Illustrated keys to the anopheline mosquitoes of Myanmar.

In the newly revised and illustrated keys to 4th instar larvae and adult female mosquitoes, the following 36 Anopheles species from Myanmar are included: Anopheles aconitus, An. aitkenii, An. annularis, An. argyropus, An. barbirostris, An. bengalensis, An. culicifacies, An. dirus, An. fluviatilis, An. gigas, An. insulaeflorum, An. jamesii, An. jeyporensis, An. karwari, An. kochi, An. kyondawensis, An. lindesayi, An. maculatus, An. majidi, An. minimus, An. nigerrimus, An. nitidus, An. pallidus, An. peditaeniatus, An. philippinensis, An. pseudojamesii, An. sinensis, An. splendidus, An. stephensi, An. subpictus, An. sundaicus, An. tessellatus, An. theobaldi, An. vagus, An. varuna, and An. willmori. The new keys presented in this paper will enable public health workers to rapidly identify mosquito vectors of malaria and to distinguish them from other species in the same genera.     

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.     

Molecular and morphological studies on the Anopheles minimus group of mosquitoes in southern China: taxonomic review,distribution and malaria vector status

Abstract. Mosquitoes of the Anopheles minimus group (Diptera: Culicidae) from nine Provinces of southern China were identified morphologically and by molecular characterization, using single‐strand conformation polymorphisms (SSCPs) and sequence data for the D3 region of the 28S ribosomal DNA and the mitochondrial COII locus. Species A and C (sensu Green et al., 1990 ) of the An. minimus complex were found to be sympatric in Yunnan Province. Species A occurs eastward from Yunnan through southern Guangxi, Hainan, Guangdong and Taiwan Provinces, whereas species C occurs northward to northern Guangxi, Guizhou and Sichuan Provinces. Morphological and molecular evidence (based on specimens from the field and four isofemale lines) shows that An. minimus forms A and B (sensu Yu & Li, 1984 ) are morphological variants of species A, which is accepted as An. minimus Theobald sensu stricto (type‐locality: Pokfulam, Hong Kong). The so‐called subspecies x of An. minimus (sensu Baba, 1950 ) is reinterpreted as An. aconitus Dönitz. The distribution and vector status of members of the An. minimus group are discussed in relation to the historical and current transmission of malaria and filariasis in China. Both An. minimus A and C have been implicated as widespread vectors of malaria, whereas only species A has been found in Hainan, where An. minimus s.l. was a vector of Bancroftian filariasis. The presence of An. aconitus in Hainan and Yunnan Provinces is confirmed, but the occurrence of An. varuna Iyengar and An. fluviatilis James, which were previously recorded in China, could not be verified.     

Six new species of the Anopheles leucosphyrus group, reinterpretation of An. elegans and vector implications

Abstract. Among Oriental anopheline mosquitoes (Diptera: Culicidae), several major vectors of forest malaria belong to the group of Anopheles (Cellia) leucosphyrus Dönitz. We have morphologically examined representative material (> 8000 specimens from seven countries) for taxonomic revision of the Leucosphyrus Group. Six new species are here described from adult, pupal and larval stages (with illustrations of immature stages) and formally named as follows: An. latens n. sp. (= An. leucosphyrus species A of Baimai et al., 1988b), An. cracens n. sp., An. scanloni n. sp., An. baimaii n. sp. (formerly An. dirus species B, C, D, respectively), An. mirans n. sp. and An. recens n. sp. Additionally, An. elegans (James) is redescribed and placed in the complex of An. dirus Peyton & Harrison (comprising An. baimaii, An. cracens, An. dirus, An. elegans, An. nemophilous Peyton & Ramalingam, An. scanloni and An. takasagoensis Morishita) of the Leucosphyrus Subgroup, together with An. baisasi Colless and the An. leucosphyrus complex (comprising An. balabacensis Baisas, An. introlatus Baisas, An. latens and An. leucosphyrus). Hence, the former Elegans Subgroup is renamed the Hackeri Subgroup (comprising An. hackeri Edwards, An. pujutensis Colless, An. recens and An. sulawesi Waktoedi). Distribution data and bionomics of the newly defined species are given, based on new material and published records, with discussion of morphological characters for species distinction and implications for ecology and vector roles of such species. Now these and other members of the Leucosphyrus Group are identifiable, it should be possible to clarify the medical importance and distribution of each species. Those already regarded as vectors of human malaria are: An. baimaii[Bangladesh, China (Yunnan), India (Andamans, Assam, Meghalaya, West Bengal), Myanmar, Thailand]; An. latens[Borneo (where it also transmits Bancroftian filariasis), peninsular Malaysia, Thailand]; probably An. cracens (Sumatra, peninsular Malaysia, Thailand); presumably An. scanloni (Thailand); perhaps An. elegans (the Western Ghat form of An. dirus, restricted to peninsular India); but apparently not An. recens (Sumatra) nor An. mirans[Sri Lanka and south-west India (Karnataka, Kerala, Tamil Nadu)], which is a natural vector of simian malarias. Together with typical An. balabacensis, An. dirus and An. leucosphyrus, therefore, the Leucosphyrus Group includes about seven important vectors of forest malaria, plus at least a dozen species of no known medical importance, with differential specific distributions collectively spanning > 5000 km from India to the Philippines.     

Multiplex PCR assay for malaria vector Anopheles minimus and four related species in the Myzomyia Series from Southeast Asia

Abstract. Mosquitoes (Diptera: Culicidae) of the Anopheles (Cellia) Myzomyia Series are important malaria vectors in Africa, India and Southeast Asia. Among 10 named species of Myzomyia known from the Oriental Region, seven form the An. minimus group. Even for expert taxonomists, the adults of these species remain difficult to identify morphologically. For technical staff of malaria control programmes, confusion may extend to misidentification of species that are not formally within the minimus group. For identification of specimens from Indochina (Cambodia, Laos, Vietnam), we describe a multiplex polymerase chain reaction (PCR) assay, based on rDNA internal transcribed spacer 2 (ITS2) sequences, that employs a cocktail of primers to identify An. minimus Theobald sibling species A and C (sensu; Green et al., 1990) and three other species in the An. minimus group (An. aconitus Dönitz, An. pampanai Büttiker & Beales, An. varuna Iyengar), as well as An. jeyporiensis James, also belonging to the Myzomyia Series. As the test is DNA‐based, it can be applied to all life stages of these mosquitoes for ecological investigations and vector incrimination studies. This PCR assay is simpler, quicker, cheaper and more readily interpreted than previous assays.     

Adult anopheline ecology and malaria transmission in irrigated areas of South Punjab, Pakistan

Surface irrigation in the Punjab province of Pakistan has been carried out on a large scale since the development of the Indus Basin Irrigation System in the late 19th century. The objective of our study was to understand how the population dynamics of adult anopheline mosquitoes (Diptera: Culicidae) could be related to malaria transmission in rural areas with intensive irrigation and a history of malaria epidemics. In this paper we present our observations from three villages located along an irrigation canal in South Punjab. The study was carried out from 1 April 1999 to 31 March 2000. Mosquitoes were collected from bedrooms using the pyrethroid spraycatch method and from vegetation and animal sheds using backpack aspirators. Overall, Anopheles subpictus Grassi sensu lato predominated (55.6%), followed by An. stephensi Liston s.l. (41.4%), An. culicifacies Giles s.l. (2.0%), An. pulcherrimus Theobald (1.0%) and An. peditaeniatus Leicester (0.1%). Most mosquitoes (98.8%) were collected from indoor resting-sites whereas collections from potential resting-sites outdoors accounted for only 1.2% of total anopheline densities, confirming the endophilic behaviour of anophelines in Pakistan. Anopheles stephensi, An. culicifacies and An. subpictus populations peaked in August, September and October, respectively. High temperatures and low rainfall negatively affected seasonal abundance in our area. There were interesting differences in anopheline fauna between villages, with An. culicifacies occurring almost exclusively in the village at the head of the irrigation canal, where waterlogged and irrigated fields prevailed. Monthly house-to-house fever surveys showed that malaria transmission remained low with an overall slide positivity rate of 2.4% and all cases were due to infection with Plasmodium vivax. The most plausible explanation for low transmission in our study area seems to be the low density of Pakistan's primary malaria vector, An. culicifacies. The role of other species such as An. stephensi is not clear. Our observations indicate that, in South Punjab, irrigation-related sites support the breeding of anopheline mosquitoes, including the vectors of malaria. As our study was carried out during a year with exceptionally hot and dry climatic conditions, densities and longevity of mosquitoes would probably be higher in other years and could result in more significant malaria transmission than we observed. To assess the overall importance of irrigation-related sites in the epidemiology of malaria in the Punjab, more studies are needed to compare irrigated and non-irrigated areas.     

Density, survival and dispersal of Anopheles gambiae complex mosquitoes in a West African Sudan savanna village

Abstract. To obtain information on adult populations of Afrotropical malaria vector mosquitoes, mark-release-recapture experiments were performed with Anopheles females collected from indoor resting-sites in a savanna area near Ouagadougou, Burkina Faso, during September 1991 and 1992. Results were used to estimate the absolute population densities, daily survival rates, and dispersal parameters of malaria vectors in that area.
In 1991 a total of 7260 female Anopheles were marked and released, of which 106 were recaptured in the release village and 6 in the neighbouring villages, a total recapture rate of 1.5%. The following year 13, 854 female Anopheles were released and 116 recaptured in Goundri and 8 in the neighbouring villages, a total recapture rate of 0.9%. Recaptures were found in three of eight villages near Goundri. Nearly all of the recaptured mosquitoes were An.gambiae s.l. Of these, molecular determination revealed that An.gambiae s.s. and An.arabiensis were present in a ratio of -2:3.
Two simple random models of dispersal were simulated and the parameters of the models determined by searching for the least-squared fit between simulated and observed distributions. The mean distance moved by individual mosquitoes, estimated in this way, ranged 350–650 m day^-1, depending on die model and the year considered. Population densities were estimated using the Lincoln Index, Fisher-Ford and Jolly's methods. The estimates of population size had high standard errors and were not particularly consistent. A 'consensus' value of 150,000–350,000 mosquitoes is believed to apply for ht An.gambiae s.l. female population. Survival was estimated to be 80–88% per day.     

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.     

Cytogenetic evidence for a complex of species within the taxon Anopheles maculatus (Diptera: Culicidae)

Two largely independent studies of chromosomes from natural populations of Anopheles maculatus provide evidence for several genetic species within the taxon. (1) Polytene chromosome variation shows four different rearrangements of arm 2 and three rearrangements of the X chromosome. There is strong evidence for three species. Two allopatric populations represent either dramatic geographic variation for two independent inversion systems within one of the genetic species, or represent two additional species. Their species status remains unresolved by this work. (2) Heterochromatic variation occurs in both X and Y chromosomes as revealed by Giemsa-banding of mitotic chromosomes from larval brains. The distribution and association of these various sex chromosomes give further evidence of a species complex. A preliminary correlation of these two kinds of chromosomal variation is given.     

Are chromosomal inversions induced by transposable elements? A paradigm from the malaria mosquito Anopheles gambiae

Chromosomal rearrangements abound in nature and can be studied in detail in organisms with polytene chromosomes. In Drosophila and in Anopheline mosquitoes most speciation processes seem to be associated with the establishment of chromosomal rearrangements, particularly of paracentric inversions. It is not known what triggers inversions in natural populations. In the laboratory inversions are commonly generated by X-rays, mutagens or after the activity of certain transposable elements (TEs). The Anopheles gambiae complex is comprised of six sibling species, each one characterized by the presence of fixed paracentric inversions on their chromosomes. Two of these, An. gambiae s.s. and An. arabiensis, are the most important vectors of human malaria and are structured into sub-populations, each carrying a characteristic set of polymorphic chromosomal inversions. We have cloned the breakpoints of the naturally occurring polymorphic inversion In(2R)d' of An. arabiensis. Analysis of the surrounding sequences demonstrated that adjacent to the distal breakpoint lies a transposable element that we called Odysseus. Characteristics of Odysseus' terminal region and its cytological distribution in different strains as well as within the same strain indicate that Odysseus is an actively transposing element. The presence of Odysseus at the junction of the naturally occurring inversion In(2R)d' suggests that the inversion may be the result of the TEs activity. Cytological evidence from Drosophila melanogaster has also implicated the hobo transposable element in the generation of certain Hawaiian endemic inversions. This picture supports the hypothesis of the important role of TEs in generating natural inversions.     

Life history consequences of larval foraging depth differ between two competing Anopheles mosquitoes

1. Anopheline larvae are surface feeders and allocate most of their time to search for food at the water surface. However, species of the Anopheles gambiae Giles complex may also show bottom feeding. The consequences of this foraging tactic for life history are unknown, yet may be relevant to understand inter‐specific competition patterns. 2. The diving ability and activity of larvae of the main African malaria vectors, An. coluzzii and An. gambiae, at two different water depths (14 and 30 cm) were assessed. We further explored the biological relevance of diving for food harvesting by monitoring key life history traits in two species treatments (single or mixed species) and two food treatments (surface or bottom feeding). 3. Overall, An. coluzzii larvae showed more diving activity than An. gambiae. When feeding at the bottom both species, and especially An. gambiae, showed a delayed emergence and a reduced emergence rate. Moreover, An. gambiae also suffered a reduced wing length. 4. Mixed‐species rearing had a detrimental effect on the life history traits of An. gambiae but not on An. coluzzii, suggesting a competitive advantage for the latter in our experimental conditions. 5. The present results confirm that anopheline larvae are able to forage for food at the bottom of their breeding site and that An. coluzzii shows a superior diving activity than An. gambiae and this at a lower cost. These behavioural differences probably reflect specific adaptations to different aquatic habitats, and may be important in shaping species distributions and the population biology of these important vector mosquitoes.     

Prominent intraspecific genetic divergence within Anopheles gambiae sibling species triggered by habitat discontinuities across a riverine landscape

The Anopheles gambiae complex of mosquitoes includes malaria vectors at different stages of speciation, whose study enables a better understanding of how adaptation to divergent environmental conditions leads to evolution of reproductive isolation. We investigated the population genetic structure of closely related sympatric taxa that have recently been proposed as separate species (An. coluzzii and An. gambiae), sampled from diverse habitats along the Gambia river in West Africa. We characterized putatively neutral microsatellite loci as well as chromosomal inversion polymorphisms known to be associated with ecological adaptation. The results revealed strong ecologically associated population subdivisions within both species. Microsatellite loci on chromosome‐3L revealed clear differentiation between coastal and inland populations, which in An. coluzzii is reinforced by a unusual inversion polymorphism pattern, supporting the hypothesis of genetic divergence driven by adaptation to the coastal habitat. A strong reduction of gene flow was observed between An. gambiae populations west and east of an extensively rice‐cultivated region apparently colonized exclusively by An. coluzzii. Notably, this ‘intraspecific’ differentiation is higher than that observed between the two species and involves also the centromeric region of chromosome‐X which has previously been considered a marker of speciation within this complex, possibly suggesting that the two populations may be at an advanced stage of differentiation triggered by human‐made habitat fragmentation. These results confirm ongoing ecological speciation within these most important Afro‐tropical malaria vectors and raise new questions on the possible effect of this process in malaria transmission.     

Polytene chromosome relationships of five species of the Anopheles dirus complex in Thailand.

Photographic maps and rearrangements of each salivary gland polytene chromosome arm of Anopheles nemophilous (species F) and of An. dirus species A, B, C, and D of the Dirus group from natural populations in Thailand are presented. Structural conformation of heterokaryotypes and comparison of chromosome banding sequences reveal 10 paracentric inversions. The data on fixed inversion of 3Rb and inversion polymorphism of the X chromosome shared by these species were used to construct a phylogeny of the five members of the An. dirus complex, thereby outlining their patterns of speciation through chromosomal rearrangements.     

Fatty acids in anopheline mosquito larvae and their habitats

Larvae of the three important Central American malaria vectors, Anopheles albimanus, An. vestitipennis, and An. darlingi, are found in distinctly different habitats broadly defined by hydrology and aquatic vegetation, but little is known about the actual food quality and quantity of these habitats. Polyunsaturated fatty acids (PUFA) are of special interest, because mosquitoes require 20:5ω3 (EPA), 20:4ω6 (ARA), and 22:6ω3 (DHA) and without an adequate supply of these PUFAs they are not able to complete their life cycle. We collected samples of larvae and their corresponding habitats and analyzed their fatty acid (FA) composition to reveal if there are any species‐specific and habitat‐specific differences in FA composition, and if habitat FA differences can be linked to differences in the mosquito FA pattern and, ultimately, mosquito performance. We also assessed how FA of wild larvae compare to the laboratory‐reared larvae. Habitats were generally low in essential PUFAs and there were no significant differences among the FA composition of habitat samples. There were significant differences in FA composition of larvae. An. darlingi contained significantly higher amounts of FA, specifically a higher content of ω‐6 PUFA, represented mainly by the linoleic acid (18:2ω‐6). Large differences were found between field‐collected and laboratory‐reared An. vestitipennis larvae, especially in the content of PUFAs. The laboratory‐reared larvae contained significantly more of the total FA, ω3 PUFA, and MUFA. The laboratory‐reared larvae contained three to five times more essential PUFAs, EPA, and DHA. However, there were no differences in the total dry weight of the 4^th instar larvae between the wild vs laboratory‐reared larvae. Total FA in both larvae and habitats of An. albimanus and An. darlingi were positively correlated with the concentration of particulate organic carbon and nitrogen (POC, PON) in their respective habitats, but no such correlation was found for An. vestitipennis. PUFA are a good indicator of nutritional quality, although factors controlling the success of anopheline development from larval habitats are likely to be more complex and would include the presence of predators, pathogens, and toxins as interacting factors.     

Malaria infection potential of anopheline mosquitoes sampled by light trapping indoors in coastal Tanzanian villages

Abstract. Anopheline mosquito populations were studied during 1992 in seven villages south of Bagamoyo, coastal Tanzania, prior to malaria control intervention using insecticide treated bednets. To collect mosquitoes, CDC light traps were used in ten houses per village fortnightly for 12 months. Anopheles females were identified and checked by ELISA for the presence of malaria sporozoite antigen and source of bloodmeal. An. funestus peaked in June-July after the long rains. Three members of the An. gambiae complex had different seasonality: An. arabiensis, An. gambiae and small numbers of An. merus were collected.
In most villages transmission was extremely high and perennial with the entomological inoculation rate reaching three to eleven infective bites per person per night in July and persisting at around 0.1 and 1 for most of the remainder of the year. Sporozoite infection rates within the An. gambiae complex ranged from 2% to 25%, with the peaks in January and July following the two rainy periods. An. funestus showed a similar pattern. The light traps were reliable, simple to operate, and proved to be satisfactory to study the mosquito vector population.     

Malaria transmission risk variations derived from different agricultural practices in an irrigated area of northern Tanzania

Malaria vector Anopheles and other mosquitoes (Diptera: Culicidae) were monitored for 12 months during 1994-95 in villages of Lower Moshi irrigation area (37 degrees 20' E, 3 degrees 21' S; approximately 700 m a.s.l.) south of Mount Kilimanjaro in northern Tanzania. Adult mosquito populations were sampled fortnightly by five methods: human bait collection indoors (18.00-06.00 hours) and outdoors (18.00-24.00 hours); from daytime resting-sites indoors and outdoors; by CDC light-traps over sleepers. Anopheles densities and rates of survival, anthropophily and malaria infection were compared between three villages representing different agro-ecosystems: irrigated sugarcane plantation; smallholder rice irrigation scheme, and savannah with subsistence crops. Respective study villages were Mvuleni (population 2200), Chekereni (population 3200) and Kisangasangeni (population approximately/= 1000), at least 7 km apart. Anopheles arabiensis Patton was found to be the principal malaria vector throughout the study area, with An. funestus Giles sensu lato of secondary importance in the sugarcane and savannah villages. Irrigated sugarcane cultivation resulted in water pooling, but this did not produce more vectors. Anopheles arabiensis densities averaged four-fold higher in the ricefield village, although their human blood-index was significantly less (48%) than in the sugarcane (68%) or savannah (66%) villages, despite similar proportions of humans and cows (ratio 1:1.1-1.4) as the main hosts at all sites. Parous rates, duration of the gonotrophic cycle and survival rates of An. arabiensis were similar in villages of all three agro-ecosystems. The potential risk of malaria, based on measurements of vectorial capacity of An. arabiensis and An.funestus combined, was four-fold higher in the ricefield village than in the sugarcane or savannah villages nearby. However, the more realistic estimate of malaria risk, based on entomological inoculation rates, indicated that exposure to infective vectors was 61-68% less for people in the ricefield village, due to the much lower sporozoite rate in An. arabiensis (ricefield 0.01%, sugarcane 0.1%, savannah 0.12%). This contrast was attributed to better socio-economic conditions of rice farmers, facilitating relatively more use of antimalarials and bednets for their families. Our findings show that, for a combination of reasons, the malaria challenge is lower for villagers associated with an irrigated rice-growing scheme (despite greater malaria vector potential), than for adjacent communities with other agro-ecosystems bringing less socio-economic benefits to health. This encourages the development of agro-irrigation schemes in African savannahs, provided that residents have ready access to antimalaria materials (i.e. effective antimalaria drugs and insecticidal bednets) that they may better afford for protection against the greater vectorial capacity of An. arabiensis from the ricefield agro-ecosystem.     

Anopheles arabiensis and An. funestus are equally important vectors of malaria in Matola coastal suburb of Maputo, southern Mozambique

Transmission characteristics of malaria were studied in Matola, a coastal suburb of Maputo, the capital City, in southern Mozambique, from November 1994 to April 1996. The local climate alternates between cool dry season (May-October) and hot rainy season (November-April) with mean annual rainfall 650-850 mm. Saltmarsh and freshwater pools provide mosquito breeding sites in Matola. Malaria prevalence reached approximately 60% among people living nearest to the main breeding sites of the vectors. Plasmodium falciparum caused 97% of malaria cases, others being P. malariae and P. ovale. Potential malaria vector mosquitoes (Diptera: Culicidae) collected at Matola during daytime indoor-resting (n = 1021) and on human bait at night (n = 5893) comprised 12% Anopheles coustani Laveran (93% biting outdoors), 46% An. funestus Giles (68% biting indoors) and 42% An. gambiae Giles sensu lato (60% biting outdoors). All 215 specimens of An. gambiae s.l. identified genetically were An. arabiensis Patton. Anopheles funestus populations remained stable throughout the year, whereas densities of the An. gambiae complex fluctuated considerably, with An. arabiensis peaking during the rainy season. No concomitant rise in malaria incidence was observed. Human landing indices of An. funestus and An. arabiensis averaged 1.8 and 3.8 per man-night, respectively. Overall Plasmodium sporozoite rates were 2.42+/-1.24% in 2181 An. funestus and 1.11+/-1.25% in 1689 An. arabiensis dissected and examined microscopically. Mean daily survival rates were 0.79 for both vector species. Estimated infective bites/person/year were 15 An. funestus and 12 An. arabiensis. Biting rates were greatest at 2100-24.00 hours for An. funestus (68% endophagic) and 21.00-03.00 hours for An. arabiensis (40% endophagic). The entomological inoculation rate (EIR) declined sharply over very short distances (50% per 90m) away from breeding-sites of the vectors. Consequently, P. falciparum prevalence among Matola residents was halved 350 m within the town. Implications for the protective effectiveness of a 'cordon sanitaire' by residual house-spraying and/or the use of insecticide-treated bednets are discussed.     

Cryptic Genetic Diversity within the Anopheles nili group of Malaria Vectors in the Equatorial Forest Area of Cameroon (Central Africa)

Background

The Anopheles nili group of mosquitoes includes important vectors of human malaria in equatorial forest and humid savannah regions of sub-Saharan Africa. However, it remains largely understudied, and data on its populations’ bionomics and genetic structure are crucially lacking. Here, we used a combination of nuclear (i.e. microsatellite and ribosomal DNA) and mitochondrial DNA markers to explore and compare the level of genetic polymorphism and divergence among populations and species of the group in the savannah and forested areas of Cameroon, Central Africa.

Principal Findings

All the markers provided support for the current classification within the An. nili group. However, they revealed high genetic heterogeneity within An. nili s.s. in deep equatorial forest environment. Nuclear markers showed the species to be composed of five highly divergent genetic lineages that differed by 1.8 to 12.9% of their Internal Transcribed Spacer 2 (ITS2) sequences, implying approximate divergence time of 0.82 to 5.86 million years. However, mitochondrial data only detected three major subdivisions, suggesting different evolutionary histories of the markers.

Conclusions/Significance

This study enlightened additional cryptic genetic diversity within An. nili s.s. in the deep equatorial forest environment of South Cameroon, reflecting a complex demographic history for this major vector of malaria in this environment. These preliminary results should be complemented by further studies which will shed light on the distribution, epidemiological importance and evolutionary history of this species group in the African rainforest, providing opportunities for in-depth comparative studies of local adaptation and speciation in major African malaria vectors.     

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.     

Thanaka (Limonia acidissima) and deet (di-methyl benzamide) mixture as a mosquito repellent for use by Karen women

The prevention and treatment of drug-resistant malaria is becoming increasingly difficult. On the Thai–Myanmar border multi-drug resistant strains of falciparum malaria are increasing and, because the malaria vector Anopheles bite outdoors during early evening, insecticide house-spraying or impregnated bednets provide only limited protection. Therefore, the protective efficacy of repellent formulations containing di-methyl benzamide (deet) and permethrin against local vectors was estimated, when applied to the skin, and their acceptability amongst pregnant Karen women who are at relatively high risk from malaria was assessed. Human landing catches of mosquitoes showed that almost complete protection was achieved using different formulations of 20% deet and 0.5% permethrin for up to 6 h. All-night collections from human subjects indicated that this repellent combination reduced exposure to malaria parasites by at least 65 and 85% for those transmitted by Anopheles minimus and An. maculatus , respectively, the two principal vectors in this area. Pregnant women in the camps preferred repellents which were mixed with 'thanaka', a root paste made from pulp of the wood apple tree, Limonia acidissima , used locally as a cosmetic. Apart from a temporary warming sensation where repellent thanaka was applied to the skin, the repellents were well tolerated. An intervention trial is currently in progress to determine whether deet mixed with thanaka can protect pregnant women against malaria in this part of the world. Bioassays using a laboratory strain of Aedes aegypti demonstrated that thanaka is itself slightly repellent at high dosages and the mixture with deet provides protection for over 10 h. This treatment would therefore also provide some personal protection against dengue, which is increasing locally, transmitted by Ae. aegypti and Ae. albopictus biting during the daytime.