Reduced susceptibility to DDT in field populations of Anopheles quadriannulatus and Anopheles arabiensis in Malawi: evidence for larval selection

Abstract.  Bioassays for insecticide resistance in adult mosquitoes were conducted on samples of Anopheles gambiae Giles s.l . (Diptera: Culicidae) species collected as larvae from breeding sites in the lower Shire Valley, Malawi. The results indicate full susceptibility to permethrin, deltamethrin and malathion, but reduced susceptibility to DDT in one sample from Thom (LT₅₀ of 8.39 min for females and 25.09 min for males). Polymerase chain reaction-based species identification of the mosquitoes assayed revealed a mixture of Anopheles arabiensis Patton and Anopheles quadriannulatus (Theobold). The LT₅₀ did not differ significantly between species. Genotyping of the L1014F and L1014S kdr alleles showed all mosquito specimens to be homozygous wild type; thus the reduced susceptibility detected is not attributable to target site insensitivity and instead is likely to be metabolic in nature. Anopheles quadriannulatus is characteristically zoophagic and exophilic. Indeed, of 82 Anopheles collected through knockdown collections within dwellings, only one was An. quadriannulatus and the rest were An. arabiensis . They are unlikely, therefore, to have been exposed to selection pressure arising from insecticide-treated net usage or to DDT indoor residual spraying. Therefore, it is suggested that this example of reduced susceptibility to DDT in An. quadriannulatus reflects selection in the larval stages.     

Effect of human odours and positioning of CO2 release point on trap catches of the malaria mosquito Anopheles gambiae sensu stricto in an olfactometer

Abstract.  The anthropophilic malaria mosquito Anopheles gambiae sensu stricto responds to CO₂ and human skin emanations. How these odorants affect the behaviour of this mosquito species is studied in an olfactometer. Carbon dioxide is released either as an homogeneous plume or in a turbulent fashion at two different positions from the trap entrance. Anopheles gambiae is deterred from entering a trap with CO₂ as the only kairomone, when presented as an homogeneous or turbulent plume. This effect is completely overcome by the addition of skin emanations to the CO₂ plume, with a high proportion of mosquitoes found in the trap with skin emanations. Rearrangement of the position of the turbulent CO₂ source so that it is 5 cm downwind of the trap entrance overcomes the deterrent effect of CO₂. Carbon dioxide alone, however, does not elicit higher proportions caught compared with clean air. Further studies with the CO₂ source positioned 5 cm downwind of the trap entrance show that skin emanations alone result in fewer mosquitoes entering the trap than CO₂+ skin emanations. Skin emanations induce more mosquitoes to fly into a trap than a synthetic blend of NH₃+ l-lactic acid when both are combined with CO₂. It is concluded that CO₂ is a poor kairomone when offered alone and that its presence in the plume at the trap entrance deters mosquitoes from entering. By contrast, when positioned just downwind of the trap entrance, CO₂ appears to guide mosquitoes to the vicinity of the trap, where skin emanations then become the principle attractant, causing the mosquito trap entry response. The results of the study have implications for the design of odour-baited traps for this mosquito species.     

Identification of five species of the Anopheles dirus complex from Thailand, using allele-specific polymerase chain reaction

The Anopheles dirus complex of mosquitoes contains some of the most important vectors of malaria in Southeast Asia. To distinguish five species of the complex that occur in Thailand, a method using the polymerase chain reaction (PCR) was developed. The method utilizes allele-specific amplification to detect fixed differences between the species in the DNA sequence of the ribosomal DNA internal transcribed spacer 2. Primers were designed to amplify fragments of diagnostic length from the DNA of the different species. The method was tested on 179 mosquitoes of the An. dirus complex from many parts of Thailand and shown to be effective. Every specimen was unambiguously identified as species A, B, C, D or F (i.e. An. dirus s.s. species B, C, D or An. nemophilous, respectively) by the PCR method, with confirmation of 58/61 identifications from polytene chromosome characteristics. For the other three specimens (3/44 from Kanchanaburi 5 locality), there was disagreement between the PCR and chromosomal methods of species identification (probably due to errors in the chromosomal identifications). Primers can be combined in a single PCR reaction providing a rapid, sensitive and straightforward method of species identification. Only small quantities of DNA are required, leaving most of the mosquito to be used for other analyses.     

Genetic population structure and introgression in Anopheles dirus mosquitoes in South-east Asia

Genetic structure and species relationships were studied in three closely related mosquito species, Anopheles dirus A, C and D in Thailand using 11 microsatellite loci and compared with previous mitochondrial DNA (mtDNA) data on the same populations. All three species were well differentiated from each other at the microsatellite loci. Given the almost complete absence of mtDNA differentiation between An. dirus A and D, this endorses the previous suggestion of mtDNA introgression between these species. The high degree of differentiation between the northern and southern population of An. dirus C (RST = 0.401), in agreement with mtDNA data, is suggestive of incipient species. The lack of genetic structure indicated by microsatellites in four populations of An. dirus A across northern Thailand also concurs with mtDNA data. However, in An. dirus D a limited but significant level of structure was detected by microsatellites over ~400 km in northern Thailand, whereas the mtDNA detected no population differentiation over a much larger area (>1200 km). There is prior evidence for population expansion in the mtDNA. If this is due to a selective sweep originating in An. dirus D, the microsatellite data may indicate greater barriers to gene flow within An. dirus D than in species A. Alternatively, there may have been historical introgression of mtDNA and subsequent demographic expansion which occurred first in An. dirus D so enabling it to accumulate some population differentiation. In the latter case the lack of migration-drift equilibrium precludes the inference of absolute or relative values of gene flow in An. dirus A and D.     

Biting patterns and host preference of Anopheles epiroticus in Chang Island,Trat Province,eastern Thailand

A study of species diversity of Anopheles mosquitoes, biting patterns, and seasonal abundance of important mosquito vectors was conducted in two villages of Chang Island, Trat Province, in eastern Thailand, one located along the coast and the other in the low hills of the central interior of the island. Of 5,399 captured female anophelines, 70.25% belong to the subgenus Cellia and remaining specimens to the subgenus Anopheles. Five important putative malaria vectors were molecularly identified, including Anopheles epiroticus, Anopheles dirus, Anopheles sawadwongporni, Anopheles maculatus, and Anopheles minimus. Anopheles epiroticus was the most commonly collected species in the coastal site, whereas An. dirus was found to be most abundant in the forest‐hill site. From both locations, a greater number of mosquitoes was collected during the dry season compared to the wet. Anopheles epiroticus showed greater exophagic and zoophilic behavior with the highest blood feeding densities occurring between 18:00 and 19:00. In contrast, An. dirus demonstrated an activity peak between midnight and 01:00. We conclude that An. epiroticus and An. dirus, in coastal and inland areas, respectively, appear to be the most epidemiologically important malaria vectors on Chang Island. As no studies of vector competency specific to Chang Island have been conducted, our conclusions that these two species play a primary role in malaria transmission are based on evidence from other localities in Thailand and mainland Southeast Asia. This information serves as a basis for designing improved vector control programs that target specific species, and if integrated with other interventions could result in the elimination of malaria transmission on the island.     

Insecticide susceptibility and resistance of larvae of the Anopheles sinensis Group (Diptera: Culicidae) from Paju, Republic of Korea

The susceptibility of members of the Anopheles sinensis Group in Korea to insecticides was evaluated under laboratory conditions using 15 insecticides currently used by local public health centers in Korea. The insecticides included eight pyrethroids, six organophosphates and a pyrazol analogue. Based on their LC₅₀ values, the order of susceptibility of An. sinensis larvae to the insecticides was bifenthrin, chlorfenapyr, α-cypermethrin and λ-cyhalothrin, with values of 0.009, 0.04, 0.06 and 0.08 p.p.m., respectively. The least susceptibility was obtained with fenitrothion, with an LC₅₀ of 7.7 p.p.m. In the comparative resistance test, the resistance ratios (RR) of 14 insecticides were compared to each other using two strains of members of the An. sinensis Group collected in the locality in 2001 and 2008. Anopheles spp. demonstrated higher RR to organophosphates such as fenthion, and low RR for the pyrethroids. Among the organophosphates, fenthion had the highest RR of 33.3 and 270.0 fold differences for LC₅₀ and LC₉₀ values, respectively. Among the pyrethroids, permethrin was observed to have the highest RR of 3.8 and 1.8 fold differences for LC₅₀ and LC₉₀ values, respectively. However, there were no significant differences in susceptibility to chlorfenapyr, chlorpyrifos, deltamethrin and fenitrothion. An. sinensis s. l. was more susceptible to the six insecticides bifenthrin, λ-cyhalothrin, α-cypermethrin, cypermethrin, cyfluthrin and pyridafenthion, showing 0.03, 0.06, 0.3, 0.3, 0.4 and 0.4 fold differences in resistance rates (RR LC₅₀), respectively.     

Population genetic evidence for species A, B, C and D of the Anopheles dirus complex in Thailand and enzyme electromorphs for their identification

Mixtures of chromosomal forms A, B, C and D in natural populations of Anopheles dirus Peyton & Harrison sensu lato in Thailand show significant positive values of Wright's fixation index for six enzyme-electromorph loci. The mean value of FIS over all loci was found to be +0.28 (SD 0.02), with a range of +0.57 (Odh) to +0.10 (Idh-2). Partitioning electromorph data for the chromosomal forms reduces the mean FIS to 0.03 (SD 0.01), which suggests that positive assortative mating is a characteristic of each form. This supports the hypothesis that the chromosomal/electrophoretic forms A, B, C and D represent four distinct biological species within the An. dirus complex. An example is given of the use of enzyme electromorphs as a means of vector identification during a malaria entomological field study involving a mixture of An. dirus species A and D. Electromorph identifications of 323 sp. A and 161 sp. D were more than 98% correct when cross-referenced to specific DNA probes.     

Responses of mosquitoes of the Anopheles farauti complex to 1-octen-3-ol and light in combination with carbon dioxide in northern Queensland, Australia

Abstract. In northern Queensland, Australia, three experiments were conducted to determine the response of mosquitoes of the Anopheles farauti complex to CDC traps baited with four attractant combinations: octenol + C0₂ and light; octenol and light; CO, and light; or C0₂ and octenol without light. A CDC-modified updraft light-trap was also trialled, but did not significantly enhance collections of An.farauti sensu lato. The combination of light, octenol and C0₂ caught significantly more An.farauti s.l. (both An.farauti No. 1 and No. 2 sibling species) when compared to C0₂ and light alone. Only small numbers of the An.farauti complex were captured when CDC traps were baited with octenol alone, i.e. no light or C0₂.     

In vitro carbon dioxide excretion from erythrocytes of two species of Antarctic fishes and its inhibition by catecholamines

This study was designed to investigate whether the blood of Pagothenia borchgrevinki , exhibits a Haldane effect, and whether activation of a Na⁺/H⁺ antiporter increases transport of intracellular protons and Bohr protons out of the erythrocytes resulting in inhibition of CO₂ excretion in both P. borchgrevinki , and Dissostichus mawsoni. When carbon dioxide dissociation curves were determined from blood samples pooled from three fish under oxygenated and deoxygenated conditions a Haldane effect was observed. Using an in vitro , CO₂ excretion assay, the rate of HCO₃⁻ dehydration was determined on blood and plasma equilibrated under an N₂atmosphere then rapidly oxygenated with air in the presence of 10⁻⁵ M noradrenaline or acetazolamide (10⁰⁴M). Whole blood and plasma from P. borchgrevinki , and D. mawsoni , were equilibrated with 0·5% CO₂ in air and assayed in the presence of 10⁻⁵ M noradrenaline. Erythrocyte CO₂ excretion rates were depressed significantly by noradrenaline in both species. The whole blood HCO₃⁻ dehydration rate was depressed significantly following rapid oxygenation in the presence of acetazolamide indicating that the pathway of CO₂ excretion included activation of intracellular carbonic anhydrase and an adrenergic receptor.     

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.     

Biological Control of Anopheles sinensis with Native Fish Predators (Aplocheilus and Aphyocypris) and Herbivorous Fish, Tilapia in Natural Rice Fields in Korea

ABSTRACT A confined field experiment was conducted to investigate biological control of malaria and inland filariasis vector, Anopheles sinensis Wied. by combined use of larvivorous fishes, Aplocheilus latipes or Aphyocypris chinensis and herbivorous, Tilapia mossambicus niloticus in natural rice fields at Banwol near Suwon, Gyeonggi province from June through October, 1989. In the presence of naturally breeding Aplocheilus at the density of 0.8 fish/m² water surface, the natural control of Anopheles larvae ranged 34.4% to 51.6% from June through August; later supplemental introduction of herbivore (Tilapia) at the release rate of 1-pair per 10 m² resulted in 67.8% increased to 80.0% control of Anopheles sinensis in 3rd and 5th week respectively. In a combined fish release at the rate of 1.0 fish/m² of Aphyocypris and 1-pair/10 m² of Tilapia produced 67.3% and 82.1% mosquito larval reduction in 3rd and 5th week periods, respectively. The similar pattern of gradual but significant suppression of mosquitoes was also evident in separate rice paddy in 4-5 week period, maintaining ca. 75'82% for the subsequent mosquito breeding season in comparison with control plot.     

Plasmodium yoelii: contribution of oocysts melanization to natural refractoriness in Anopheles dirus

It is well known that Anopheles dirus is naturally refractory to rodent malaria parasite, Plasmodium yoelii, but the mechanism is still largely unknown. Here, we found that some P. yoelii taken into An. dirus could develop into oocysts, but oocysts were partially melanized at 7 days and completely melanized at 15 days post-infectious blood meal. Transmission electronic microscopy could find the melanized P. yoelii oocysts in An. dirus as early as 5 days post-infection, with a few haemocytes attaching to the melanized oocysts, indicating a typical humoral melanization reaction. Although the change of protein pattern at 24h post-infection suggested that other unknown mechanisms and/or factors might be involved in killing ookinetes, our data implied that oocysts melanization was one of the mechanisms of An. dirus to block P. yoelii development. In addition, activity of phenoloxidase, such as monophenol oxidase and o-diphenoloxidase, in haemolymph of An. dirus fed on infectious blood meal was much higher than that of mosquitoes fed on 5% glucose or normal mouse blood (p<0.05), implying the possible role of PO in oocysts melanization by An. dirus.     

Complex population history of two Anopheles dirus mosquito species in Southeast Asia suggests the influence of Pleistocene climate change rather than human-mediated effects

Anopheles dirus and Anopheles baimaii are closely related species which feed on primates, particularly humans, and transmit malaria in the tropical forests of mainland Southeast Asia. Here, we report an in-depth phylogeographic picture based on 269 individuals from 21 populations from mainland Southeast Asia. Analysis of 1537 bp of mtDNA sequence revealed that the population history of A. baimaii is far more complex than previously thought. An old expansion (pre-300 kyr BP) was inferred in northern India/Bangladesh with a wave of south-eastwards expansion arriving at the Thai border (ca 135-173 kyr BP) followed by leptokurtic dispersal very recently (ca 16 kyr BP) into peninsular Thailand. The long and complex population history of these anthropophilic species suggests their expansions are not in response to the relatively recent (ca 40 kyr BP) human expansions in mainland Southeast Asia but, rather, fit well with our understanding of Pleistocene climatic change there.     

Effects of physico-chemical treatments on hemagglutination activity of Anopheles gambiae haemolymph and midgut extract

Abstract. Anopheles gambiae midgut extracts and haemolymph possessed agglutinins, titre 1:16 to 1:256, against human red blood cells (RBCs). Subjection of both tissues to protein precipitation reagents, organic chemical and selected protease, neuraminidase and other glycosidic hydrolase treatments revealed the haemagglutinins to be protein, most likely glycoprotein, in nature - not lipoprotein, lipid, glycolipid or nucleic acid. An.gambiae agglutinins were thermo-labile >40^oC, affected by freezing and thawing treatments, and contained disulphide and hydrogen bonds on the basis of sensitivity following exposure to dithio-threitol and urea respectively. Optimum haemagglutination depended generally on slightly acid to neutral pH conditions and agglutinin activity was Ca²⁺ ion, albeit to a lesser extent Mg²⁺ ion, dependent. The midgut extract agglutinin subunit molecule had a relative molecular weight (M_r) of 65kDa whilst that of haemolymph was 40kDa.
This study presents the first report on selected physico-chemical properties, the glycoproteinaceous nature and tentative subunit M_r of mosquito midgut extract and haemolymph anti-RBC agglutinin(s).     

Laboratory colonization of Anopheles quadriannulatus from sympatry with other sibling species of the Anopheles gambiae complex in Zimbabwe

Abstract. A laboratory colony of the mosquito Anopheles quadriannulatus was established from a wild population occurring sympatrically with An.arabiensis in Zimbabwe. These sibling species are members of the An.gambiae Giles complex and were distinguished primarily by means of their specific polytene chromosome banding patterns. By using an ox-baited trap, we sampled selectively for the more zoophilic An.quadriannulatus. It was confirmed that An.quadriannulatus has the diagnostic slow allozyme of aspartate aminotransferase (AAT^95/95). In a mixed population under laboratory conditions, An.arabiensis displaced An.quadriannulatus within eight generations, without introgression. Colonization of An.quadriannulatus was facilitated by pooling the progeny from wild-caught mothers of confirmed identity and by using a specially adapted cage to promote mating.     

Pyrethroid-impregnated hessian curtains for protection against mosquitoes indoors in South India

Abstract. Hessian curtains impregnated with deltamethrin 50 mg/m² were hung in the eaves and doorways of eight one-roomed huts in Madurai, Tamil Nadu State, South India. Statistically significant reductions of indoor-resting and man-biting densities of the mosquitoes Anopheles subpictus and Culex quinquefasciatus were observed for 14 weeks, in two field trials. Bioassays on curtains in the field showed over 50% mortality of Cx quinquefasciatus and An. stephensi for up to 8 weeks. The curtains were highly acceptable to the community, and cost approximately Rs.33.15 (US1.05) for material and Rs.10 (0.32) for deltamethrin per hut, totalling Rs 53.15 (1.70) for two impregnations giving 6 months protection. Comparative costs of house-spraying with residual insecticides are estimated as Rs.1.92 (0.06) for two rounds of DDT at lg/m², or Rs.40 (1.27) for three rounds of malathion at 2g/m². Therefore the relative annual cost of protection using deltamethrin-impregnated hessian curtains is 28× or 1.3× more than for house-spraying with DDT or malathion, respectively (excluding operational expenditure).     

中国大劣按蚊复合体生物分类的研究(英文)

大劣按蚊（广义）是东南亚和我国南方的高效传疟媒介,其复合体不同成员种的传疟作用尚未清楚阐明,探索近缘种的正确鉴别为蚊虫生物分类的重要基础。本文报道对我国大劣按蚊复合体（Anophelesdiruscomplex）生物分类的研究结果,应用染色体核型、卵的扫描结构、核糖体DNA间隔2区序列及PCR鉴别检测等方法,确认该复合体A种分布于海南省,D种分布于云南省。文内并对蚊虫生物分类的发展方向及其对疟疾防治的重要性作了讨论。     

Development of a polymorphic strain of Plasmodium vivax in monkeys.

A strain of Plasmodium vivax from Thailand with a polymorphic repeat unit of the circumsporozoite protein was established in Saimiri sciureus boliviensis and 3 species of Aotus monkeys. All 11 attempts to transmit infection via sporozoite inoculation, 4 times to splenectomized S. sciureus boliviensis, 2 times to splenectomized Aotus nancymai, and 5 times to intact Saimiri monkeys, were successful. Anopheles freeborni, Anopheles stephensi, Anopheles dirus, and Anopheles gambiae mosquitoes were infected by feeding on parasitemic blood from a chimpanzee and an Aotus azarae boliviensis monkey. Our results indicate that this strain may be useful in antisporozoite vaccine trials.     

Malaria vector composition and insecticide susceptibility status in Guinea Conakry,West Africa

This study provides data on malaria vector species composition and insecticide susceptibility status from three localities in Guinea Conakry. A total of 497 mosquitoes were collected resting indoors and morphologically identified as belonging to the Anopheles gambiae complex. The majority of these were An. gambiae s.s. (99.6%), but a small percentage (0.4%) were identified as Anopheles arabiensis. Thirty‐four Anopheles funestus s.s. were also collected. The molecular S form of An. gambiae s.s. was predominant over the M form in Siguiri (95%) and Boffa (97.4%), whereas at Mt Nimba the M form was more abundant (61.4%) than the S form (38.1%). One hybrid M/S specimen was recorded from Mt Nimba. Siguiri populations showed high levels of resistance to DDT, dieldrin and bendiocarb. Anopheles gambiae from Boffa were largely susceptible to the insecticides tested. At Mt Nimba, resistance to DDT and bendicocarb was detected. Biochemical enzyme analysis showed that an altered acetylcholinesterase is operating in the field at low levels. The frequency of the 1014F kdr allele in the An. gambiae S form was 0.24 at Siguiri and 0.14 at Mt Nimba. A single RR specimen was found in the M form. The heterogeneity in species composition and resistance profiles between sites requires vector control interventions to be tailored to each site based on the data collected from ongoing monitoring and surveillance.     

Adaptation of the AMRU-1 strain of Plasmodium vivax to Aotus and Saimiri monkeys and to four species of anopheline mosquitoes.

A chloroquine-resistant strain of Plasmodium vivax (AMRU-1) from Papua New Guinea has been adapted to grow in 4 species of Aotus monkeys (Aotus lemurinus griseimembra, Aotus vaciferans, Aotus nancymai, and Aotus azarae boliviensis), hybrid Aotus monkeys, and Saimiri boliviensis monkeys. Whereas it was possible to infect Saimiri monkeys with this parasite by inoculation of parasitized erythrocytes, only 42% of Saimiri monkeys became infected, compared to 92% of Aotus monkeys attempted. Comparative mosquito feedings showed that only A. vociferans, A. l. griseimembra, and Saimiri boliviensis monkeys produced infections in mosquitoes. Oocysts were observed on the guts of the 4 species of mosquitoes used (Anopheles gambiae, Anopheles stephensi, Anopheles freeborni, and Anopheles dirus), but sporozoite transmission was effected only with the intravenous inoculation of sporozoites from An. dirus into an A. l. griseimembra monkey.