malaria parasite incidence during 2007 was recorded for the two locations offering the early primaquine treatment relative to other locations using the delayed primaquine treatment.     

Biting and resting behaviour of anophelines in western Venezuela and implications for control of malaria transmission

A study was conducted in three villages in western Venezuela between February 1988 and October 1989 to determine the biting and resting activity of anophelines in relation to human habits, rainfall and fenitrothion spraying of houses. Mosquitoes were collected landing on humans inside and outside experimental huts throughout the night. Only three mosquitoes were found resting in the huts in the morning, but 2470 were collected resting on vegetation in the early morning. The collections yielded eleven species of anopheline, the most abundant being Anopheles nuneztovari, comprising over 75% of the total anophelines collected, followed by An.triannulatus, An.albitarsis s.l. and An.oswaldoi. The four most abundant species showed different diel patterns of biting. For An.nuneztovari the peak of activity was close to midnight indoors and outdoors, for An.triannulatus between 19.00 and 20.00 hours outdoors, for An.albitarsis mainly before midnight indoors and outdoors and for An.oswaldoi outdoors at 19.00 hours, with an additional smaller peak indoors at midnight. Most of the human population use bednets, go to bed before 22.00 hours and are therefore most exposed to mosquitoes that bite outdoors early in the night. Fenitrothion house-spraying failed to prevent large mosquito populations developing in the wet season, presumably because of their exophilic resting habits. The possible advantages of impregnation of existing bednets with pyrethroids, and provision of nets for people who do not have them, are discussed.     

The impact of permethrin-impregnated bednets on malaria vectors of the Kenyan coast

Abstract. The effects of introducing permethrin-impregnated bednets on local populations of the malaria vector mosquitoes Anopheles funestus and the An.gambiae complex was monitored during a randomized controlled trial at Kilifi on the Kenyan coast. Pyrethrum spray collections inside 762 households were conducted between May 1994 and April 1995 after the introduction of bednets in half of the study area. All-night human bait collections were performed in two zones (one control and one intervention) for two nights each month during the same period. PCR identifications of An.gambiae sensu lato showed that proportions of sibling species were An.gambiae sensu stricto > An.merus > An.arabiensis.
Indoor-resting densities of An.gambiae s.l. and the proportion of engorged females decreased significantly in intervention zones as compared to control zones. However, the human blood index and Plasmodium falciparum sporozoite rate remained unaffected. Also vector parous rates were unaltered by the intervention, implying that survival rates of malaria vectors were not affected. The human-biting density of An.gambiae s.l. , the predominant vector, was consistently higher in the intervention zone compared to the control zone, but showed 8% reduction compared to pre-intervention biting rates - versus 94% increase in the control zone.
Bioassay, susceptibility and high-performance liquid chromatography results all indicated that the permethrin content applied to the nets was sufficient to maintain high mortality of susceptible vectors throughout the trial. Increased rates of early outdoor-biting, as opposed to indoor-biting later during the night, were behavioural or vector composition changes associated with this intervention, which would require further monitoring during control programmes employing insecticide-treated bednets.     

Morphological and chromosomal descriptions of new species in the Anopheles subpictus complex

Abstract. Anopheles subpictus Grassi is shown to comprise four reproductively distinct species, designated A, B, C and D, occurring sympatrically in villages of Pondicherry, southeast India.
Adult females were reared individually from wild larvae and examined for their morphological and chromosomal characters. Paracentric fixed inversions on the X-chromosome serve to distinguish the species cytogenetically, with no inversion heterozygotes (i.e. no interspecific hybrids) among totals of 717 species A (X+^a,+^b), 1863 species B (Xa, b), 869 species C (Xa,+^b) and 1365 species D (X +^a,b) identified.
Morphologically, diagnostic characters for each of the four species are seen in the egg float ridge number, larval mesothoracic seta 4, pupal seta 7-1 and the palpi of female adults. Species A. C and D immatures inhabit freshwater, whereas the malaria vector species B breeds in saltwater and was found only in coastal villages.     

Egg-float ridge number in Anopheles stephensi: ecological variation and genetic analysis

Eight Indian laboratory stocks of Anopheles stephensi Liston could be grouped into three categories with, respectively, 14-22, 12-17 and 9-15 ridges on the egg-floats. The mode number of ridges among the eggs laid by individual females in these stocks was 16-19, 13-16 and 10-14, respectively. The category with the highest egg-float ridge number corresponded with the type-form and the lowest with var. mysorensis Sweet and Rao; the new egg-float category with ridge number modes of thirteen to sixteen was designated as 'intermediate'. All three forms, i.e. type-form, intermediate and myosorensis were observed in semi-urban areas while only intermediate and mysorensis were seen in rural areas. Breeding experiments indicated no post-copulatory barriers between the populations. Likelihood analysis of the results of crosses and back crosses indicated that variation in ridge number is controlled by more than one genetic factor. The stocks with different ridge numbers are best considered as 'ecological variants'.     

Pteridine fluorescence for age determination of Anopheles mosquitoes

The age structure of mosquito populations is of great relevance to understanding the dynamics of disease transmission and in monitoring the success of control operations. Unfortunately, the ovarian dissection methods currently available for determining the age of adult mosquitoes are technically difficult, slow and may be of limited value, because the proportion of diagnostic ovarioles in the ovary declines with age. By means of reversed-phase HPLC this study investigated the malaria vectors Anopheles gambiae and An. stephensi to see if changes in fluorescent pteridine pigments, which have been used in other insects to determine the age of field-caught individuals, may be useful for age determination in mosquitoes. Whole body fluorescence was inversely proportional to age (P < 0.001, r2 > 91%) up to 30 days postemergence, with the regression values: y = 40580-706x for An. gambiae, and y = 52896-681x for An. stephensi. In both species the main pteridines were 6-biopterin, pterin-6-carboxylic acid and an unidentified fluorescent compound. An. gambiae had only 50-70% as much fluorescence as An. stephensi, and fluorescent compounds were relatively more concentrated in the head than in the thorax (ratios 1:0.8 An. gambiae; 1:0.5 An. stephensi). The results of this laboratory study are encouraging. It seems feasible that this simpler and faster technique of fluorescence quantification could yield results of equivalent accuracy to the interpretation of ovarian dissection. A double-blind field trial comparing the accuracy of this technique to marked, released and recaptured mosquitoes is required to test the usefulness of the pteridine method in the field.     

Multiple host feeding in field populations of Anopheles culicifacies and An. subpictus in Sri Lanka

A histological technique was used to detect the multiple blood-feeding of female mosquitoes, within the same gonotrophic cycle, in field populations of the malaria vectors Anopheles culicifacies and An. subpictus (Diptera: Culicidae) at a village in the low country of Sri Lanka, during 1994-96. Among 3306 An. culicifacies and 871 An. subpictus engorged females examined, respectively, 34.4% and 30.4% were multiple-fed. In these two species, double meals accounted for 92.7% and 89.5%, and triple meals for 7.3% and 10.5% of multiple meals, respectively. More females of An. culicifacies (53.7%) than An. subpictus (44.5%) multiple-fed on different nights. Multiple feeding rates in the two species were independent of sample size and field abundance. In An. culicifacies, but not An. subpictus, these rates were proportional to ambient temperature but not to humidity. Bloodmeal ELISA showed that 8.3% of 242 An. culicifacies were human-fed and 80% of these (i.e. 6.6%) were concurrently bovid-fed. Combining the histological and ELISA data, it was estimated that multiple feeding involving a human host had occurred in 7.2% of the blood-feeding An. culicifacies population at this study site. Only 1.2% of 406 An. subpictus were human-fed and all of these were concurrently bovid-fed. Multiple bloodfeeding within the same gonotrophic cycle was attributed to a local 'frequent feeding strategy' in these primarily zoophagic and endophilic malaria vectors. Unfortunately this strategy enhances their human-biting rate and hence their vectorial capacity.     

Isolation and characterization of polymorphic microsatellite markers from Asian malaria mosquito Anopheles sinensis (Diptera: Culicidae)

Microsatellite-containing region were isolated and characterized in Anopheles sinensis, a primary vector of malaria parasites in Asia. An enrichment protocol yielded 252 microsatellite sequences. We designed primers to amplify 20 unique microsatellites, 14 of which amplify cleanly and were polymorphic. A survey of 24 individuals showed that 12 loci are highly variable with the number of alleles ranging from two to 11, and expected heterozygosity ranging from 0.116 to 0.903. These markers will be useful for population genetic studies and genome mapping in A. sinensis.     

Probing behaviour and sporozoite delivery by Anopheles stephensi infected with Plasmodium berghei

We observed that Plasmodium berghei sporozoite-infected Anopheles stephensi was not impaired in its ability to locate blood on a host. When probing rats, infected mosquitoes took as long as non-infected mosquitoes to locate blood. Contrary to previous suggestions, infective mosquitoes delivered sporozoites into mineral oil even after extensively probing a vertebrate host. We observed that, in mosquitoes having probed a host, both the mean number of sporozoites ejected over 3 min into oil (35.9 v. 31.7 sporozoites) and the proportion of mosquitoes delivering sporozoites (60% v. 50%) were similar to mosquitoes not having probed. We then developed a model of sporozoite delivery, taking into account observations that sporozoites are clumped in the lumen of the glands as well as upon delivery, and that output is uneven and inconsistent. We conclude that clumping optimizes transmission, if a threshold of infection exists and the mean number of sporozoites per clump is greater than the threshold.     

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。