Geographical distribution of Anopheles minimus species A and C in western Thailand

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1 Vol. 30, no. 2 Journal of Vector Ecology 225 Geographical distribution of Anopheles minimus species A and C in western Thailand Ampornpan Kengluecha 2, Pornpimol Rongnoparut 1,4, Soamrutai Boonsuepsakul 1, Ratana Sithiprasasna 2, Prinyada Rodpradit 1, and Visut Baimai 3,4 1 Department of Biochemistry, Faculty of Science, Mahidol University, Bangkok, Thailand 2 Department of Entomology, Armed Forces Research Institute of Medical Sciences, Bangkok, Thailand 3 Department of Biology, Faculty of Science, Mahidol University, Bangkok, Thailand 4 Center for Vectors and Vector-Borne Diseases, Faculty of Science, Mahidol University, Bangkok, Thailand Received 4 January 2005; Accepted 3 April 2005 ABSTRACT: Elucidating vector distribution based on an accurate species identification is important to understanding the nature of the species complex in order to achieve vector control. Morphologically, An. minimus s.l. is difficult to distinguish from both its species complex and its closely related species. A polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) technique and a single multiplex-allele specific PCR developed for species identification were applied in this study in comparison with morphological identification. Both methods were used, combining with geographical information systems to determine the distribution of An. minimus species A and C. The investigation on the breeding habitats was performed in the malarious area of western Thailand. Anopheles larvae were collected from 36 bodies of water among five districts (Sangkhaburi, Thong Pha Phum, Si Sawat, Muang, and Sai Yok) of Kanchanaburi Province, Thailand. In this study, An. minimus A larvae were present in all study districts but the association differed when focusing on study sites within each district. Although there were many reports of An. minimus A in Ban Phu Rat and Ban Phu Toei villages in Sai Yok District, we did not find the breeding sites of species A in those two areas. An. minimus A and C were found in Ban Phu Ong Ka village in Sai Yok District. The breeding habitats of An. minimus C were present covering km of distance in northern part of Sai Yok and this species was also found in the central and southern parts of Si Sawat District. Journal of Vector Ecology 30 (2): Keyword Index: Anopheles minimus, species distribution, molecular identification, malaria, Thailand. INTRODUCTION Thailand, a developing country in Southeast Asia confronting endemic malaria, needs reliable surveillance programs to understand and manage its malaria problems. Understanding the spatial and temporal changes in anopheline mosquito abundance, quantification of transmission potential of vector populations, and description of distributions of host (human) populations are necessary prerequisites for predicting high-risk malaria areas. Anopheles minimus sensu lato (subgenus Cellia, Myzomyia Series and Funestus Group, Minimus subgroup) (Harbach 2004) is a major malaria vector throughout its distribution, extending from Uttar Pradesh in India to Southeast Asia, China, Taiwan, and Japan. In Thailand, its distribution is reduced in the peninsula and was not observed in central plains but remains abundant in forested hilly areas (Harrison 1980). This species plays an important role in malaria transmission during the dry season and during much of rainy season when populations of Anopheles dirus are low at the Thai-Myanmar border (Sithiprasasna et al. 2003). It is known as a species complex comprising two species on mainland Southeast Asia, informally designated An. minimus A and C (Harbach 2004). Species A has been found predominantly in Thailand and has been recorded in Vietnam, Laos, Cambodia, and China (Chen et al. 2002, Green et al. 1990, Kengne et al. 2001, Van Bortel et al. 1999). Species C was only recorded in three provinces in western and northern Thailand (Kanchanaburi, Tak, and Chiang Mai) (Green et al. 1990, Sharpe et al. 1999), northern Vietnam (Kengne et al. 2001, Van Bortel et al. 1999) and south-central provinces of China (Chen et al. 2002). The differences between the two species in their biting and resting behavior have been studied. Species A is more anthropophilic and endophilic than species C in northern Vietnam (Van Bortel et al. 1999), while in Thailand both species tend to feed from cows rather than humans (Rwegoshora et al. 2002). Misidentification using morphological characters to separate An. minimus s.l. from closely related species could occur (Harrison 1980). Moreover, An. minimus A and C are isomorphic and cannot be distinguished by their morphology (Green et al. 1990). Green et al. (1990) found that a humeral pale (HP) wing spot was present more often in An. minimus C than in An. minimus A in western Thailand. However, the presence of HP spots cannot be used with any degree of confidence to differentiate species C from species A (Chen et al. 2002). Allozyme electrophoresis was used to identify species of the An. minimus complex and related species in

2 226 Journal of Vector Ecology December 2005 An. minimus group, including An. aconitus (Green et al. 1990, Van Bortel et al. 1999). Recently, PCR-based methods have been developed to identify this species complex. These include random amplified polymorphic DNA-PCR, allele-specific amplification (ASA), single-strand conformation polymorphism (SSCP), PCR-restriction fragment length polymorphism (RFLP), and multiplex PCR of RAPD and internal transcribed spacer 2 (ITS2) (Garros et al. 2004, Kengne et al. 2001, Sharpe et al. 1999, Sucharit and Komalamisra 1997, Van Bortel et al. 2000). The aim of this study was to identify species of An. minimus complex by using morphological characters and molecular techniques. MATERIALS AND METHODS Mosquito collection Anopheles larvae were collected from different sites in five districts including: Muang, Sangkhaburi, Si Sawat, Sai Yok, and Thong Pha Phum of Kanchanaburi Province (Figure 1) from February to June The study area is in a malaria endemic area of western Thailand near the Myanmar border. Larvae were collected from breeding habitats and reared to adults for species identification by morphological and molecular techniques. Morphological identification Adult mosquitoes were identified using the morphological key (Harrison 1980). Specimens identified as Anopheles minimus s.l. were scored for the presence or absence of the humeral pale (HP) spot on the wings. An. minimus specimens with ambiguous morphology were identified as An. minimus s.l. All specimens were stored at -20 ºC for until verification by molecular techniques. Molecular identification Genomic DNA of individual mosquito was extracted using Ready Amp TM Genomic DNA Purification System kit (Promega Corporation, Madison, WI, U.S.A.), following the manufacturer s instructions. Isolated genomic ssdna was subjected to PCR amplification using both multiplex allelespecific PCR assay (Garros et al. 2004) and PCR-RFLP (Van Bortel et al. 2000) to confirm species. The Polymerase Chain Reaction-Restriction Fragment Length Polymorphism (PCR- RFLP) assay based on the ITS2 marker and developed by Van Bortel et al. (2000) was used for the identification of An. minimus A, An. minimus C, and closely related species (i.e., An. aconitus). PCR primers, PCR conditions, and restriction digestion with MspI and Sau96I (New England Biolabs, Beverly, MA) were carried out following the previously described method (Van Bortel et al. 2000). Multiplex allelespecific PCR assay (Garros et al. 2004) was performed to confirm the identification of the members of the An. minimus species complex. PCR primers, ITS2A, MIA, and MIC were used to identify An. minimus A and C. The primer sequences were according to those previously described (Garros et al. 2004). PCR amplification using AmpliTaq Gold DNA polymerase (Applied Biosystems, Branchburg, NJ, U.S.A.) was performed as described (Garros et al. 2004). RESULTS Approximately 350 of some 600 Anopheles specimens collected between February and June 2004 from five districts of Kanchanaburi were identified as being from the Myzomyia series of the subgenus Cellia. Of these, 238 specimens morphologically identified as An. minimus were confirmed by both PCR-RFLP and multiplex allele-specific PCR (Garros et al. 2004) to distinguish them from their closely related species group. As shown on Table 1, a total of 114 field specimens from Muang, Sangkhaburi, Si Sawat, Sai Yok and Thong Pha Phum were identified as An. minimus A, while 123 specimens from Sai Yok, and Si Sawat were An. minimus C. One specimen from Sangkhaburi, identified as An. aconitus, was morphologically misidentified as An. minimus A (Table 1). When using molecular identification, 84% of An. minimus A and 89% of An. minimus C of morphologically identified mosquitoes were in agreement for species identification. A portion (18% of total specimens) of ambiguously identified An. minimus s.l. by their morphology was identified by molecular technique (among these, ~70% were An. minimus A and ~30% were species C). We did not find any natural hybrids between An. minimus A and C in our study, although Van Bortel et al. (1999) first reported the hybrid (< 1% of samples) in the village of Khoi in northern Vietnam. A total of 36 larval breeding habitats categorized into five types among the five study districts (Table 1) were mapped with data on the district locations and are shown in Figure 1. An. minimus A was found in stream margins, stream pools, swamps, and ditches, while the breeding sites of An. minimus C were distributed in stream margins, stream pools and ground pools. All of the 78 An. minimus larvae collected from 16 water bodies in Sangkhaburi, Thong Pha Phum, and Muang were identified as An. minimus A. Although both 36 An. minimus A and 123 An. minimus C were found in Sai Yok and Si Sawat, they were found in sympatry only in the Ban Phu Ong Ka and Sai Yok districts. Three larval collection sites of An. minimus A and two larval collection sites of An. minimus C were found in Ban Phu Ong Ka (Table 1). In this study, An. minimus A was associated with all study districts. However, the association differed by focusing on study sites in the districts. Figure 1 shows localities of breeding habitats of An. minimus A, An. minimus C, and both A and C around the five districts, with flags representing the location of each village. No breeding sites of species A were found in Ban Phu Rat or Ban Phu Toei in Sai Yok. The An. minimus species complex population appeared to be separated in the area. The breeding habitats of An. minimus C were present in Ban Tha Thung Na and Ban Phu Rat, covering the distance of km in the northern part of Sai Yok. It is clear that An. minimus C is predominant in the Sai Yok district. An. minimus C was also found in the central parts of Si Sawat District (Figure 1). Figure 2 depicts the number of malaria patients by subdistrict from October 2001 to May The bar graphs show infection with Plasmodium falciparum, P. vivax, mixed

3 Vol. 30, no. 2 Journal of Vector Ecology 227 Table 1. Localities, habitat types, and numbers of An. minimus A, An. minimus C, and An. aconitus identified by molecular techniques (multiplex PCR and PCR-RFLP). Larval habitat types (ditch, stream margin, stream pool, and swamp) are represented by Dit, Stm, Stp, and Sw, respectively. District Village Name Collection No. Habitat type An.minimus A An.minimus C An. aconitus Muang Ban Chan Ui K-62 Stm 10 Muang Ban Thap Si La K-37 Sw 6 Muang Ban Thap Si La K-57 Stm 4 Muang Ban Tha Wi K-65 Stp 19 Sai Yok Ban Tha Sao K-41 Sw 4 Sai Yok Ban Phu Rat K-09 Stm 6 Sai Yok Ban Phu Rat K-10 Stm 4 Sai Yok Ban Phu Rat K-08 Stm 6 Sai Yok Ban Phu Rat K-05 Stm 6 Sai Yok Ban Phu Rat K-31 Stm 24 Sai Yok Ban Phu Ong Ka K-36 Stm 8 Sai Yok Ban Phu Ong Ka K-34 Sw 9 Sai Yok Ban Phu Ong Ka K-55 Stm 5 Sai Yok Ban Phu Ong Ka K-56 Stm 12 Sai Yok Ban Phu Toei K-02 Stm 6 Sai Yok Ban Phu Toei K-04 Stm 4 Sai Yok Ban Phu Toei K-03 Stm 7 Sai Yok Ban Phu Toei K-01 Stm 2 Sai Yok Ban Tha Thung Na K-27 Stp 6 Sai Yok Ban Dong Sak K-26 Stm 9 Sangkhaburi Ban Chong Ua K-22 Stm 1 Sangkhaburi Ban Chong Ua K-24 Stm 7 Sangkhaburi Ban Chong Ua K-23 Sw 2 Sangkhaburi Ban Chong Ua K-20 Stm 4 Sangkhaburi Ban Chong Ua K-21 Stm 10 1 Si Sawat Ban Pong Wai K-44 Stm 20 Si Sawat Ban Dan Mae Chalaep K-46 Stm 6 Thong Pha Phum Ban Huai Khayeng K-18 Stm 1 Thong Pha Phum Ban U Long K-19 Stm 2 Thong Pha Phum Ban Tha Khanun K-16 Stm 1 Thong Pha Phum Ban Nong Bang K-12 Dit 4 Thong Pha Phum Ban Nong Bang K-11 Stm 3 Thong Pha Phum Ban Nong Bang K-13 Stm 1 Thong Pha Phum Ban Nong Bang K-14 Stm 3

4 228 Journal of Vector Ecology December 2005 Kanchanaburi Figure 1. Thematic map showing localities of breeding habitats of An. minimus A (black dots), An. minimus C (white dots), both A and C (asterisks) around the five districts. A flag represents the location of each village. The insert map shows the map of Thailand indicating Kanchanaburi Province, the area covered by the present research. infection of both falciparum and vivax, and gametocyte. The number of malaria infections showed no correlation with the distribution of An. minimus A and C. DISCUSSION An. minimus species A and species C were correctly identified morphologically at 84% and 89% and misidentified or ambiguously identified at 16% and 11%, respectively. The percentage of morphological identification error when compared with the PCR-based methods in this study agrees with those reported in previous studies that An. minimus A and C cannot be distinguished accurately by morphology due to character variations (Chen et al. 2002, Green et al. 1990). Green et al. (1990) investigated population genetics of An. minimus A and C in Thailand and suggested that the identification using morphological characters can lead to 37% error. Chen et al. (2002) found that the morphological identification for both species A and C females from southern China could be unreliable. Morphological identification of closely related species in the Minimus group, including An. aconitus, An. varuna, and An. minimus can be easily mixed up due to overlapping morphological characters (Harrison 1980). However, in the present study only one An. aconitus was morphologically misidentified as An. minimus A (Table 1). Our previous studies also revealed that 1% (2 of 200) of the specimens collected from Mae Sot, Tak Province and identified morphologically as An. minimus was proven to be An. varuna by the PCR-RFLP method (unpublished data). This is in contrast with a previous study by Van Bortel et al. (2001) that found as much as 96% (72 of 75) of morphologically identified An. minimus A were misidentified.

5 Vol. 30, no. 2 Journal of Vector Ecology 229 Figure 2. Thematic map showing the number of malaria patients from October May 2004 for all species of malaria parasite by subdistrict. In the pie chart, infections with Plasmodium falciparum, Plasmodium vivax, mixed infection of both P. falciparum and P. vivax, and gametocytes are shown. They were identified by the PCR-RFLP method as An. varuna, An. aconitus, and An. pampanai. An. minimus s.l. has been reported to oviposit primarily in clear, running streams in forested areas (Harrison 1980). In this study, stream margin was the main breeding habitat for both An. minimus A and C. We were unable to identify which key environmental factors were associated with species A or C since there were a variety of habitat types. Alternatively, the results imply that these species could be associated with locations of habitat rather than types of habitat. An. minimus A is known to occur throughout Thailand (Green et al. 1990). This is consistent with our finding of An. minimus A in the five districts of Kanchanaburi Province. The occurrence of An. minimus C is restricted to the Sai Yok district. Charlwood and Edoh (1996) explained the distribution of An. gambiae Giles complex in that An. gambiae Giles and An. arabiensis Patton differ in location of breeding site rather than host availability. The association of An. minimus C distribution with more humid areas in Ban Phu Toei has been reported by Sucharit et al. (1988). Beside Ban Phu Toei and Ban Phu Rat, we also found An. minimus C in Ban Tha Thung Na, Ban Dong Sak, and Ban Phu Ong Ka of Sai Yok district (Table 1; Figure 1). It is possible that Ban Phu Ong Ka is the area that separates breeding sites of An. minimus C in the northern part from An. minimus A in the southern part of Sai Yok (Figure 1). An. minimus C extends its distribution eastward into Si Sawat and this is the first report of species C found in Si Sawat district (Figure 1). Further investigation in the factors that may influence the spatial distribution pattern of An. minimus A and C, including satellite imagery of landuse/land-cover classification and proximity analysis of breeding habitats of An. minimus A and C, is underway. Acknowledgments This work was supported by the TRF/BIOTEC Special Program for Biodiversity Research and Training and CNRS (France) grant BRT R

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