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Keywords:

  • Anopheles nigerrimus;
  • metaphase karyotypes;
  • hybridization experiments;
  • ITS2;
  • COI;
  • COII

ABSTRACT

  1. Top of page
  2. ABSTRACT
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgments
  8. REFERENCES CITED

Thirteen isoline colonies of Anopheles nigerrimus were established from individual wild-caught females collected from cow-baited traps at locations in Thailand and Cambodia. Three types of X (X1, X2, X3) and 4 types of Y (Y1, Y2, Y3, Y4) chromosomes were recovered, according to differing amounts of extra heterochromatin. Four karyotypic forms were designed depending upon apparently distinct figures of X and Y chromosomes, i.e., Form A (X1, X2, X3, Y1), B (X2, X3, Y2), C (X1, Y3), and D (X3, Y4). Forms C and D were new metaphase karyotypes discovered in this study. Form A appeared to be common in both Thailand and Cambodia. Forms B and D were found to be rather specific to southern and northeastern Thailand, respectively, whereas Form C was confined to Cambodia. Hybridization experiments among the eight isoline colonies, which were representative of four karyotypic forms of An. nigerrimus, demonstrated genetic compatibility in giving viable progenies and synaptic salivary gland polytene chromosomes through F2-generations. These results elucidated the conspecific relationship, comprising four cytological forms within this taxon. Supportive evidence was confirmed further by very low intraspecific sequence variations (average genetic distance = 0.002–0.007) of the nucleotide sequences in ribosomal DNA [second internal transcribed spacer (ITS2)] and mitochondrial DNA [cytochrome c oxidase subunit I (COI) and subunit II (COII)].


INTRODUCTION

  1. Top of page
  2. ABSTRACT
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgments
  8. REFERENCES CITED

Anopheles (Anopheles) nigerrimus belongs to the Nigerrimus Subgroup and Hyrcanus Group of the Myzorhynchus Series, and is distributed widely in Thailand and other countries, i.e., India (Assam, Bihar and Punjab), Sri Lanka, Bangladesh, China (Hainan Island), Myanmar, Laos, Cambodia, Vietnam, Malaysia (Malaysian Peninsular, Sabah and Sarawak), Indonesia (Java and Sumatra), and Brunei (Reid 1968, Scanlon et al. 1968, Harrison and Scanlon 1975, Knight and Stone 1977, Harbach 2012). Regarding medical importance, An. nigerrimus was incriminated as a suspected vector of Plasmodium vivax in Thailand (Baker et al. 1987, Harbach et al. 1987, Gingrich et al. 1990, Rattanarithikul et al. 1996) and P. falciparum and P. vivax in Bangladesh (Alam et al. 2010, 1998). Recently, it was incriminated as a secondary or incidental vector of Wuchereria bancrofti in Asia (Manguin et al. 2010). Additional experiments indicated that this anopheline species could serve as a potential vector of the filarial nematode, nocturnally subperiodic Brugia malayi, as determined by 50–65% susceptibility rates and 4.20–9.77 average number of L3 larvae per infected mosquito (Saeung et al. 2013).

Cytologically, two karyotypic forms of An. nigerrimus, Form A (X1, X2, Y1) and B (X1, X2, Y2), were obtained from Ubon Ratchathani and Ayutthaya Provinces, respectively (Baimai et al. 1993). These two karyotypic variants appeared to result from a gradual increase in the extra heterochromatin on X and Y chromosomes (Baimai 1998). Genetic variation at the chromosomal level, within the taxon Anopheles, potentially results in the existence of species complex and causes difficulty in exactly identifying sibling species (isomorphic species) and/or subspecies (cytological forms) members that result from identical morphology or minimal morphological distinction. Additionally, those members may differ in biological characteristics (e.g., microhabitats, resting and biting behaviors, sensitivity or resistance to insecticides, susceptible or refractory to pathogens, etc.), which can be used to determine their potential for transmitting pathogenic agents. Inability to identify individual members in the complexes of Anopheles vectors may result in the failure to differentiate between a vector and non-vector species, and result in unsuccessful vector control (Subbarao 1998, Van Bortel et al. 2001, Singh et al. 2010). Regarding the above information, very little is known about the genetic proximities among karyotypic variants of An. nigerrimus. Thus, we first report two new karyotypic forms [C (X1, Y3) and D (X3, Y4)] of An. nigerrimus and determine the genetic proximity among its four karyotypic variants by hybridization experiments related to the comparative DNA sequencing of the second internal transcribed spacer (ITS2) of ribosomal DNA (rDNA), cytochrome c oxidase subunit I (COI), and cytochrome c oxidase subunit II (COII) of mitochondrial DNA (mtDNA).

MATERIALS AND METHODS

  1. Top of page
  2. ABSTRACT
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgments
  8. REFERENCES CITED

Field collections and establishment of isoline colonies

Wild-caught, fully engorged female mosquitoes of An. nigerrimus were collected from cow-baited traps at four allopatric locations in Thailand (Lampang Province, northern region; Ubon Ratchathani Province, northeastern region; Songkhla and Nakhon Si Thammarat Provinces, southern region), and one location in Cambodia (Ratanakiri Province) (Figure 1, Table 1). A total of 13 isolines were established successfully and main­tained in our insectary using the techniques described by Choochote and Saeung 2013. Exact species identification was performed by using intact morphology of egg, larval, pupal, and adult stages from the F1-progenies of isolines, following standard keys (Reid 1968, Harrison and Scanlon 1975, Rattanarithikul et al. 2006). These isolines were used for studies on the metaphase karyotype, hybridization experiment, and molecular analysis.

image

Figure 1. Map of Thailand and Cambodia showing five provinces where samples of An. nigerrimus were collected and the number of isolines of the four karyotypic forms (A-D) detected in each location.

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Table 1. Locations in Thailand and Cambodia, code of isolines, four karyotypic forms (A-D) of An. nigerrimus and their GenBank accession numbers.
  Location (Geograpical coordinate)Code of isolineaKaryotypic formGenBank accession numberReference
ITS2COICOII 
  1. a: used in crossing experiments.

Thailand      
  Lampang (17° 53′ N, 99° 20′ E)Lp1AaA (X1, Y1)AB778774AB778787AB778800This study
  Ubon Ratchathani (15° 31′ N, 105 ° 35′ E)Ur1AaA (X2, Y1)AB778775AB778788AB778801This study
 Ur7AA (X2, Y1)AB778776AB778789AB778802This study
 Ur20DaD (X3, Y4)AB778777AB778790AB778803This study
 Ur26AA (X3, Y1)AB778778AB778791AB778804This study
  Nakhon Si Thammarat (08° 29′ N, 100 ° 0′ E)Ns1BaB (X2, Y2)AB778779AB778792AB778805This study
 Ns2BB (X3, Y2)AB778780AB778793AB778806This study
 Ns3AaA (X2, Y1)AB778781AB778794AB778807This study
  Songkhla (07° 13′ N, 100 ° 37′ E)Sk2AaA (X2, Y1)AB778782AB778795AB778808This study
 Sk3AA (X1, Y1)AB778783AB778796AB778809This study
Cambodia      
  Ratanakiri (13° 44′ N, 107° 0′ E)Rt2CaC (X1, Y3)AB778784AB778797AB778810This study
 Rt3CC (X1, Y3)AB778785AB778798AB778811This study
 Rt4AaA (X1, Y1)AB778786AB778799AB778812This study
Kalimantan, Indonesia      
Hyrcanus GroupK13HM488261Paredes-Esquivel et al. 2011
 K22HM488263Paredes-Esquivel et al. 2011
 K26HM488267Paredes-Esquivel et al. 2011
An. belenraeEU789794Park et al. 2008a
An. crawfordiSk1BB (X3, Y2)AB779152AB779181AB779210Saeung et al. unpublished data
An. kleiniEU789793Park et al. 2008a
An. lesteriEU789791Park et al. 2008a
 ilG1AB733028AB733036Taai et al. 2013a
An. nitidusUr2DD (X3, Y4)AB777782AB777803AB777824Songsawatkiat et al. unpublished data
An. paraliaeSk1BB (X1, Y2)AB733487AB733503AB733519Taai et al. 2013b
An. peditaeniatusRbBB (X3, Y2)AB539061AB539069AB539077Choochote 2011
An. pullusEU789792Park et al. 2008a
 AY444348AY444347Park et al. 2003
An. sinensisi2ACMA (X, Y1)AY130473Min et al. 2002
 AY444351Park et al. 2003
 i1BKRB (X, Y2)AY130464Min et al. 2002

Metaphase karyotype preparation

Metaphase chromosomes were prepared from ten samples of the early 4th-instar larval brains of F1-progeny of each isoline, using techniques previously described by Saeung et al. (2007). Identification of karyotypic forms fol­lowed the standard cytotaxonomic systems of Baimai et al. (1993).

Hybridization experiment

The eight laboratory-raised isolines of An. nigerrimus were selected arbitrarily from the 13 isoline colonies, which were representative of four karyotypic forms, i.e., Form A [Lp1A (X1, Y1), Ur1A (X2, Y1), Ns3A (X2, Y1), Sk2A (X2, Y1), Rt4A (X1, Y1)], B [Ns1B (X2, Y2)], C [Rt2C (X1, Y3)], and D [Ur20D (X3, Y4)] (Table 1). These isolines were used for crossing experiments in order to determine post-mating barriers by employing the techniques previously reported by Saeung et al. (2007).

DNA extraction and amplification

Three molecular markers (ITS2, COI, COII) were used to determine intraspecific sequence variation within the taxon An. nigerrimus. DNA was extracted from individual adult female F1-progeny of each isoline of An. nigerrimus using the DNeasy® Blood and Tissue Kit (QIAGEN). Primers for the amplification of ITS2, COI, and COII regions followed previous studies by Saeung et al. (2007). The ITS2 region of the rDNA was amplified using ITS2A and ITS2B primers (Beebe and Saul 1995). The universal LCO1490 and HCO2198 barcoding primers of Folmer et al. 1994 were used to amplify the 658 bp COI gene fragment. The mitochondrial COII region was amplified using LEU and LYS primers, as recommended by Sharpe et al. (2000). Each PCR reaction was carried out in a total volume of 20 µl containing 0.5 U Ex Taq (Takara), 1X Ex Taq buffer, 2 mM of MgCl2, 0.2 mM of each dNTP, 0.25 µM of each primer, and 1 µl of the extracted DNA. Regarding ITS2, the conditions for amplification consisted of initial denaturation at 94° C for 1 min, 30 cycles at 94° C for 30 s, 55° C for 30 s, and 72° C for 1 min, and a final extension at 72° C for 5 min. Thermal cycling conditions for COI and COII were initial denaturation at 94° C for 1 min, 30 cycles at 94° C for 30 s, 50° C for 30 s, and 72° C for 1 min, and a final extension at 72° C for 5 min. PCR products were visualized by ethidium bromide staining after electrophoresis in 1.5% agarose gel, and finally purified using the QIAquick® PCR Purification Kit (QIAGEN). Sequencing reactions were conducted in both directions using the BigDye® V3.1 Terminator Cycle Sequencing Kit and 3130 genetic analyzer (Applied Biosystems). The sequences obtained were submitted to the DDBJ/EMBL/GenBank nucleotide sequence database under accession numbers AB778774-AB778812 (Table 1). All sequence data generated from this study were compared with those available in GenBank, using BLAST (http://blast.ncbi.nlm.nih.gov/Blast.cgi).

Sequencing alignment and phylogenetic analysis

The DNA sequence data were edited manually in BioEdit version 7.0.5.3 (Hall 1999) and aligned using CLUSTAL W (Thompson et al. 1980). Gap sites were excluded from the following analysis. The Kimura two-parameter (K2P) model was employed to calculate genetic distances (Kimura 1980), which were used to construct neighbor-joining trees (Saitou and Nei 1987) and bootstrap 1,000 replicates with the MEGA version 4.0 program (Tamura et al. 2007). Bayesian analysis was conducted with MrBayes 3.2 (Ronquist et al. 2012) by using two replicates of one million generations with the nucleotide evolutionary model. The best-fit model was chosen for each gene separately using the Akaike Information Criterion (AIC) in MrModeltest version 2.3 (2004, program distributed by the author). The general time-reversible (GTR) with gamma distribution shape parameter (G) was selected for ITS2, whereas the GTR+I+G was the best-fit model for combining sequences of COI and COII. Bayesian posterior probabilities were calculated from the consensus tree after excluding the first 25% of trees as burn-in.

RESULTS

  1. Top of page
  2. ABSTRACT
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgments
  8. REFERENCES CITED

Metaphase karyotypes

Cytogenetic investigations of F1-progenies of the 13 isolines of An. nigerrimus revealed different types of sex chromosomes due to the addition of extra heterochromatin. There were three types of X (submetacentric X1, large submetacentric X2, and small metacentric X3) and four types of Y chromosomes (large subtelocentric Y1, submetacentric Y2, small telocentric Y3, and small subtelocentric Y4). The X3, Y3, and Y4 chromosomes were discovered in the present study. Based on the figures of X3 and Y3 chromosomes, they were probably represented the ancestral forms of X and Y chromosomes, respectively (Figure 2). These types of X and Y chromosomes formed four karyotypic forms on the basis of X and Y chromosome configurations, which were designated as Forms A (X1, X2, X3, Y1), B (X2, X3, Y2), C (X1, Y3), and D (X3, Y4). Forms C and D were new karyotypic forms discovered in the present investigation. The number of isolines of these karyotypic forms occurring in different locations of Thailand and Cambodia are demonstrated in Figure 1 and Table 1. Form A appeared to be common in both Thailand and Cambodia. Forms B and D were found to be rather specific to southern and northeastern Thailand, respectively, whereas Form C was confined somewhat to Cambodia.

image

Figure 2. Metaphase karyotypic forms of An. nigerrimus. (a) Form A (X1, Y1: Lampang); (b) Form A (X2, Y1: Ubon Ratchathani); (c) Form A (X2, Y1: Songkhla); (d) Form A (X3, Y1: Ubon Ratchathani); (e) Form B (X2, Y2: Nakhon Si Thammarat); (f) Form B (X3, Y2: Nakhon Si Thammarat); (g) Form C (X1, Y3: Ratanakiri); (h) Form D (X3, Y4: Ubon Ratchathani); (i) Form A (heterozygous X2, X3: Ubon Ratchathani); diagrams of representative meta­phase karyotype of Form C (j) and Form D (k).

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Hybridization experiment

The parental, reciprocal, and F1-hybrid crosses among the eight isolines of An. nigerrimus yielded viable progenies through the F2-generations. The hatchability, pupa­tion, emergence rates, and ratio of adult female/male of parental, reciprocal, and F1-hybrid crosses were 79.06–94.05%, 91.14–100%, 94.64–100%, and 0.81–1.12; 71.97–92.01%, 85.07–100%, 87.23–100%, and 0.89–1.14; 67.86–92.97%, 87.00–100%, 89.82–98.17%, and 0.71–1.27, respectively. No evidence of genetic incompatibility and/or post-mating reproductive isolation was observed among these crosses. The salivary gland polytene chromosomes of the 4th instar larvae of F1-hybrids from all crosses showed synapsis without inversion loops along the whole length of all autosomes and the X chromosome (Figure 3).

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Figure 3. Synapsis in all arms of salivary gland polytene chromosome of F1-hybrid larvae of An. nigerrimus. (a) Lp1A female x Ur1A male; (b) Lp1A female x Sk2A male; (c) Lp1A female x Ns3A male; (d) Lp1A female x Rt4A male; (e) Lp1A female x Ns1B male, note: small gap of homosequential asynapsis was found on chromosome 3R (small arrow); (f) Lp1A female x Rt2C male; (g) Lp1A female x Ur20D male.

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DNA sequences and phylogenetic analysis

Sequences generated from 13 isolines of An. nigerrimus Forms A-D from Thailand and Cambodia showed the same length as those of ITS2 (508 bp), COI (658 bp), and COII (685 bp). Comparison of ITS2 sequences of ten and three isolines from Thai and Cambodian An. nigerrimus, respectively, were performed. Among them, eight were identical and the remaining five (Ns1B, Ns2B, Ns3A, Sk2A and Sk3A) from the southern region of Thailand shared the same nucleotide sequences, but they differed from the other eight by only two nucleotide substitutions (A[LEFT RIGHT ARROW]G at position 70 and T[LEFT RIGHT ARROW]G at position 481). The evolutionary relationships were constructed among the four karyotypic forms using neighbor-joining and Bayesian trees. Both phylogenetic methods showed similar tree topologies, thus, only the Bayesian tree was shown for all regions (Figures 4 and 5). The average genetic distances within and between the four karyotypic forms exhibited no significant difference in three DNA regions (0.002–0.007) of both Thai and Cambodian populations. Therefore, the 13 isolines were placed within a single species, namely An. nigerrimus. Additionally, three ITS2 retrieved sequences (428 bp) from GenBank, previously identified as the Hyrcanus Group by Paredes-Esquivel et al. 2011, were grouped together with four karyotypic forms of An. nigerrimus (average genetic distances = 0.003). These ITS2 sequences were nearly identical to our sequences, and only two nucleotide substitutions were found among them. In support, the phylogenetic trees for ITS2, COI, and COII of these isolines, representing Forms A-D, were clearly different from other species of the Hyrcanus Group (Figures 4 and 5).

image

Figure 4. Bayesian phylogenetic relationships among the 13 isolines of An. nigerrimus from Thailand and Cambodia based on ITS2 sequences compared with nine species of the Hyrcanus Group and three Hyrcanus-group specimens from Kalimantan, Indonesia (Paredes-Esquivel et al. 2011). Numbers on branches are bootstrap values (%) of NJ analysis and Bayesian posterior probabilities (%). Only the values higher than 70% both on bootstrap values and posterior probabilities are shown. Branch lengths are proportional to genetic distance (scale bar).

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image

Figure 5. Bayesian phylogenetic relationships among the 13 isolines of An. nigerrimus from Thailand and Cambodia, based on combined sequences of COI and COII, compared with seven species of the Hyrcanus Group. Numbers on branches are bootstrap values (%) of NJ analysis and Bayesian posterior probabilities (%). Only the values higher than 70% both on bootstrap values and posterior probabilities are shown. Branch lengths are proportional to genetic distance (scale bar).

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DISCUSSION

  1. Top of page
  2. ABSTRACT
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgments
  8. REFERENCES CITED

Metaphase chromosome investigations of four An. nigerrimus isolines from two allopatric locations (two isolines/each location) in Thailand (Nachaluai District, Ubon Ratchathani, and Bangpa-in District, Ayutthaya Provinces) were performed first by Baimai et al. (1993). The results revealed that this anopheline species exhibited karyotypic variation via a gradual increase in the extra heterochromatin on X (X1, X2) and Y (Y1, Y2) chromosomes. In this study, a total of 13 An. nigerrimus isolines obtained from four and one locations in Thailand and Cambodia, respectively, demonstrated three types of X (X1, X2, X3) and four types of Y (Y1, Y2, Y3, Y4) chromosomes, thus forming four karyotypic forms, which were designated as Form A (X1, X2, X3, Y1), B (X2, X3, Y2), C (X1, Y3), and D (X3, Y4). The newly discovered Forms C and D from Ratanakiri, Cambodia, and Ubon Ratchathani, northeastern Thailand, were based on the unique characteristics of small telocentric Y3 and small subtelocentric Y4 chromosomes, respectively, which were clearly different from the former two types of Y chromosomes (large subtelocentric Y1, submetacentric Y2) previously reported by Baimai et al. (1993). Apparently, the four distinct karyotypic forms of An. nigerrimus were due to the gradual addition of extra heterochromatin on sex chromosomes. Thus, the accumulation of heterochromatin in the genome elucidates the possible cytological mechanism for karyotypic evolution of Oriental anophelines as proposed by Baimai (1998). Regarding the distribution of An. nigerrimus cytological forms, it is worth noting that a new karyotypic Form C was detected in only two isoline colonies from Ratanakiri, Cambodia, whereas Form A was common in both Thailand and Cambodia. Interestingly, Form B and D were recovered specifically in Nakhon Si Thammarat Province, southern region and Ubon Ratchathani Province, northeastern region of Thailand, respectively. However, additional surveys are needed in order to obtain greater numbers of isolines from both countries, and this would bring about understanding of the exact distribution pattern of An. nigerrimus cytological forms.

Hybridization experiments using isoline colonies of Anopheles mosquitoes, which relate to data of cytogenetic and molecular investigations to elucidate post-mating barriers, have been proven to be robust traditional techniques for recognizing sibling species and/or subspecies members within the taxon Anopheles (Kanda et al. 1981, Baimai et al. 1987, Subbarao 1998, Junkum et al. 2005, Somboon et al. 2005, Saeung et al. 2007, 1987, Thongwat et al. 2008, Suwannamit et al. 2009, Thongsahuan et al. 2009, Choochote 2011, Saeung et al. 2012). The genetic diversity at the chromosomal level of An. nigerrimus found in this study warrants intensive hybridization experiments among the four karyotypic forms. The results showed no post-mating reproductive isolation. All crosses yielded viable progenies through F2-generations and synaptic salivary gland polytene chromosomes, suggesting conspecific nature, comprised four cytological forms within this taxon. The low intraspecific sequence variations (average genetic distance = 0.002–0.007) of the nucleotide sequences in ribosomal DNA (ITS2) and mitochondrial DNA (COI and COII) of the four karyotypic forms in both Thai and Cambodian An. nigerrimus populations were good supportive evidence. The present results are in accordance with hybridization experiments among karyotypic forms of other Anopheles species, including An. vagus (Choochote et al. 2002), An. pullus (= An. yatsushiroensis) (Park et al. 2003), An. sinensis (Choochote et al. 1998, Min et al. 2002, Park et al. 2008b), An. aconitus (Junkum et al. 2005), An. barbirostris species A1 and A2 (Saeung et al. 2007, Suwannamit et al. 2009), An. campestris-like taxon (Thongsahuan et al. 2009) and An. peditaeniatus (Choochote 2011, Saeung et al. 2012).

Misidentification of species can lead to failure in controlling target vectors, especially the sibling species and/or subspecies members of Anopheles species complexes in sympatric areas. Several studies reported misidentification of malaria vectors due to overlapping and/or variations based on morphological characters (Van Bortel et al. 2001, Singh et al. 2010). Paredes-Esquivel et al. 2011 reported that field workers had misidentified mosquitoes of the Hyrcanus Group as belonging to species of the Barbirostris Group. In order to overcome unresolved taxonomic questions on members of the An. hyrcanus group, the evidence from molecular markers was combined with morphological and hybridization experiments that identified the exact species status of four karyotypic forms of An. nigerrimus for the first time in two different geographical localities. Furthermore, three ITS2 published sequences of specimens from south Kalimantan, Indonesia (K13: GenBank accession number HM488261, K22: GenBank accession number HM488263, K26: GenBank accession number HM488267) were identified as the Hyrcanus Group by Paredes-Esquivel et al. 2011 and retrieved and compared with sequences of this study. It was interesting to note that these sequences were placed within the same clade of the Thai and Cambodian An. nigerrimus in the phylogenetic tree and the low level of intra-specific divergence (0.001–0.006) was found among them. This study confirms the presence of An. nigerrimus in Kalimantan, Indonesia, which corresponds to previous findings by O’Connor 1980.

Acknowledgments

  1. Top of page
  2. ABSTRACT
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgments
  8. REFERENCES CITED

This work was supported by The Thailand Research Fund to W. Choochote and A. Saeung (TRF Senior Research Scholar: RTA5480006) and Royal Golden Jubilee Ph.D. Program to W. Choochote and S. Songsawatkiat (Grant No. PHD/0356/2552) and Faculty of Medicine Endowment Fund, Chiang Mai University, Chiang Mai, Thailand. The authors would like to thank Dr. Wattana Navacharoen, Dean of the Faculty of Medicine, Chiang Mai University, for his interest in this research. We are grateful to Dr. Wannapa Suwonkerd, Office of Disease Prevention and Control 10, Chiang Mai, Mr. Kritsana Taai, Department of Parasitology, Faculty of Medicine, Chiang Mai University, Chiang Mai, Thailand, and Dr. Tho Sochanta, National Center for Malaria Control, Parasitology and Entomology, Phnom Penh, Cambodia, for their field work assistance.

REFERENCES CITED

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  2. ABSTRACT
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgments
  8. REFERENCES CITED
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