Medical and Veterinary Entomology

Tỉnh Bắc KwaZulu/Natal (KZN) của Nam Phi nằm giáp Mozambique ở phía nam, giữa Swaziland và Ấn Độ Dương. Để kiểm soát các vector gây bệnh sốt rét tại KZN, các ngôi nhà được phun hàng năm với DDT tồn dư 2 g/m² cho đến năm 1996 khi việc xử lý chuyển sang deltamethrin 20–25 mg/m². Tại Ndumu (27°02′ S, 32°19′ E), tỷ lệ bệnh sốt rét được ghi nhận đã tăng gấp sáu lần từ 1995 đến 1999. Các khảo sát Entomological cuối năm 1999 phát hiện nhóm muỗi Anopheles funestus (Diptera: Culicidae) trú trong các ngôi nhà đã phun ở một số khu vực của Ndumu. Loài vector rất nội trú này đã bị loại khỏi Nam Phi bởi phun DDT trong những năm 1950, khiến An. arabiensis Patton, ít nội trú hơn, trở thành vector quan trọng duy nhất tại KZN. Các ngôi nhà được phun deltamethrin tại Ndumu đã được kiểm tra tính hiệu quả của thuốc diệt côn trùng bằng phương pháp sinh học với An. arabiensis dễ nhiễm bệnh (được nuôi tại phòng thí nghiệm) và cho thấy tỷ lệ chết 100%. Các thành viên của nhóm An. funestus từ các ngôi nhà ở Ndumu (29 con đực, 116 con cái) đã được xác định bằng phương pháp rDNA PCR và phát hiện có bốn loài: 74 An. funestus Giles sensu stricto, 34 An. parensis Gillies, bảy An. rivulorum Leeson và một An. leesoni Evans. Trong số các con cái An. funestus s.s., 5,4% (4/74) dương tính với Plasmodium falciparum qua các xét nghiệm ELISA và PCR. Để kiểm tra kháng pyrethroid, những con muỗi trưởng thành được tiếp xúc với liều phân biệt permethrin và tỷ lệ tử vong được ghi chép 24 giờ sau khi tiếp xúc: tỷ lệ sống sót của đực mạnh bắt tự nhiên là 5/10 An. funestus, 1/9 An. rivulorum và 0/2 An. parensis; tỷ lệ sống sót của thế hệ trưởng thành nuôi từ 19 con cái An. funestus trong phòng thí nghiệm trung bình 14% (sau 1 giờ tiếp xúc với 1% permethrin 25 : 75 cis : trans trên giấy trong bộ thử nghiệm WHO) và 27% (sau 30 phút trong chai chứa 25 μg permethrin 40 : 60 cis : trans). Những Anopheles funestus có tỷ lệ sống sót > 20% trong hai phương pháp thử nghiệm khả năng kháng này gồm 5/19 và 12/19. Thế hệ từ 15 gia đình được thử nghiệm trên giấy tẩm DDT 4% và cho kết quả tử vong 100%. Việc phát hiện tỷ lệ kháng pyrethroid này, gắn liền với sự gia tăng bệnh sốt rét tại Ndumu, có ý nghĩa nghiêm trọng đối với hoạt động kiểm soát vector truyền bệnh sốt rét ở miền nam châu Phi.

Samples of the dengue vector mosquito Aedes aegypti (L.) (Diptera: Culicidae) were collected from 13 localities between 1995 and 1998. Two laboratory strains, Bora (French Polynesia) and AEAE, were both susceptible to DDT and permethrin; all other strains, except Larentuka (Indonesia) and Bouaké (Ivory Coast), contained individual fourth‐instar larvae resistant to permethrin. Ten strains were subjected to a range of biochemical assays. Many strains had elevated carboxylesterase activity compared to the Bora strain; this was particularly high in the Indonesian strains Salatiga and Semarang, and in the Guyane strain (Cayenne). Monooxygenase levels were increased in the Salatiga and Paea (Polynesia) strains, and reduced in the two Thai strains (Mae Kaza, Mae Kud) and the Larentuka strain. Glutathione S‐transferase activity was elevated in the Guyane strain. All other enzyme profiles were similar to the susceptible strain. The presence of both DDT and pyrethroid resistance in the Semarang, Belem (Brazil) and Long Hoa (Vietnam) strains suggested the presence of a knock‐down resistant (kdr)‐type resistance mechanism. Part of the S6 hydrophobic segment of domain II of the voltage‐gated sodium channel gene was obtained by RT‐PCR and sequenced from several insects from all 13 field strains. Four novel mutations were identified. Three strains contained identical amino acid substitutions at two positions, two strains shared a different substitution, and one strain was homozygous for a fourth alteration. The leucine to phenylalanine substitution that confers nerve insensitivity to pyrethroids in a range of other resistant insects was absent. Direct neurophysiological assays on individual larvae from three strains with these mutations demonstrated reduced nerve sensitivity to permethrin or lambda cyhalothrin inhibition compared to the susceptible strains.

The permethrin tolerance (PT) of a population of the mosquito Anopheles gambiae (Diptera: Culicidae) increased following the introduction of permethrin‐impregnated nets for malaria control in certain villages near Kisumu, western Kenya. Using a biochemical test that indirectly measures oxidases associated with permethrin resistance, we found that this population had higher oxidase levels than a comparison population from villages without impregnated nets. Mosquitoes from a colony of An. gambiae selected for PT, the RSP (reduced susceptibility to permethrin) strain, were exposed to permethrin with or without the oxidase inhibitor piperonyl butoxide (PB). Significantly higher mortality rates occurred when permethrin was synergized by PB, presumably by suppression of oxidases responsible for PT. An unselected (UNS) colony of An. gambiae that was more susceptible than RSP in a permethrin‐susceptibility bioassay (i.e. LT₅₀ 22 min for UNS, vs. 42 min for RSP) was compared with the RSP colony for levels of oxidases and esterases. The levels of both enzymes were very significantly higher in the RSP strain (P < 0.0001). We speculate that use of impregnated nets selected for higher oxidase and esterase levels in An. gambiae to metabolize permethrin acquired from the nets. Both oxidase and esterase mechanisms could confer cross‐resistance to other pyrethroids.

Abstract.  The mosquito Anopheles stephensi Liston (Diptera: Culicidae) is the urban vector of malaria in several countries of the Middle East and Indian subcontinent. Extensive use of residual insecticide spraying for malaria vector control has selected An. stephensi resistance to DDT, dieldrin, malathion and other organophosphates throughout much of its range and to pyrethroids in the Middle East. Metabolic resistance mechanisms and insensitivity to pyrethroids, so‐called knockdown resistance (kdr), have previously been reported in An. stephensi. Here we provide molecular data supporting the hypothesis that a kdr‐like pyrethroid‐resistance mechanism is present in An. stephensi. We found that larvae of a pyrethroid‐selected strain from Dubai (DUB‐R) were 182‐fold resistant to permethin, compared with a standard susceptible strain of An. stephensi. Activities of some enzymes likely to confer pyrethroid‐resistance (i.e. esterases, monooxygenases and glutathione S‐transferases) were significantly higher in the permethrin‐resistant than in the susceptible strain, but the use of synergists — piperonyl butoxide (PBO) to inhibit monooxygenases and/or tribufos (DEF) to inhibit esterases — did not fully prevent resistance in larvae (permethrin LC₅₀ reduced by only 51–68%), indicating the involvement of another mechanism. From both strains of An. stephensi, we obtained a 237‐bp fragment of genomic DNA encoding segment 6 of domain II of the para type voltage‐gated sodium channel, i.e. the putative kdr locus. By sequencing this 237 bp fragment, we identified one point mutation difference involving a single A–T base change encoding a leucine to phenylalanine amino acid substitution in the pyrethroid‐resistant strain. This mutation appears to be homologous with those detected in An. gambiae and other insects with kdr‐like resistance. A diagnostic polymerase chain reaction assay using nested primers was therefore designed to detect this mechanism in An. stephensi.

Abstract. Mosquito responses to carbon dioxide were investigated in Noungou village, 30 km northeast of Ouagadougou in the Sudan savanna belt of Burkina Faso, West Africa. Species of primary interest were the main malaria vectors Anopheles gambiae S.S. and An.arabiensis, sibling species belonging to the An.gambiae complex. Data forAn.finestus, An.pharoensis, Culex quinquefasciatus and Mansonia uniformis were also analysed.

Carbon dioxide was used at concentrations of 0.04‐0.6% (cf. 0.03% ambient concentration) for attracting mosquitoes to odour‐baited entry traps (OBETs). The ‘attractiveness’ of whole human odour was also compared with CO₂, emitted at a rate equivalent to that released by the human bait. In a direct choice test with two OBETs placed side‐by‐side, the number of An.gambiae s. I. entering the trap with human odour was double the number trapped with CO₂, alone (at the human equivalent rate), but there was no significant difference between OBETs for the other species of mosquitoes. When OBETs were positioned 20 m apart, again CO₂, alone attracted half as many An.gambiae s.l. and only 40% Anlfunestus, 65% Ma.uniformis but twice as many An.pharoensis compared to the number trapped with human odour.

The dose‐response for all mosquito species was essentially similar: a linear increase in catch with increasing dose on a log‐log scale. The slopes of the dose‐response curves were not significantly different between species, although there were significant differences in the relative numbers caught. If the dose‐response data are considered in relation to a standard human bait collection (HBC), however, the behaviour of each species was quite different. At one extreme, even the highest dose of CO₂, did not catch more An.gambiae s.1. than one HBC. At the other extreme, the three highest doses of CO₂, caught significantly more Ma.unifonnis than did one HBC. An.pharoensis and Cx quinquefasciatus showed a threshold response to CO₂, responding only at doses above that normally released by one man. An.funestus did not respond to CO₂, alone at any dose in sufficient numbers to assess the dose response. Within the An.gambiae complex, An.arabiensis 'chose' the CO₂,‐baited trap with a higher probability than An.gambiae S.S. Also An.arabiensis, the less anthropophilic of the two species, was more abundant in CO₂,‐baited OBETs than in human bait collections.

AbstractStudies were carried out in Zimbabwe of the responses of tsetse to cattle treated with deltamethrin applied to the parts of the body where most tsetse were shown to land. Large proportions ofGlossina pallidipesAusten (Diptera: Glossinidae) landed on the belly (∼ 25%) and legs (∼ 70%), particularly the front legs (∼ 50%). Substantial proportions ofGlossina morsitans morsitansWestwood landed on the legs (∼ 50%) and belly (25%), with the remainder landing on the torso, particularly the flanks (∼ 15%). Studies were made of the knockdown rate of wild, femaleG. pallidipesexposed to cattle treated with a 1% pour‐on or 0.005% suspension concentrate of deltamethrin applied to the (a) whole body, (b) belly and legs, (c) legs, (d) front legs, (e) middle and lower front legs, or (f) lower front legs. The restricted treatments used 20%, 10%, 5%, 2% or 1% of the active ingredient applied in the whole‐body treatments. There was a marked seasonal effect on the performance of all treatments. With the whole‐body treatment, the persistence period (knockdown > 50%) ranged from ∼ 10 days during the hot, wet season (mean daily temperature > 30 °C) to ∼ 20 days during the cool, dry season (< 22 °C). Restricting the application of insecticide reduced the seasonal persistence periods to ∼ 10–15 days if only the legs and belly were treated, ∼ 5–15 days if only the legs were treated and < 5 days for the more restricted treatments. The restricted application did not affect the landing distribution of tsetse or the duration of landing bouts (mean = 30 s). The results suggest that more cost‐effective control of tsetse could be achieved by applying insecticide to the belly and legs of cattle at 2‐week intervals, rather than using the current practice of treating the whole body of each animal at monthly intervals. This would cut the cost of insecticide by 40%, improve efficacy by 27% and reduce the threats to non‐target organisms and the enzootic stability of tick‐borne diseases.

Abstract. The oral susceptibility of 22 South African livestock associated Culicoides species to infection with bluetongue virus serotype 1 (BTV‐1) and its replication rate in C. imicola Kieffer and C. bolitinos Meiswinkel (Diptera: Ceratopogonidae) over a range of different incubation periods and temperatures are reported. Field‐collected Culicoides were fed on sheep blood containing 7.5 log₁₀TCID₅₀/mL of BTV‐1, and then held at constant different temperatures. Virus replication was measured over time by assaying individual flies in BHK‐21 cells using a microtitration procedure. Regardless of the incubation temperatures (10, 15, 18, 23.5 and 30°C) the mean virus titre/midge, infection rates (IR) and the proportion of infected females with transmission potential (TP = virus titre/midge ≥ 3 log₁₀ TCID₅₀) were found to be significantly higher in C. bolitinos than in C. imicola. Results from days 4–10 post‐infection (dpi), at 15–30°C, shows that the mean IR and TP values in C. bolitinos ranged from 36.7 to 87.8%, and from 8.4 to 87.7%, respectively; in C. imicola the respective values were 11.0–13.7% and 0–46.8%. In both species the highest IR was recorded at 25°C and the highest TP at 30°C. The time required for the development of TP in C. bolitinos ranged from 2 dpi at 25°C to 8 dpi at 15°C. In C. imicola it ranged from 4 dpi at 30°C to 10 dpi at 23.5°C; no individuals with TP were detected at 15°C. There was no evidence of virus replication in flies held at 10°C. When, at various points of incubation, individual flies were transferred from 10°C to 23.5°C and then assayed 4–10 days later, virus was recovered from both species. The mean virus titres/midge, and proportion of individuals with TP and IR, were again significantly higher in C. bolitinos than in C. imicola. Also the infection prevalence in C. magnus Colaço was higher than in C. imicola. Low infection prevalences were found in C. bedfordi Ingram & Macfie, C. leucostictus Kieffer, C. pycnostictus Ingram & Macfie, C. gulbenkiani Caeiro and C. milnei Austen. BTV‐1 was not detected in 14 other Culicoides species tested; however, some of these were tested in limited numbers. The present study indicates a multivector potential for BTV transmission in South Africa. In C. imicola and C. bolitinos the replication rates are distinct and are significantly influenced by temperature. These findings are discussed in relation to the epidemiology of bluetongue in South Africa.

Triatoma infestans (Hemiptera: Reduviidae) from 22 Andean localities in Bolivia (n=968) and Peru (n=37) were analysed by multi‐locus enzyme electrophoresis. Among 12 gene–enzyme systems analysed, GPD, 6GPD and PGM were polymorphic, ACON, G6PD, GPI, 1DH, LAP, MDH, ME, PEP‐A and PEP‐B were monomorphic. Allozyme frequencies were analysed in relation to geographical and climatic factors, and the presence or absence of Trypanosoma cruzi infection. At one locality (Vallegrande, Bolivia), the frequency of 6Pgd‐1 was significantly higher in infected (41% of 85) than in uninfected (17% of 83) adult T. infestans, although no such difference was found among nymphs (n = 347). From other localities, only insects infected with T. cruzi were subjected to isozyme analysis. Populations of T. infestans within villages showed panmixia, while genetic differentiation of T. infestans between villages was correlated with the distance between them. The genetic structure of T. infestans natural populations followed an ‘isolation by distance’ model, involving a series of founder effects followed by genetic drift, rather than adaptation in response to differential selection pressures. This conforms with circumstantial evidence that T. infestans spread, mainly in association with recent human migrations, from a source, probably in southern Bolivia. Isoenzyme characterization of populations of T. infestans could be used to infer sources of re‐infestation during the surveillance phase of control programs.

ABSTRACT. The visceral leishmaniasis (VL) vector Phlebotomus argentipes Annandale & Brunetti is widely distributed throughout the Indian sub‐continent and S.E. Asia. The absence of VL in areas such as Sri Lanka has been attributed to the zoophilic nature of P.argentipes, since they were not recorded biting man.

Field studies on P. argentipes were undertaken in the central highlands of Sri Lanka, near Kandy, in May 1988. Male sandflies outnumbered females on cows by 19:1, and were regularly spaced at all densities. This behaviour is considered analagous to swarming in other Nematocera. However, all‐night human‐biting catches show the biting rate to be similar (mean=8.4, range 2–25 bites per night over ten consecutive nights) to that in N.E. India where VL is endemic. This anthropophagy was maintained during laboratory colonization.