Genetic Diversity, Haplotype Relationships, and kdr Mutation of Malaria Anopheles Vectors in the Most Plasmodium knowlesi-Endemic Area of Thailand

Abstract

Plasmodium knowlesi, a malaria parasite that occurs naturally in long-tailed macaques, pig-tailed macaques, and banded leaf monkeys, is currently regarded as the fifth of the human malaria parasites. We aimed to investigate genetic diversity based on the cytochrome c oxidase subunit I (COI) gene, detect Plasmodium parasites, and screen for the voltage-gated sodium channel (VGSC)-mutation-mediated knockdown resistance (kdr) of Anopheles mosquitoes in Ranong province, which is the most P. knowlesi-endemic area in Thailand. One hundred and fourteen Anopheles females belonging to eight species, including An. baimaii (21.05%), An. minimus s.s. (20.17%), An. epiroticus (19.30%), An. jamesii (19.30%), An. maculatus s.s. (13.16%), An. barbirostris A3 (5.26%), An. sawadwongporni (0.88%), and An. aconitus (0.88%), were caught in three geographical regions of Ranong province. None of the Anopheles mosquitoes sampled in this study were infected with Plasmodium parasites. Based on the sequence analysis of COI sequences, An. epiroticus had the highest level of nucleotide diversity (0.012), followed by An. minimus (0.011). In contrast, An. maculatus (0.002) had the lowest level of nucleotide diversity. The Fu’s Fs and Tajima’s D values of the Anopheles species in Ranong were all negative, except the Tajima’s D values of An. minimus (0.077). Screening of VGSC sequences showed no presence of the kdr mutation of Anopheles mosquitoes. Our results could be used to further select effective techniques for controlling Anopheles populations in Thailand’s most P. knowlesi-endemic area.

1. Introduction

Four species of malaria parasite have long been known to cause human health issues, including Plasmodium vivax, P. ovale, P. malariae, and P. falciparum [1]. Plasmodium knowlesi, a malaria parasite that occurs naturally in long-tailed macaques (Macaca fascicularis), pig-tailed macaques (Ma. nemestrina), and banded leaf monkeys (Presbytis melalophos), is now regarded as the fifth human malaria parasite [2,3]. The first naturally acquired human infection was documented in 1965 when a traveler acquired P. knowlesi after a brief stay in peninsular Malaysia [4]. Human P. knowlesi infections are prevalent in Southeast Asian countries such as Thailand, Cambodia, Myanmar, the Philippines, Singapore, Vietnam, Malaysia, and Indonesia. [3]. In addition, this zoonotic malaria parasite has also been reported in other regions after being carried by travelers who visited Southeast Asian countries such as Malaysia [5,6] and Thailand [7,8].

Mosquitoes of the genus Anopheles are responsible for spreading malaria to humans. Although wild Plasmodium-infected Anopheles mosquitoes have been extensively surveyed to validate their role as malaria vectors, few studies have been able to confirm vectors of P. knowlesi due to a lack of appropriate molecular tools [9]. In 1961, Anopheles hackeri was identified as the natural vector of simian malaria P. knowlesi in peninsular Malaysia, based on sporozoites inoculated into a rhesus monkey [10]. However, this Anopheles species cannot transmit P. knowlesi to humans because it feeds mainly on monkeys and does not attack humans. The confirmation of P. knowlesi vectors using molecular techniques was begun in 2006 by Vythilingam et al. [11], who discovered that An. latens is a vector of P. knowlesi in Sarawak, Malaysia, using a nested polymerase chain reaction (PCR) assay. In 2011, Marchand et al. [12] reported P. knowlesi infections in An. dirus sensu stricto (s.s.) in Southern Vietnam. Jiram et al. [13] confirmed that An. cracens is a vector of P. knowlesi in Kuala Lipis in peninsular Malaysia. In 2009, P. knowlesi infections were also found in An. sundaicus sensu lato (s.l.) in Katchal Island, India [14]. Recently, An. balabacensis and An. donaldi were identified as vectors of P. knowlesi in Lawas, Northern Sarawak, Malaysian Borneo, based on the detection of Plasmodium DNA in the salivary glands of wild Anopheles mosquitoes using a nested PCR assay [9]. As noted earlier, Anopheles mosquitoes, confirmed to be P. knowlesi vectors in the past, are only found in Malaysia, with single reports from Vietnam and India. Therefore, other countries should continue to investigate Anopheles mosquito vectors to control knowlesi malaria effectively. Since Anopheles vectors behave differently in different regions, a malaria vector in one region may not be a malaria vector in another [15].

Thailand is a malaria-epidemic country, especially in border areas, caused by P. vivax and P. falciparum [16]. Nevertheless, the trend of P. vivax and P. falciparum malaria cases is one of annual decrease. Thus, Thailand’s Ministry of Public Health set a goal of eliminating malaria by 2024. However, the surge of knowlesi malaria cases may hinder Thailand’s efforts to eliminate malaria. In 2004, the first case of a human P. knowlesi infection was reported in Thailand. The patient had a travel history that included a few weeks in the forest in Prachuap Khiri Khan province [17]. After the first case was reported, there continued to be a few humans infected per year (<10 cases) until 31 cases were reported in 2018. The following year, P. knowlesi infection rates remained high (19 cases in 2019), and they began to increase in 2020 (22 cases) and 2021 (72 cases). In January–October 2022, 140 P. knowlesi infected patients were reported [18]. Although the number of P. knowlesi infected patients is currently on the rise, there is no information available on the natural vectors of P. knowlesi in Thailand, which makes controlling the disease difficult.

Ranong is one of Thailand’s southern provinces, near the Myanmar border, and is the most P. knowlesi-endemic area in Thailand, with 53 infected patients in 2022 (accounting for 96.36% of total cases during January–October 2022). In contrast, other malaria infections are rare (one case of P. vivax and another of P. falciparum in 2022). A substantial portion of Ranong is forested area, a vital habitat for the primary malaria vectors in Thailand, including An. dirus, An. minimus, and An. maculatus [15,16]. Meanwhile, a portion of Ranong is coastal area, which is the habitat of An. epiroticus, a secondary malaria vector in Thailand [19]. For malaria control to be successful, comprehensive knowledge of Anopheles vectors is necessary [15]. However, in-depth information on Anopheles mosquitoes in Thailand’s most P. knowlesi-endemic area is still lacking.

The genetic diversity of insect vectors in endemic areas is critical, providing useful information about the taxonomic status of species and the spatial limits of natural populations [20]. This knowledge permits researchers to understand and predict the epidemiology, distribution, and transmission dynamics of vector-borne diseases based on the basic biology of the vectors [20]. The cytochrome c oxidase subunit I (COI) gene is a frequently utilized marker in molecular studies on the genetic diversity of insects, including Anopheles mosquitoes, due to its high accuracy [21,22,23].

In addition, genetic monitoring of Anopheles mosquitoes also allows for more effective vector control strategies. Malaria vector control via the use of insecticide-treated nets (ITNs), long-lasting insecticide nets (LLINs), and indoor residual spraying (IRS) of insecticides is the primary technique for reducing malaria transmission [24]. However, insecticide-resistant Anopheles mosquitoes have been reported in many countries [25]. The voltage-gated sodium channel (VGSC) is the main target for both pyrethroid and dichlorodiphenyltrichloroethane (DDT) insecticides [25]. Molecular studies can help to examine the polymorphisms associated with the resistance of several insects, including Anopheles mosquitoes, against pyrethroids and DDT, also called knockdown resistance (kdr), based on genetic mutations of codon 1014 in the VGSC gene [26,27,28].

To optimize entomological information for vector control strategies in Thailand’s most P. knowlesi-endemic area, in-depth molecular information on Anopheles mosquitoes is required. The present study aimed to investigate genetic diversity based on COI, detect Plasmodium parasites, and screen for VGSC-mutation-mediated knockdown resistance of Anopheles mosquitoes in Ranong province, which is Thailand’s most P. knowlesi-endemic area.

2. Materials and Methods

2.1. Ethics Statement

The current investigation was conducted in compliance with the conditions outlined in the guidelines for animal care and usage in research developed by the Suan Sunandha Rajabhat University in Thailand. The Institutional Animal Care and Use Committee of the Suan Sunandha Rajabhat University in Bangkok, Thailand, reviewed and approved all experimental procedures and fieldwork beforehand (Animal Ethics Permission number: IACUC 64-010/2021).

2.2. Study Sites and Sample Collection

We conducted our study in Ranong province, Thailand’s most P. knowlesi-endemic area [18]. Ranong is the northernmost province on Thailand’s Andaman coast and shares a border with Myanmar. It is located around 580 km from Bangkok, Thailand. Three different locations in Ranong province were selected for Anopheles collection, including northern (10°45′48.3″ N, 98°53′31.4″ E), central (9°57′20.1″ N, 98°42′05.3″ E), and southern (9°22′06.8″ N, 98°27′52.1″ E) areas. The northern part of Ranong province includes the northernmost Kraburi and La-un districts and is covered by mountains and forests. The central part includes high forested hills on the right bank of a large reservoir (Hat Som Paen reservoir), the left bank of which is adjacent to the Andaman Sea. In addition, the central area includes large islands such as Koh Chang and Koh Phayam. The southern area includes the southernmost districts of the province, Kapur and Suk Samran, boundaried on the left side by the Andaman coast and on the right side by high mountain and forest areas. Many natural water sources on the left bank of the central and southern sampling areas are brackish water sources. All three areas of Ranong are knowlesi malaria outbreak zones (a total of 8 cases in 2020–2022 for the northern area or Kraburi and La-un districts; 24 cases for the central area or Mueang Ranong district; and 46 cases for the southern area or Kapur and Suk Samran districts), according to a malaria report from Thailand’s Ministry of Public Health [18].

We conducted adult Anopheles collections once every two months between January and June 2022 in accordance with the survey plan of the Ranong Vector Borne Disease Control Center. Anopheles mosquitoes from three different locations in Ranong province (Figure 1) were collected throughout the night between 18:00 and 6:00 over five nights, using 12 BG-Pro CDC-style traps (BioGents, Regensburg, Germany) with BG-lure cartridges (BioGents, Regensburg, Germany) and solid carbon dioxide (dry ice). The mosquito bags were removed from the traps in the morning (6:00 a.m.) and kept in the freezer at −20 °C until the mosquitoes died. Then, the gathered mosquito samples were brought to the College of Allied Health Sciences laboratory at Suan Sunandha Rajabhat University in Thailand and stored in the freezer at −20 °C until further use.

2.3. Morphological and Molecular Species Identification

The initial identification of wild-caught Anopheles mosquitoes at the species/group level was performed via morphological examination under a stereo microscope (Nikon Corp., Tokyo, Japan), using an illustrated key of adult Anopheles from Thailand [29]. Each morphologically identified Anopheles specimen was kept individually in a 1.5 mL micro-centrifuge tube with silica gel (one specimen/tube) and stored at −20 °C until required. Next, all Anopheles specimens were reconfirmed using molecular methods to distinguish sibling species and prevent operator mistakes. Genomic DNA was extracted from the legs of individual Anopheles mosquitoes using the FavorPrep™ mini kit (Favorgen Biotech, Ping-Tung, Taiwan) according to the manufacturer’s protocol. Multiplex allele-specific PCR (MAS-PCR) assays based on the internal transcribed spacer 2 (ITS2) region of DNA were used to identify the following: (1) five sibling species of the Dirus complex, including An. dirus s.s., An. baimaii, An. cracens, An. nemophilous, and An. scanloni; (2) five species of the Maculatus group, including An. maculatus s.s., An. dravidicus, An. pseudowillmori, An. rampae, and An. sawadwongporni; and (3) five species of the Funestus group, including An. minimus s.s., An. harrisoni, An. aconitus, An. pampanai, and An. varuna, according to the previous protocols of Walton et al. [30], Walton et al. [31], and Garros et al. [32], respectively. For the molecular identification of other Anopheles species, we compared COI Anopheles sequences to the barcode reference library.

2.4. Detection of Malaria-Infected Anopheles Mosquitoes

For screening of Plasmodium sporozoites in Anopheles mosquitoes, the fast COX-I PCR method was used, as described previously by Echeverry et al. [33]. We extracted Plasmodium DNA from the head and thorax of each female Anopheles mosquitoes. An approximately 520 bp segment of the Plasmodium DNA COI region was amplified using the primer pair COX-IF (5′ AGA ACG AAC GCT TTT AAC GCC TG 3′) and COX-IR (3′ ACT TAA TGG TGG ATA TAA AGT CCA TCC wGT 5′). The PCR amplifications were conducted using a thermal cycler (Biometra TOne Series, Germany) in a total volume of 25 μL, containing 4 μL of DNA template, a 1 µM concentration of each primer, 1x blood Phusion PCR Master Mix (Thermo Scientific, Waltham, MA, USA), and distilled water up to 25 μL. The PCR reaction conditions were as follows: initial steps at 98 °C at 4 min followed by 70 cycles of 98 °C at 1 s, 69 °C at 5 s, and 72 °C at 35 s, with a final extension at 72 °C at 10 min. Each PCR contained negative (water without DNA) and positive (DNA of P. falciparum from culture) controls. PCR products were spread by electrophoresis on 1% agarose gels stained with Midori Green DNA stain (Nippon Gene, Tokyo, Japan), under an ImageQuant LAS 500 imager (GE Healthcare Japan Corp., Tokyo, Japan). A specimen showing a clear DNA band size of 540 bp on agarose gel was considered infectious (Plasmodium-genus-positive). If a positive sample had been found, PCR products would have been sent to a service company for DNA sequencing, and then those sequences would have been used for species assessments of Plasmodium parasites by comparing them to reference sequences in the Barcode of Life Data System database (BOLD).

2.5. Polymerase Chain Reaction (PCR) and Sequencing of COI and VGSC Genes

Genomic DNA derived from the legs of each Anopheles specimen was used to amplify COI and VGSC gene fragments. The PCR amplification of approximately 709 bp of the COI gene was performed using two primers, including forward primer COI_F (5′-GGA TTT GGA AAT TGA TTA GTT CCT T-3′) and reverse primer COI_R (5′-AAA AAT TTT AAT TCC AGT TGG AAC AGC-3′) [34]. PCR was conducted according to the previously reported procedure of Chaiphongpachara et al. [35].

The PCR amplification of an approximately 300 bp fragment flanking codon 1014 of the VGSC gene was performed using two primers, including forward primer AgF_kdr (5′-GAC CAT GAT CTG CCA AGA TGG AAT-3′) and reverse primer An_kdr_R2 (5′-GAG GAT GAA CCG AAA TTG GAC-3′) [26]. The 25 µL PCR reaction consisted of 1U Platinum Taq DNA polymerase (Invitrogen, Carlsbad, CA), a 0.4 µM concentration of each primer, 1× reaction buffer, 1.5 mM MgCl₂, 0.2 mM dNTPs, 4 μL of DNA template, and distilled water up to 25 μL. PCR amplifications were performed in a thermal cycler with the following temperature cycles: 94 °C at 5 min, 45 °C at 30 s, and 72 °C at 30 s, followed by 36 cycles of 94 °C at 30 s, 50 °C at 45 s, and 72 °C at 1 min; 35 cycles of 94 °C at 40 s, 54 °C at 60 s, and 72 °C at 1 min.

PCR amplification products of COI and VGSC were visualized on 1% agarose gels stained with Midori Green DNA stain (Nippon Gene, Tokyo, Japan) under an ImageQuant LAS 500 imager (GE Healthcare Japan Corp., Tokyo, Japan) for quality evaluation, before being sent to Solgent Company in Daejeon, South Korea, for the purification of PCR products and DNA sequencing.

2.6. Molecular Analyses

The trace files of COI and VGSC sequences for Anopheles specimens were manually aligned, checked, and edited, and consensus sequences were created from forward and reverse sequences using the BioEdit version 7.2 [36]. Afterward, COI and VGSC consensus sequences were aligned and manually edited using Clustal X [37] in the MEGA X (Molecular Evolutionary Genetics Analysis) software [38].

We compared the COI sequences of our Anopheles specimens to those available in GenBank to confirm species identification using the Basic Local Alignment Search Tool (BLAST, available online http://blast.ncbi.nlm.nih.gov/Blast.cgi (accessed on 10 October 2022) and the National Center for Biotechnology Information (NCBI) and BOLD (https://www.boldsystems.org/ (accessed on 10 October 2022) databases. Acceptance of Anopheles specimens required ≥98% nucleotide sequence identity for the available species sequences in the databases [39]. In addition, the intraspecific and interspecific genetic distances of all Anopheles species were calculated using the Kimura two-parameter distance algorithm (K2P) in MEGA X. We constructed a phylogenetic tree based on maximum likelihood (ML) with Tamura three-parameter plus gamma distribution plus invariable site model (best-fit substitution model) for COI sequences, using bootstrapping values defined for 1000 repetitions in MEGA X, in order to examine the evolutionary relationships among Anopheles species.

We used DNA Sequences Polymorphism (DnaSp) 6 software [40] to calculate the number of polymorphic (segregating) sites (s), nucleotide diversity (π), number of haplotypes (h), haplotype diversity (Hd), the average number of nucleotide differences (k), and statistical tests of neutrality, namely Tajima’s D test [41] and Fu’s Fs test [42], based on the mitochondrial COI gene, to investigate the genetic diversity of Anopheles mosquitoes in each species. In addition, haplotype networks of each Anopheles species were created using the median-joining network method in PopArt 1.7 to visualize the relationships among Anopheles individuals. For screening of kdr mutations in the VGSC gene, we investigated the VGSC sequences of all the specimens to find known resistant mutations (L1014C, L1014F, and L1014S).

3. Results

3.1. Anopheles Mosquitoes

In this study, 114 Anopheles females were caught in three geographical regions of Ranong province. Molecular identification revealed that the Anopheles specimens represented eight species, including An. baimaii (21.05%), An. minimus s.s. (20.17%), An. epiroticus (19.30%), An. jamesii (19.30%), An. maculatus s.s. (13.16%), An. barbirostris A3 (5.26%), An. sawadwongporni (0.88%), and An. aconitus (0.88%) (

Table 1. Species and numbers of Anopheles sampled in the geographical region of Ranong province, Thailand (January–June 2022), identified using molecular methods.

Anopheles Species	Ranong Province			Total (%)
	Northern Part	Central Part	Southern Part
An. aconitus	0	0	1	1 (0.88)
An. baimaii	4	14	6	24 (21.05)
An. barbirostris A3	0	0	6	6 (5.26)
An. epiroticus	0	0	22	22 (19.30)
An. jamesii	6	0	16	22 (19.30)
An. maculatus s.s.	0	14	1	15 (13.16)
An. minimus s.s.	22	0	1	23 (20.17)
An. sawadwongporni	1	0	0	1 (0.88)
Total (%)	33 (28.95)	28 (24.56)	53 (46.49)	114 (100)

). Their distributions in Ranong province, as obtained in this study, are presented in Figure 1.

The southern part of Ranong had the highest number of Anopheles mosquito species (n = 7), followed by the northern (n = 4) and central (n = 2) parts, respectively. Anopheles baimaii was the only Anopheles species found across all three parts of Ranong province. In contrast, An. aconitus and An. sawadwongporni were extremely rare, with just a single specimen found in the southern and northern parts of the province, respectively. Thus, An. aconitus and An. sawadwongporni, represented by only one specimen each, were excluded from genetic diversity analyses.

3.2. Malaria Parasite Detection

According to the fast COX-I PCR method results, none of the 114 Anopheles mosquitoes examined were infected with Plasmodium parasites.

3.3. Nucleotide Sequences

One hundred and fourteen COI sequences of Anopheles mosquitoes were submitted to the GenBank database under accession numbers OP253978–OP254091 (

Table 2. GenBank accession numbers of COI sequences obtained in this study.

Anopheles Species	n	Location	GenBank Accession Numbers
An. Aconitus	1	Southern part of Ranong	OP253978
An. Baimaii	4	Northern part of Ranong	OP253979–OP253982
	14	Central part of Ranong	OP253983–OP253996
	6	Southern part of Ranong	OP253997–OP254002
An. barbirostris A3	6	Southern part of Ranong	OP254003–OP254008
An. epiroticus	22	Southern part of Ranong	OP254009–OP254030
An. jamesii	6	Northern part of Ranong	OP254031–OP254036
	16	Southern part of Ranong	OP254037–OP254052
An. maculatus	14	Central part of Ranong	OP254053–OP254066
	1	Southern part of Ranong	OP254067
An. minimus	22	Northern part of Ranong	OP254068–OP254089
	1	Southern part of Ranong	OP254090
An. sawadwongporni	1	Northern part of Ranong	OP254091

) and were used for COI sequence analyses.

Intraspecific genetic divergences of Anopheles mosquitoes ranged from 0.2 to 1.3%, with an average value of 0.7% (

Table 3. Interspecific and intraspecific K2P genetic distances of Anopheles species were collected based on COI sequences in this study.

Anopheles Species	% Mean Sequence Divergence (Min–Max)
	1	2	3	4	5	6	7	8
1. An. aconitus	NA
2. An. baimaii	11.5% (11.1–11.7)	0.5% (0.0–1.3)
3. An. barbirostris A3	11.1% (10.9–11.2)	9.6% (9.3–10.1)	0.3% (0.0–0.7)
4. An. epiroticus	10.7% (9.9–11.2)	12% (10.9–12.9)	11.6% (10.9–12.4)	1.3% (0.0–2.7)
5. An. jamesii	10% (9.8–10.4)	11.3% (10.7–12.4)	13.6% (13.1–14.6)	11.8% (11.1–12.6)	0.5% (0.0–1.9)
6. An. maculatus	10.2% (10.1–10.4)	8.9% (8.3–9.8)	10.2% (9.9–10.4)	11.6% (10.8–12.4)	9.5% (9.3–9.8)	0.2% (0.0–0.7)
7. An. minimus	8.2% (7.9–8.4)	10.9% (10.4–11.2)	11.2% (10.6–11.6)	11.5% (10.6–12.7)	11% (10.6–12.1)	10% (9.5–10.4)	1.1% (0.0–2.9)
8. An. sawadwongporni	10.6% (10.6–10.6)	11.5% (11.2–11.9)	10.6% (10.4–10.7)	11.5% (10.9–11.9)	11.7% (11.6–12.1)	6.6% (6.5–6.6)	9.9% (9.5–10.3)	NA

). The highest intraspecific divergence was observed in An. epiroticus (1.3%), followed by An. minimus (1.1%), An. baimaii (0.5%), and An. jamesii (0.5%). In contrast, interspecific genetic divergences of Anopheles mosquitoes varied from 6.5 to 14.6%, with an average value of 10.7% (Table 3). The highest interspecific divergence was observed between An. jamesii and An. barbirostris A3 (13.6%), followed by those between An. epiroticus and An. baimaii (12%) and between An. jamesii and An. epiroticus (11.8%). Based on the genetic distances of all individuals, the maximum intra- and minimum interspecific genetic values were nonoverlapping; thus, all Anopheles species in this study could be correctly differentiated by COI sequence analysis.

3.4. Phylogenetic Analysis

The ML phylogenetic tree showed that the species of Anopheles mosquitoes identified in this study was clearly separated into clades, supported by perfect bootstrap values (100%) (Figure 2). The An. minimus clade was sister to the An. aconitus clade, with the An. jamesii clade and the An. epiroticus clade far away, respectively. The An. barbirostris A3 and An. baimaii clades, which were sister clades, had the most distant relationships with other species. Sister clades of the An. maculatus group were positioned between sister clades of An. barbirostris A3 and An. baimaii, and the An. epiroticus clade. In addition, the phylogenetic analysis based on COI sequences indicated that the An. minimus and An. epiroticus clades were split into two distinct subclades and the An. jamesii clade was split into three distinct subclades.

3.5. Genetic Diversity

One hundred and twelve sequences were used to estimate the genetic diversity (the single sequences of An. aconitus and An. sawadwongporni were excluded). The nucleotide and haplotype diversity values of six Anopheles species are depicted in

Table 4. Genetic diversity indices and neutrality tests of Anopheles species in Ranong.

Anopheles Species	n	s	π (±SD)	h	Hd (±SD)	k	Neutrality Tests
							Fu’s Fs	Tajima’s D
An. aconitus	1	–	–	1	–	–	–	–
An. baimaii	24	22	0.005 ± 0.001	17	0.938 ± 0.039	3.616	−10.476 *	−1.621
An. barbirostris A3	6	5	0.003 ± 0.001	4	0.800 ± 0.172	2.067	−0.439	−0.315
An. epiroticus	22	33	0.012 ± 0.001	17	0.974 ± 0.022	8.797	−4.804 *	−0.220
An. jamesii	22	20	0.005 ± 0.001	11	0.900 ± 0.041	3.667	−2.544 *	−1.233
An. maculatus	15	6	0.002 ± 0.000	5	0.743 ± 0.090	1.695	−0.214	−0.285
An. minimus	23	29	0.011 ± 0.002	12	0.925 ± 0.032	8.016	−0.211	0.077
An. sawadwongporni	1	–	–	1	–	–	–	–

Anopheles aconitus and An. sawadwongporni, represented by only one specimen each, were excluded from the analyses. An asterisk (*) after Fu’s Fs and Tajima’s D values represents the statistical difference at p < 0.05. Abbreviations: n = number of sequences; s = number of polymorphic (segregating) sites; π = nucleotide diversity; h = number of haplotypes; Hd = haplotype diversity; k = average number of nucleotide differences.

.

The genetic diversity index assessment showed that An. epiroticus had the highest level of nucleotide diversity (0.012 ± 0.001 SD), followed by An. minimus (0.011 ± 0.002 SD), An. baimaii (0.005 ± 0.001 SD), An. jamesii (0.005 ± 0.001 SD), An. barbirostris A3 (0.003 ± 0.001 SD), and An. maculatus (0.002 ± 0.000 SD) (Table 4). The highest level of haplotype diversity was observed in An. epiroticus (0.974 ± 0.022 SD/h = 17), followed by An. baimaii (0.938 ± 0.039 SD/h = 17), An. minimus (0.925 ± 0.032 SD/h = 12), An. jamesii (0.900 ± 0.041 SD/h = 11), An. barbirostris A3 (0.800 ± 0.172 SD/h = 4), and An. maculatus (0.743 ± 0.090 SD/h = 5).

The Fu’s Fs and Tajima’s D values of six Anopheles species in Ranong were almost all negative, except Tajima’s D values of An. minimus (0.077, Table 4). These results suggested a high number of low-frequency mutations and that Anopheles populations in Ranong are undergoing demographic expansion. Significantly negative Fu’s FS values support this finding; however, Fu’s FS was significant in An. baimaii, An. epiroticus, and An. jamesii, whereas Tajima’s D was not significant in all species.

3.6. Haplotype Relationships

The frequencies of and relationships between 112 haplotypes of An. baimaii, An. barbirostris A3, An. epiroticus, An. jamesii, An. maculatus, and An. minimus identified in Ranong based on COI sequences are shown in median-joining haplotype networks (Figure 3).

The network analysis of An. baimaii revealed that H1 was the central haplotype that was highly connected to haplotype lines, and was the only one found in all localities of Ranong (northern, central, and southern parts). The haplotype network of An. minimus showed two distinct genetic lineages, A and B, based on mutation steps on the haplotype lines, similar to the An. epiroticus network, which showed two lineages.

The central haplotype of An. minimus and An. epiroticus could not be identified because their frequencies were not clearly different. The haplotype network of An. jamesii showed that H8 was the most common haplotype and H1 was a shared haplotype between the northern and southern populations. Based on mutation steps, three genetic lineages of An. jamesii were identified. The haplotype networks of An. barbirostris A3 showed that H1 was the most frequent haplotype, and all haplotypes were connected in a straight line, whereas the An. maculatus network showed that H2 was the most frequent haplotype and H3 was the shared haplotype, including samples from the central and southern parts.

3.7. Screening VGSC-Mutation-Mediated Knockdown Resistance

One hundred and fourteen DNA sequences of the VGSC gene fragment from the Anopheles specimens were checked for screening of knockdown resistance mutations. All the sequenced specimens presented only the L1014 wild-type allele in the VGSC gene (

Table 5. Screening for kdr mutations in the voltage-gated sodium channel (VGSC) gene in specimens of eight Anopheles species obtained in this study.

Species	Allelic Frequency
	L1014 Wild			L1014C (TGT)	L1014F (TTT)	L1014S (TCA)
	TTA	TTG	CTA
An. Aconitus	1	0	0	0	0	0
An. baimaii	0	0	24	0	0	0
An. barbirostris A3	0	6	0	0	0	0
An. epiroticus	22	0	0	0	0	0
An. jamesii	22	0	0	0	0	0
An. maculatus	15	0	0	0	0	0
An. minimus	23	0	0	0	0	0
An. sawadwongporni	1	0	0	0	0	0
Total	84	6	24	0	0	0

). No kdr-resistant alleles (L1014C, L1014F, or L1014S) were found in any of the 114 Anopheles specimens screened (Figure 4, Table 5).

4. Discussion

In this study, specimens of eight species of Anopheles mosquitoes collected from Ranong province, which is Thailand’s most P. knowlesi-endemic area, were subjected to species confirmation using molecular methods; the species included An. aconitus, An. baimaii, An. barbirostris A3, An. epiroticus, An. jamesii, An. maculatus s.s., An. minimus s.s., and An. sawadwongporni. In the northern part of Ranong, a total of four Anopheles species were found: An. baimaii, An. jamesii, An. sawadwongporni, and An. minimus s.s. as the dominant species. In the central area, An. baimaii and An. maculatus s.s. were found to be the dominant species. Most An. minimus mosquitoes live in forest edge areas, so they are common in the northern part of Ranong where there are many forest edge areas. Meanwhile, the Hat Som Paen reservoir area, which consists of densely forested high mountains in the central part of Ranong, is a suitable habitat for An. baimaii. We also found An. maculatus in the reservoir area, which is likely their habitat, although previous reports indicated that they were predominantly distributed along the edge of the forest [15,16]. In the southern part of Ranong, An. epiroticus is the dominant species because their habitat is coastal areas.

Unfortunately, no Anopheles mosquitoes in this survey were infected with Plasmodium parasites. However, some species of Anopheles mosquitoes require special entomological surveillance, based on previous reports of P. knowlesi infections in other countries. Several previous studies in Malaysia have reported that Anopheles mosquitoes in the Leucosphyrus group are important vectors of P. knowlesi [43,44,45]. Anopheles baimaii (previously known as An. dirus species D) is a species member in the Dirus complex and belongs to the Leucosphyrus group [46]. This Anopheles species is considered the primary vector of human malaria in Thailand [47]. Our study results indicated that they are distributed in forested areas throughout Ranong. In addition, An. nemophilous, belonging to the Leucosphyrus group, has also been reported in Ranong province [48]. A previous study reported P. knowlesi infections in An. sundaicus s.l. in Katchal Island, India [14]. Anopheles epiroticus (previously known as An. sundaicus species A) is a common Anopheles species found near coastal areas in Ranong and other provinces of Thailand [49,50]. This Anopheles species belongs to the Sundaicus complex and is considered Thailand’s secondary vector of malaria [48,51]. Anopheles barbirostris species A3 is a cryptic species in the Barbirostris complex belonging to the Barbirostris subgroup [52]. In Thailand, this mosquito species has previously only been reported in Kanchanaburi province [52]. Our study is the first to demonstrate the additional distribution of An. barbirostris A3 in Thailand. However, An. barbirostris A3 is another species that should not be overlooked because An. donaldi, a member species in the Barbirostris subgroup, has been reported to carry P. knowlesi infections in Lawas, Northern Sarawak, Malaysian Borneo [9]. In addition, investigations of blood meal sources of malaria vector mosquitoes using specific PCR assays should be continued in the future to determine their anopheline anthropophilic, zoophilic, or zoo-anthropophilic origin. The host preferences of Anopheles species are very important pieces of information in evaluating their ability to transmit simian malaria to humans [53].

The genetic distances between the eight Anopheles taxa based on 114 COI sequences showed that the maximum intra- and minimum interspecific genetic values were nonoverlapping, indicating the existence of a distinct barcode gap. The presence of a barcoding gap confirms the success of DNA barcoding for species identification [54]. Recently, Chaiphongpachara et al. [35] succeeded in identifying several mosquito species in Thailand based on nucleotide differences in the COI gene, except for An. dirus and An. baimaii. Our study results provide supporting evidence that DNA barcoding based on COI can be used to identify mosquito species. However, other DNA markers, such as ITS2, must be used for species identification in cases where Anopheles mosquitoes in the Dirus complex are found [55].

The nucleotide diversity (π) and nucleotide diversity (Hd) are important genetic indicators used to measure genetic diversity among populations. The nucleotide diversity values of An. baimaii, An. barbirostris A3, An. epiroticus, An. jamesii, An. maculatus, and An. minimus in Ranong were lower than the haplotype diversity values, indicating a recent Anopheles population expansion to a small effective population size after a bottleneck [56]. This demographic event occurred long enough ago for the haplotypes to increase through mutation, but not enough for the accumulation of large sequence differences [57]. Furthermore, a high level of haplotype diversity results from a large population and different environments and living habits suitable for their rapid development in nature [58].

Our results showed that genetic diversity values were similar in some species and different in others, for multifactorial reasons. Mosquito genetic diversity has both internal and external causes [59]. Internal causes of genetic diversity are genetic mutations or changes, whereas external factors are strongly related to the ecological environment of the mosquitoes [59]. Genetic diversity is an important factor that allows natural populations to adapt to and survive long-term changes or adverse environmental conditions [60]. Ranong is one of the provinces in southern Thailand with unique ecological features. The area is covered by mountains and fertile forests, and is adjacent to the Andaman Sea. It is also the wettest province in Thailand. It has been previously reported that Anopheles mosquito populations can swiftly adapt to alterations in environmental conditions, which may impact the genetic diversity within species at the population level and their gene flow [56,61]. However, a limitation of our study was the assessment of Anopheles vectors in only one endemic area, which provided insufficient data for this answer.

The Fu’s Fs and Tajima’s D values of An. baimaii, An. barbirostris A3, An. epiroticus, An. jamesii, and An. maculatus in Ranong all showed negative values, supporting population size expansion in Ranong. If a population is selectively neutral and at equilibrium between genetic drift and selectively neutral mutation, the Tajima’s D value is expected to be zero. Positive Tajima’s D values indicate a sudden decrease in population size and/or balancing selection, whereas negative Tajima’s D values indicate population size expansion after a recent bottleneck or mutational selection [41]. Our results were consistent with the population structure of An. baimaii in Thailand, indicating that the population is expanding [56].

The ML phylogenetic tree and haplotype network results revealed two distinct genetic lineages, A and B, of An. minimus and An. epiroticus in Ranong. Recently, Bunmee et al. [56] reported the existence of A and B lineages for An. minimus s.s. in Thailand, which agrees with our research findings. In many of Thailand’s malaria transmission areas, such as Tak, Surat Thani, Yala, Chanthaburi, and Trat, there are two lineages of An. minimus, which are often found together [56]. However, it is unclear whether the two lineages have the potential to transmit malaria or other different behaviors. In addition, our study is the first to reveal two distinct genetic lineages of An. epiroticus based on the COI gene, which shows genetic variation and local adaptation. Syafruddinid et al. [62] explained that the COI gene is suitable for assessing genetic variation within populations of An. epiroticus because mtDNA has a high mutation rate. However, this gene cannot be used as a molecular marker to differentiate between An. epiroticus and its other sibling members [62]. Although three genetic lineages of An. jamesii were identified, only one group had many samples, whereas the other two groups had only one member each. Consequently, future genetic studies on this species should be conducted.

The early detection and surveillance of VGSC-mutation-mediated knockdown resistance (kdr) in Anopheles populations can provide entomological data on the causes of pyrethroid resistance in insects to inform the development of strategies to control malaria vectors [63]. The present study showed no presence of kdr mutation in the VGSC gene among Anopheles mosquitoes from Thailand’s most P. knowlesi-endemic area. This result is similar to those of previous Anopheles investigations in Ubon Ratchathani province, northeastern Thailand [64]. However, this study is limited by a lack of information on the susceptibility of the mosquito samples tested. Therefore, we do not know the true state of insecticide resistance in these Anopheles populations. Further entomological investigations into the susceptibility of adult mosquito vectors to insecticides are required.

5. Conclusions

Our study demonstrated the genetic diversity of Anopheles mosquitoes in Thailand’s most P. knowlesi-endemic area. Our genetic diversity analysis will contribute to a more comprehensive genetic profile of Anopheles vectors in Thailand. In addition, our attempts to detect P. knowlesi infection in Anopheles mosquitoes did not reveal infected specimens. However, three Anopheles species, including An. baimaii, An. barbirostris A3, and An. epiroticus, should be kept under special surveillance as P. knowlesi infections have been found in these species in other countries. This entomological information could lead to the selection of appropriate methods for controlling these Anopheles populations, such as insecticide-treated nets (ITNs) and indoor residual spraying (IRS), in order to control the spread of monkey malaria to humans in Thailand’s most P. knowlesi-endemic area. In addition, educating the population about vector breeding sites and strategies for protection against Anopheles mosquitoes, such as applying mosquito repellent or wearing protective clothes when entering forests where monkeys reside, are crucial ways to help reduce the incidence of knowlesi malaria.