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.

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.

Abstract. 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 3min 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.

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.

Culicoides imicola Kieffer is considered to be the main vector of bluetongue disease (BT) and African horse sickness (AHS) in the Mediterranean basin. It has been assumed that this midge species is exophilic and, consequently, that stabling of livestock should provide effective protection against these diseases. This study presents the results of sampling surveys for C. imicola carried out both inside and outside stables on three farms in mainland Spain. The number of C. imicola captured varied as a function of the populations sampled and trap location (inside vs. outside). The daily mean number captured inside during the sampling of each farm population was directly correlated with the daily mean number captured outside, but daily correlation of captures was not observed. By contrast with previous studies, the mean catch of C. imicola inside was consistently higher than that outside. No clear effect of stable characteristics on the degree of entry was detected. In addition, proportions of males and age‐graded female groups varied among populations and with trap location. Proportionately more males and fewer engorged females were captured outside than inside, although the proportions varied among stables. These results contrast with those of previous studies, and with the assumed pronounced exophilic behaviour of C. imicola, and raise important questions about the vector activity of this species in the study area and its implications for the epidemiology of BT and/or AHS.

Light microscopy photographs, line illustrations and scanning electron micrographs are provided for first instars of Chrysomya albiceps (Wiedemann), Chrysomya megacephala (Fabricius) and Protophormia terraenovae (Robineau‐Desvoidy) (all: Diptera: Calliphoridae). The following morphological structures are documented: pseudocephalon; antennal complex; maxillary palpus; facial mask; thoracic and abdominal spinulation; spiracular field; posterior spiracles, and cephaloskeleton. New diagnostic features of the cephaloskeleton and the spinulation of abdominal segments are described. Verification of earlier descriptions revealed major discrepancies among published data. The present results allow for the clarification, correction and, especially, complementation of existing information provided by numerous authors. Finally, an identification key for first‐instar larvae of European and Mediterranean Chrysomyinae of forensic importance is presented.

First instars of Lucilia ampullacea Villeneuve, Lucilia caesar Linnaeus, Lucilia cuprina Weidemann, Lucilia richardsi Collin, Lucilia sericata Meigen and Lucilia silvarum Meigen (Diptera: Calliphoridae) are thoroughly documented with scanning electron microscopy images, light microscopy photographs and line drawings. The following morphological structures are documented: pseudocephalon, antennal complex, maxillary palpus, facial mask, cephaloskeleton, thoracic and abdominal spinulation, spiracular field, and posterior spiracles. New diagnostic features of the cephaloskeleton are presented and the spinulation of the abdominal segments is described. Earlier descriptions are summarized and major discrepancies with the current study are discussed. The present results allow for the clarification, correction and, especially, complementing existing information provided by numerous authors. The first instar larva of L. richardsi is described for the first time and an identification key to the first instars of European species of Lucilia Robineau‐Desvoidy of forensic importance is presented.

ABSTRACT. Chorionic structure and size can be of great value in the identification of the eggs of British blowflies of forensic importance. The most useful features are the shape and structure of the plastronic area between the hatching pleats. Correct identification of the eggs of the species considered here would be of use in forensic investigations, not only in Britain, but also in the wide area of the Holarctic region.

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.

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.