Entomological surveys of malaria in Khammouane Province,Lao PDR,in 1999 and 2000

Anopheline mosquitos and their relation to malaria transmission were studied 3 times: in July and August, 1999; in December, 1999; and in August and September, 2000. The studies took place in the malaria endemic villages of Khammouane Province, southeast of Lao PDR. A total of 28 species were collected using human and animal bait. Human bait attracted predominantly Anopheles dirus and An. minimus, which were identified as vectors by the detection of sporozoites by dissection, PCR, and enzyme-linked immunosorbent assays for Plasmodium falciparum and P. vivax. The vectorial capacity of An. dirus was 0.009-0.428, while that of An. minimus was 0.048-0.186. The inoculation rate of An. dirus was 0.052-0.137 (Boualapha; August, 2000). An. nivipes and its sister species, An. philippinensis, were principally zoophilic, although a considerable number of the females were also attracted to human bait in the villages of the paddy field areas. An. philippinensis infected with oocysts of P. vivax was detected in a specimen collected by animal bait. These two species were considered as vectors in Khammouane Province. Four species, An. notanandai, An. sawadwongporni, An. willmori, and An. hodgkini, had not been recored before in Lao PDR. Information is provided on host preference and the nocturnal biting activities of common species and the incidence of malaria in the study areas.     

A rapid polymerase chain reaction based method for identification of the Anopheles dirus sibling species

A simple polymerase chain reaction (PCR) based method was developed to differentiate the Anopheles dirus, species A, B, C and D in Thailand using specific primers designed from species specific sequences. The PCR protocol was optimized to obtain products of 120 bp, 75 bp, 60 bp and 172 bp for species A, B, C and D, respectively. This method used a cocktail of four primer sets to identify these An. dirus sibling species. The method is very sensitive as only a small portion of mosquito was required allowing the rest of the mosquito to be used for other analyses. Specimens also kept for up to 14 years could be analyzed unambiguously from either larvae or adult. This method is advantageous over other PCR-based methods for identification of malaria vectors because it does not require any specific DNA extraction. A mosquito specimen was homogenized in 1x PCR buffer, then the supernatant directly used for PCR identification, allowing a large number of samples to be processed at the same time. It provides a simple and rapid practical method for screening An. dirus species, which is essential in malaria vector epidemiological studies in Southeast Asia.     

Cytogenetic studies of some species complexes of Anopheles in Thailand and Southeast Asia

Recent studies on cytogenetics, behavioral, geographical and distinct morphological characters on adult, pupal and larval stages have revealed that "balabacensis" is a species complex. Anopheles dirus the mainland species, is distributed widely in Thailand and is renowned for its role as primary vector of human malarial parasites. Further, evidence from cytogenetic and taxonomic studies suggests that "An. dirus" is a species complex comprising at least four distinct species provisionally designated: dirus A, B, C and D. These cryptic species are distinguishable only partially morphologically, but can be separated on the basis of metaphase chromosomes using the Giemsa and Hoechst 33258 staining techniques. Apparently, these siblings show distinct patterns of geographic distribution in Thailand and Peninsular Malaysia. The recognition of dirus as a complex of species in Thailand and Peninsular Malaysia requires a re-evaluation of the role that the individual members of this complex have in the transmission of malaria parasites in this region. Cytological analysis of gene rearrangements in ovarian polytene chromosomes has shown that An. maculatus is a sibling-species complex consisting of at least four species in Thailand provisionally designated: maculatus A, B, C and G. These siblings are sympatric in some populations. Furthermore, species B is so highly polymorphic for chromosome rearrangements that four geographic forms can be recognized. It is not known whether these four forms are subspecies or yet further species within the species B complex. These sibling-species must be differentiated in order to understand any differential capabilities in their transmission of human malaria parasites. Anopheles nivipes was elevated from synonymy under An. philippinensis to full species status by Reid, a decision recently confirmed by cross mating experiments. The Thailand Malaria Division does not differentiate these two species and only identifies An. philippinensis, yet, An. nivipes is by far the most common of the two species in Thailand. Furthermore, preliminary surveys of the ovarian polytene chromosomes of several widely separated populations of An. nivipes in Thailand have revealed at least two distinct chromosomal types of nivipes based on fixed inversions on the X chromosomes.     

Population cytogenetics of the malaria vector Anopheles leucosphyrus group

Until recently, very little was known of Anopheles species complexes and their relationships to epidemiology and malaria transmission in Southeast Asia. During the past eight years, extensive studies on the genetics of natural populations of anopheline mosquitoes in this region, involving the interdisciplinary efforts of taxonomists, operational entomologists and biologists, have revealed groups of cryptic species of Anopheles vectors, particularly the An. leucos phyrus group. This species group comprise seventeen species and two subspecies widely distributed in the forested areas of Southeast Asia. Among these species. An. dirus Peyton and Harrison, has been shown by cytogenetic and morphological studies to be a complex of at least seven isomorphic species, provisionally designated species A, B, C, D, E, F and takasagoensis, on the Southeast Asian mainland. Cytological identification of these species is based on distinct banding patterns of salivary gland polytene chromosomes as well as heterochromatin differences in mitotic karyotypes. The five species found in Thailand (A-D, F) exhibit distinct geographic distributions. Species A is widespread throughout Thailand except in the south. Species B had been found in sympatry with species C in southern Thailand and both seem to show north-south clinal geographic variation. Species D is common on the west side of southern Thailand and along the Thai-Burmese border in sympatry with species A. Species F, An. nemophilous Peyton and Ramalingam, has been found in a population at the Thai-Malaysian border in this study although it was known to be common in southern and western Thailand and Peninsular Malaysia. Species E is known only from western India. The five species found in Thailand also exhibit seasonal variation in relative abundance and different nocturnal biting cycles. Chromosomal polymorphisms have been observed in mitotic and polytene chromosomes of An. dirus A and D. Species B and C also show heterochromatin variation in the sex chromosomes, but are monomorphic for the standard sequence in polytene chromosomes. These biological characteristics of the An. dirus complex may have implications for understanding the epidemiology of malaria in Southeast Asia. Recent cytogenetic studies of wild-caught samples of An. leucosphyrus from Sumatra, Kalimantan and southern Thailand have revealed the presence of two distinct species within this taxon. Species A is widely distributed in southern Thailand, East Malaysia and Kalimantan, while species B is confined to Sumatra. The two isomorphic species are vectors of human malaria within their range of distribution.(ABSTRACT TRUNCATED AT 400 WORDS)     

Epidemiological observations on malaria in some parts of Tengakhat PHC, Dibrugarh district, Assam.

A three year study carried out in 44 villages (pop. 17,938) reveals seasonal transmission of malaria i.e., from June to November. The incidence of malaria was high in the year 1988 in comparison to that of 1987 and 1989. P. falciparum was the predominant species. The population living in forest fringe areas were more prone to malaria than the rest. Anopheles nigerrimus, An. kochi, An. karwari and An. philippinensis were most abundant among the 16 anopheline species collected. Known vectors of malaria such as An. minimus, An. philippinensis, An. annularis and An. dirus were detected. An. dirus was incriminated as vector during this study.     

Illustrated keys to the mosquitoes of Thailand. IV. Anopheles

Illustrated keys for the identification of the larvae and adult female Anopheles mosquitoes of Thailand are presented along with distribution maps, tabulated bionomics information, and a checklist. A total of 73 species are treated, including 71 previously and newly described species (An. cracens = dirus B, An. scanloni = dirus C, An. baimaii = dirus D, An. latens = leucosphyrus A, and An. epiroticus = sundaicus A). Also, two undescribed species are included, i.e., An. minimus C and a new species near An. gigas. Thirty-four chromosomal forms of 14 species are discussed, with suggestions provided for resolving their taxonomic status.     

Malaria transmission by Anopheles dirus in Attapeu Province, Lao PDR

A study was carried out in four malaria-endemic villages in Attapeu Province, in the southern region of Lao PDR. All-night human landing collections were carried out in May, August, and October 2002, to determine malaria vectors. At the same time, mass blood surveys were also carried out in the same villages. Anopheles dirus was the predominant species in three of the study villages. Sporozoites were found only in An. drius from Phou Hom. However, in Beng Phoukham, An. dirus was positive for oocysts. The distribution of malaria cases was highest in Phou Hom and this correlated well with the vectorial capacity of An. dirus. The risk for infection from An. dirus was also high, at 0.99.     

Behavioural heterogeneity of Anopheles species in ecologically different localities in Southeast Asia: a challenge for vector control

In Southeast Asia the biodiversity of Anopheles species in the domestic environment is very high. Only few species are considered major vectors throughout the region, whereas the vector status of other species varies from area to area. Often it is difficult to identify an Anopheles species as a malaria vector in areas with low malaria incidence. The behaviour of Anopheles species largely determines their vector status, and insights into their behaviour are essential to evaluate the appropriateness of vector control measures. This study was conducted in six ecologically different localities in Southeast Asia to rank the different Anopheles species in terms of anthropophily and endophagy in order to estimate their current epidemiological importance. Concurrently, the biting and resting behaviour of the vectors was analysed to evaluate the appropriateness of insecticide-impregnated bed nets and residual house spraying in vector control. Anopheles dirus A was highly anthropophilic at all sites where it occurred. By contrast, the degree of anthropophily exhibited by An. minimus A depended on availability of cattle. Anopheles campestris, An. nimpe, An. sinensis, An. maculatus, An. aconitus showed a high degree of anthropophily in certain villages, indicating their potential of participating in malaria transmission, although the actual incidence of malaria in the study villages can be fully explained by transmission of the major vectors (An. dirus A, An. minimus A and An. sundaicus). Late biting of An. minimus A and biting activity throughout the night of An. sundaicus favour bed nets as a control method for these species, whilst exophilic and outdoor biting in combination with early feeding behaviour of An. dirus A will make both insecticide-impregnated bed nets and indoor residual spraying less suitable for controlling this species. Spatial variation in biting and resting behaviour was observed within almost all Anopheles species. These heterogeneities may result in the differences in epidemiological importance and in response to vector control of Anopheles species in different areas. Moreover, environmental changes and changes in human practice are expected to influence the behaviour, hence the role of the different species in malaria transmission. The effect of environmental changes on vector behaviour should be followed up carefully.     

Molecular characterization and species identification of the Anopheles dirus and An. minimus complexes in north-east India using r-DNA ITS-2

The sibling species composition of the Anopheles minimus and Anopheles dirus complexes is poorly known in the highly malarious north-eastern region of India where these two vector taxa are accountable for most of the malaria transmission among 30.7 million inhabitants. Prevalent members of these two complexes in this part of India were identified using sequences for the second internal transcribed spacer (ITS2) of ribosomal DNA. Anopheles baimaii (species D) of the An. dirus complex and An. minimus s.s. (species A) of the An. minimus complex were detected in Arunachal Pradesh, Assam, Meghalaya and Nagaland states. No intraspecific variation was observed in the ITS2 sequence (479bp) of An. baimaii whereas a single substitution was detected in the ITS2 sequence (372bp) of An. minimus from Nagaland state.     

Natural human humoral response to salivary gland proteins of Anopheles mosquitoes in Thailand

During blood feeding, arthropod vectors inject saliva into vertebrate hosts. The saliva is biochemically complex and pharmacologically active, and may play an important role in pathogen transmission. To examine whether mosquito saliva could elicit humoral immune response in humans under natural conditions, we have collected sera from malaria patients, healthy villagers, and people from a non-malarious region in Thailand. Here we have demonstrated that anti-Anopheles salivary protein antibodies occurred predominantly in patients with acute Plasmodium falciparum or P. vivax malaria, whereas people from a non-malarious area had no such antibodies. Besides, antibody levels against mosquito salivary proteins in malaria patients were highly variable, which may be related to the levels of mosquito exposure. Despite variability, patients' sera with high IgG titers consistently detected several proteins in Anopheles dirus salivary gland protein extracts. Immunohistochemical staining of Anopheles salivary glands with human sera showed that the salivary gland-specific IgGs reacted strongly with the median lobe. Comparison using Anopheles and Aedes salivary proteins suggests that the anti-salivary protein antibodies detected in malaria patients were Anopheles-specific, consistent with the major malaria vector status of An. dirus in this area.     

Malaria transmission and major malaria vectors in different geographical areas of Southeast Asia

During the last decade, major progress in malaria control has been achieved in Vietnam, Laos and Cambodia. However, malaria is still a potentially fatal disease in some hilly-forested areas and continues to be endemic in a few coastal foci. To estimate the risk that stems from the major vectors after a decade of intensive malaria control, an entomological study based on human landing collections was conducted between April 1998 and November 2000 in six study villages (four in Vietnam, one in Cambodia and one in Laos) located in different physio-geographical areas. Five villages were selected in places where new cases of malaria still occurred. In the sixth village, in the northern hilly area of Vietnam, no case of malaria was detected during the past 3 years. In three study villages of the hilly forested areas of Cambodia and central Vietnam, Anopheles dirus A still played an important role in malaria transmission and maintain perennial transmission inside the villages despite its low density. Anopheles minimus A was found in all study villages except in the southern coastal village of Vietnam. Its role in malaria transmission, however, varied between localities and surveys. In one study village of central Vietnam it was almost absent (one specimen collected over 480 man nights), and in another village sporozoite positive specimens (2.8%) were only observed during the first two surveys whereas this species disappeared from the collections from November 1998 onwards (six surveys: 360 man nights). In the northern study site An. minimus A and C were found in all collections, but no local malaria transmission occurred. However, the constant presence of these two species associated with a high longevity (parous rate up around 80% and 65%, respectively), suggests that transmission can occur at almost any time if parasite reservoirs are reintroduced in the area. The proper management of malaria cases and population movement is, therefore, important to prevent outbreaks and the reintroduction of malaria in northern Vietnam. In the study site of the Mekong delta, An. sundaicus occurred at high densities (up to 190 bites/man/night). The recent changes in land use from rice cultivation to shrimp farming probably explains the increase of this brackish water breeding species during the study period. However, none of the 11,002 specimens was positive for Plasmodium circumsporozoite protein (CSP). The relative low survival rate as estimated by the parous rate (around 47%) may reflect its low vectorial status that could explain the very low malaria incidence (1.9 case/100 persons/year) in this study site. A calculated sporozoite rate of maximum 1/300,000 is enough to explain this low malaria incidence. Despite the successes in malaria control, the vector An. dirus A continues to play an important role in malaria transmission, whereas An. minimus A showed temporal and spatial variation in its role as vector. The role of An. sundaicus as vector could not be confirmed because of the low incidence in the coastal study village. Other Anopheles species may be locally involved, but in the five study villages where malaria is still present they probably do not contribute significantly to malaria transmission. The study also points towards the fact that in Southeast Asia it will become increasingly difficult to incriminate Anopheles species in malaria transmission while the risk for malaria transmission still persist.     

The importance of Anopheles dirus (A. balabacensis) as a vector of malaria in northeast India

Anopheles dirus (A. balabacensis) is the most prevalent among twelve anopheline species collected during the course of entomological studies carried out in Changlang district of Arunachal Pradesh during July and August, 1988. It is observed to be a forest species and is found to breed in small stagnant water pools completely or partially shaded by plants in the open jungle. Sporozoites have been detected in several specimens of A. dirus (A. balabacensis) supporting its role in transmission of malaria in forest areas.     

Soil analysis around anopheline breeding habitats in north-western Thailand

The epidemiology of malaria is largely dependent on its vector habitat. Each species of Anopheles larvae has a specific habitat requirement for its development. Anopheline mosquitoes are common throughout Thailand and utilize a wide variety of habitats. The dominant malaria vectors in Thailand are An. dirus, An. maculatus, and An. minimus. The relationship between soil chemical components and the particular species of anopheline in their specific aquatic habitats was studied from September 2002 to July 2003 at Ban Khun Huay, Ban Pa Dae, and Ban Tham Seau in the Mae Sot district, Tak Province, Thailand. Mapping of each habitat was performed using a Global Positioning System unit. A total count of 2,130 laboratory reared adult Anopheles were collected from 138 habitats categorized into 11 different types identified into 18 species from larval sampling in three villages. An. dirus, An. maculatus, and An. minimus were found 5.26%, 10.70%, and 55.31%, respectively, along with other minor species. Drainage and/or season seemed to be associated with the presence of An. dirus, An. maculatus, An. minimus, An. jamesii, An. sawadwongporni, and An. peditaeniatus. Chemical tests: pH, aluminum, magnesium, calcium, and ferric iron showed some associations with the presence of Anopheles. Only drainage was found to be a parameter associated with the presence of An. minimus.     

Incrimination of Anopheles dirus as a vector of malaria in Dibrugarh district, Assam

Anopheles nigerrimus, A. vagus, A. dirus, A. karwari and A. kochi were the most abundant species among 12 anopheline species collected during the course of entomological studies carried out from July to September, 1988 in an area endemic for malaria under Tengakhat PHC of Dibrugarh district, Assam. Sporozoites were detected in the salivary glands of one specimen of A. dirus, Peyton & Harrison (A. balabacensis) which constituted about 18.11 per cent of the total anopheline collection.     

2001-2008年海南省恶性疟发病率与大劣按蚊捕获数关系

目的分析海南省恶性疟发病率与大劣按蚊捕获数的相关性,为有针对性地开展海南省恶性疟防控工作提供依据。方法计算2001-2008年五指山市和琼中县恶性疟发病率和捕获的大劣按蚊数,采用Cochran-Armitage趋势检验分析两个地区恶性疟发病率的变化趋势;采用Spearman秩相关分析,探讨两个地区恶性疟发病率与大劣按蚊数量的相关性。结果 2001-2008年两个地区恶性疟发病率存在波动,但总体呈显著下降的趋势（Z五指山=-6.16,P〈0.01;Z琼中=-7.17,P〈0.01）;两个地区大劣按蚊捕获量均存在较大波动,并且恶性疟发病率与大劣按蚊捕获量均无显著相关性（r五指山=0.36,P〉0.05;r琼中=0.67,P〉0.05）。结论恶性疟的防控不仅要加强灭蚊工作,更要从环境治理、易感人群保护等多个角度入手,开展全方位的防控工作。     

Malaria prevalence and a brief entomological survey in a village surrounded by rice fields in Khammouan province, Lao PDR

We surveyed Nongceng, a village in a south-eastern province of Lao PDR, for malaria and its vectors. Nongceng is situated in a basin and surrounded by rice fields. In February 1998 (dry season), 28.6% of 126 villagers were infected with malaria, and in September 1998 (rainy season), 16.3% of 147 villagers. The prevalence of malaria infection was consistently high in children under 10, and the predominant malaria species was Plasmodium falciparum. In brief surveys of the mosquitoes performed on the same day as the malaria surveys, 2007 Anopheles females from 12 species were collected by means of human bait, animal bait and resting collections. Of the vector species known to be important in transmitting malaria in neighbouring Thailand - An. minimus, An. dirus, and An. maculatus groups - only An. minimus was found. Its density was, however, very low in both seasons and it was therefore unlikely to be the vector. In fact, An. nivipes accounted for more than 65% of all mosquitoes collected and was the most common species collected from human baits. The results of this study show that endemic areas of malaria in Lao PDR are not necessarily related to forest. Rather, An. nivipes is suspected to be the most important vector.     

用聚合酶链反应区分我国大劣按蚊复合体A和D种

目的：建立一种鉴别大劣按蚊复合体Ａ和Ｄ种的ＰＣＲ检测技术。方法：根据大劣按蚊Ａ和Ｄ种核糖体ＤＮＡ第二内转录间隔区的序列差异，设计种特异引物，通过ＰＣＲ扩增出种特异长度（Ａ种为３７４ｂｐ，Ｄ种为６６３ｂｐ）的片段区分蚊种。结果：该法敏感性达１／１６００单蚊抽提ＤＮＡ或１／５单条蚊腿水匀浆ＤＮＡ。应用该法检测大劣按蚊ＡＦＲＩＭＳ实验室品系１０例，ＨＮ实验室品系２０例，以及采自海南省白沙、和平、罗葵、毛阳等地野外样本１４８例，均显示Ａ种特异带；云南省勐腊地区野外样本３０例，全部为Ｄ种特异带。结论：提供一种简便可靠的蚊种鉴别ＰＣＲ法，确认我国海南和云南的大劣按蚊分别为Ａ种和Ｄ种，为深入研究我国大劣按蚊复合体不同成员种的地理分布、生态习性和传疟作用创造了条件。     

Estimation of vectorial capacity of Anopheles dirus (Diptera: Culicidae) in a forest-fringed village of Assam (India)

To estimate the vectorial capacity of Anopheles dirus, the main vector of forest malaria in the northeastern region of India, in order to gain an understanding of entomological factors related to malaria transmission in forest-fringe areas of Assam, India, an isolated village in the tropical rain forest-fringed area in the district of Dibrugarh, Assam, under the influence of An. dirus alone was studied. Data on various entomological variables required for computation of the vectorial capacity were generated in each month from June 1999 to May 2000 in the field using standard techniques. Malaria prevalence was also studied during the same period in the study village and correlated with the estimated vectorial capacity of An. dirus. Vectorial capacity of An. dirus was highest, 0.779 for Plasmodium vivax (Pv) and 0.649 for Plasmodium falciparum (Pf), during the hot-monsoon season (June-September) and decreased to 0.08 (Pv) and 0.07(Pf) in the temperate postmonsoon season (October-November) before attaining zero values in the cool-dry season (December-February). With increasing temperature in the temperate premonsoon season (March-May), vectorial capacity recorded was 0.119 and 0.82 for Pv and Pf, respectively. Significant positive correlation was seen between the estimated vectorial capacity of An. dirus and the number of new Pf (r = 0.86, p < 0.001) and Pv (r = 0.69, p < 0.02) cases in the study village in different months. Thus, this study highlights the pattern of malaria transmission by An. dirus in a forest-fringe area of Assam that begins in March, peaks in July/August, subsides by November, and remains interrupted between December and February. Measures for controlling malaria in forest-fringe areas should be scheduled accordingly.     

Status of malaria in Thailand

Despite decades of control success and a competent network of country-wide health infrastructure, malaria remains an important health threat in rural Thailand. All 4 known human malaria parasites have been reported present, with Plasmodium falciparum and Plasmodium vivax predominant. The expansion and intensity of multi-drug resistant Plasmodium falciparum is the most serious development to occur the last several decades. Members of 3 anopheline species complexes, Anopheles dirus, Anopheles minimus, and Anopheles manculatus, are considered to be primary malaria vectors in the country. Representatives within all 3 taxa are difficult or impossible to separate morphologically from one another, and insufficient information exists about population genetics between sibling species and vector status. Vector control in Thailand has been the primary means of malaria control, mainly by the use of routine residual insecticide spray inside houses. The use of DDT in vector control has resulted in measurable successes to interrupt malaria transmission in many parts of the country. Since 1949, DDT has been the predominant compound used: however, its public health use has continued to decline as a result of perceived operational difficulties, political issues and environmental concerns. The increased use of pyrethroids to impregnate bednets and for intradomiciliary spraying are generally more accepted by rural populations and are rapidly replacing the use of DDT. Organized malaria control activities have reduced malaria morbidity from 286/1,000 population in 1947 to 1.5/1,000 population by 1996. Despite encouraging trends in dramatically reducing malaria, the rates of disease may be re-emerging in the country as evidence from an increased annual parasite index from 1.78/1,000 in 1997 to 2.21 in 1998. The possible reasons for the apparent increase in incidence are discussed in terms of the technical, operational and social obstacles in malaria control in Thailand.     

Confirmation of Anopheles varuna in vietnam, previously misidentified and mistargeted as the malaria vector Anopheles minimus.

Malaria control programs in Southeast Asia are faced with several questions concerning vector behavior and species identification, which need to be answered to consolidate and further improve the results of control practices. The vector system in Southeast Asia is complex because of the number of species potentially involved in malaria transmission. Additionally, the follow-up and evaluation of preventive control measures are hampered by the misidentification of vectors due to overlapping morphological characters of the female mosquitoes. In central Vietnam, control practices are aimed at 2 main species, Anopheles dirus s.l. and Anopheles minimus s.l. These reputed vectors were studied in an area of Binh Thuan Province of south-central Vietnam. Different collection methods were used to capture mosquitoes quarterly during a 1-year period. Mosquitoes were identified in the field and later subjected to detailed morphological examination and polymerase chain reaction-restriction fragment length polymorphism analysis. What was thought to be an unusual morphotype of An. minimus was shown to be Anopheles varuna, and most specimens identified as the former species in the field proved to be the latter species. Very few An. minimus individuals were found during the study period. The population of An. varuna was found to be highly zoophilic, and based on this behavior, it cannot be considered a vector in Vietnam. Because this species was previously being misidentified as An. minimus, a nonvector was mistargeted as a malaria vector in Binh Thuan Province. Anopheles dirus, which was found positive for Plasmodium falciparum circumsporozoite via enzyme-linked immunosorbent assay, is clearly the main vector in this area. Despite the fact that several potential secondary vectors were found during the study, the primary target for vector control in the region should be An. dirus.