A multiplex PCR assay for the identification of five species of the Anopheles barbirostris complex in Thailand

Background The Barbirostris Complex comprises six formally described species that cannot be differentiated based on morphology alone. Out of these six species, two have been reported as putative malaria vectors, An. campestris and An. wejchoochotei. Five species are present in Thailand, An. barbirostris, An. campestris, An. dissidens, An. saeungae and An. wejchoochotei, while An. vanderwulpi occurs in Indonesia. As these species cannot be accurately differentiated by morphological characters, there is a crucial lack of information on their bionomics and role in the transmission of malaria and filariasis agents. Results For differentiating the six species, an allele-specific amplification (AS-PCR) based on the second internal transcribed spacer (ITS2) sequence was developed. From 862 mosquitoes in the Barbirostris Complex collected in 23 provinces throughout Thailand, the AS-PCR was able to identify five species and its validation was undertaken on 185 specimens. Conclusions This multiplex-PCR assay is potentially able to definitely identify all six species of the Barbirostris Complex and was validated on five species present in Thailand.


Background
Anopheles (Anopheles) barbirostris belongs to the Barbirostris Complex within the Barbirostris Group of the Myzorhynchus Series [1]. Recently, Taai & Harbach [2] described within the Barbirostris Complex three new species, An. dissidens, An. saeungae and An. wejchoochotei, which accounts for six formally named species including An. barbirostris, An. vanderwulpi and An. campestris, the latter one being recognized as a member of this complex [2]. Four species are reported as primarily zoophilic throughout their geographic range, although they may bite humans in the absence of their usual hosts (typically bovids). The two others, An. wejchoochotei and An. campestris, are known for their greater anthropophilic behavior, especially the latter species that more readily bites humans than any other members of the Barbirostris Complex [2,3]. Anopheles barbirostris (s.l.) is widely distributed in Thailand [4,5] and more globally in the Asian region [2,[6][7][8]. It has been reported as a vector of Plasmodium falciparum and Plasmodium vivax in Sri Lanka, Bangladesh, Indonesia (Sumatra, Sulawesi, Flores), Timor Leste, as well as a secondary vector on the island of Borneo [9] and a putative malaria vector in the Aranyaprathet District, Sa Kaeo Province, southeastern Thailand [10,11]. More specifically, An. barbirostris (s.l.), An. campestris and An. wejchoochotei (former 'campestris-like' , see Table 1) have been incriminated as vectors of P. falciparum and P. vivax [2][3][4][11][12][13][14][15][16][17]. However, the lack of reliable methods to identify the species within the complex has hampered precise evaluation of the specific role of each member in transmission of malaria and other pathogens, e.g. Brugia timori and Brugia malayi in Indonesia [12,29]. The aim of this study was to develop a rapid and accurate identification method to distinguish the known species of the Barbirostris Complex. The principle of allele-specific PCR (AS-PCR) methodology was selected based on species-specific differences within the sequences of the internal transcribed spacer 2 (ITS2), a ribosomal DNA gene (rDNA) widely used to differentiate cryptic species of Anopheles, particularly those belonging to Asian complexes and groups [18][19][20][21][22].

Primer design based on ITS2 sequences
Allele-specific primers were designed from the rDNA ITS2 sequences from previous studies [2,[23][24][25]. ITS2 sequences were aligned using Multalin version 5.4.1 [26] to obtain a consensus sequence for each species, which was used to determine specific primers. Primers were designed manually and using the Primer3 input (version 0.4.0) program [27]. The melting temperatures of the primers were kept similar to each other so that they could be combined readily in a single PCR set-up. The oligonucleotide primers were synthetized by Eurogentec (Belgium).

Multiplex allele-specific PCR
To identify the six species of the Barbirostris Complex, nine primers were designed. To avoid high competition between the primers and according to melting temperatures, a double multiplex PCR was developed.

Mosquito collection, morphological identification and DNA extraction for PCR assay validation
During December 2016 and March 2017, 862 specimens of the Barbirostris Complex were collected in 23 provinces of Thailand ( Fig. 1). Mosquito collections were done between 18:00 and 24:00 h by human-landing catches or cow-baited trapping (tents or landing catches) depending on the locality. Females were individually placed in 1.5 ml Eppendorf tubes and preserved by desiccation with silica gel. Morphological identification of mosquitoes was performed at Kasetsart University using standard illustrated keys allowing the separation of three taxa, An. barbirostris, An. campestris, and a third one called "unknown species" [23]. Genomic DNA was extracted from whole individual adult mosquitoes based on routine procedures [18]. Of 434 samples amplified by AS-PCR, 43 samples were subsequently sequenced for species confirmation and PCR assay validation.

Primer design
Primer design was based on the ITS2 sequences of five out of six species available in GenBank ( Table 2); the ITS2 sequence of An. campestris is not available. Primer names, sequences, size of the PCR products and respective melting temperatures (Tm) are presented in Table 3. Due to the close similarity of the ITS2 sequences of the six members, primer design was difficult with some species having similar individual size bands (bp), thus a dual PCR assay was developed in order to more reliably separate all species. Based on nucleotide alignment of the ITS2 region, nine primers were designed. A forward primer common to five species, except An. campestris (fBDSVW), then two species-specific forward primers (fBar, fCamp), and six reverse primers (rBar&Van, rVan&Dis, rCamp, rSaue1, rSaue2, rWej). Anopheles barbirostris is interrogated by two forward primers (fBDSVW and fBar) and one reverse primer (rBar&Van) providing 2 bands at 388 bp and 208 bp in PCR1 (Table 3). It was necessary to design a speciesspecific forward primer (fBar) for An. barbirostris in order to differentiate it from An. vanderwulpi, which shares the same two other primers (fBDSVW and rBar&Van). Nucleotide alignment of the amplified ITS2 region for the six species and the nine primer sites are shown in Fig. 2. The specificity of each primer was tested and the results presented in Fig. 3.    Table 3 were confirmed in the AS-PCR ( Table 4).

Sequencing of the ITS2 region
The complete ITS2 sequence length of each species ranges between 1500-1850 bp (Table 1). Within the 43 sequenced samples, three profiles were obtained from 31 specimens that matched the ITS2 sequences of An. wejchoochotei (n = 14), An. saeungae (n = 10) and An. dissidens (n = 7), respectively (Table 5). For the 12 additional sequences, a small part of the ITS2 sequence was amplified, which could not allow any definitive species identification.

Distribution of the Barbirostris Complex species in Thailand
Anopheles wejchoochotei was identified in 54.6% of the specimens and in 19 of 23 sampled provinces indicating a wide distribution in Thailand (Fig. 1, Table 6). The second most common species was An. saeungae with 18.9% of the specimens collected in 15 provinces located throughout Thailand, excluding the southernmost region. Anopheles dissidens was found in 13 provinces, representing 24.3% of the collected specimens that were mainly located in the western and southern areas of Thailand. Anopheles barbirostris, with only 1.6% of the specimens identified, appeared confined to Chiang Mai Province, northern Thailand; while only one specimen (0.5%) of An. campestris was collected in Chanthaburi Province, eastcentral Thailand.

Discussion
Populations of An. barbirostris (s.l.) have been identified across a wide geographic range including India and Sri Lanka, throughout most of Southeast Asia, in particular Malaysia and Indonesia where it extends from Sumatra, Java, Bali in the west, to Kalimantan, Sulawesi, and throughout the Lesser Sunda Islands and Timor-Leste in the east [7]. Their role in transmission of both malaria and Brugian filariasis has been documented in   Sulawesi, Flores and Timor [12,13,[29][30][31][32], and mentioned as putative malaria vectors in Sri Lanka [33], possibly Bangladesh [34] and Thailand [10,11,17]. However, all of these reports on the natural vectorial role of An. barbirostris (s.l.) are based on unreliable morphological identifications and a period before the Barbirostris Complex was recognized in 2001 [35]. The complex now includes six formally named sibling species [1], and while almost identical (isomorphic) in adult morphology, their respective roles in malaria and filarial transmission differs drastically (Table 1). In Thailand, five formal species have been identified, while a sixth one, An. barbirostris species A3, has been reported from Kanchanaburi Province [25,36]. Not formally characterized and unnamed, this species has a much smaller ITS2 sequence of 1070 bp [25]. Due to the lack of specimens, species A3 has not been included in this study, although primers have been designed to differentiate it from the other species of the complex. Considering the wide geographical distribution of the Barbirostris Complex and the involvement of some members in plasmodia and filarial transmission, it is crucial to better understand the full diversity of species that constitute the complex and which ones are vectors of public health importance so as to better target control efforts to maximize suppression of transmission and increase the likelihood of achieving malaria elimination [37]. Therefore, a reliable identification technique using molecular methods was an essential development to differentiate between species, especially those that occur in sympatry, that would allow further investigation on their specific bionomics, distribution and role in pathogen transmission.
For correct identification at the species level, molecular markers are used to separate sibling species, especially the rRNA ITS2 gene that is widely used to differentiate within many Asian Anopheles complexes [18][19][20][21][22]. The developed AS-PCR was able to differentiate all five species of the Barbirostris Complex present in Thailand. Primers to identify the species occurring in Indonesia, An. vanderwulpi, also found in sympatry with An. barbirostris [8], and An. barbirostris species A3 from Thailand have been designed and awaiting validation analysis of field samples when available. The application of this AS-PCR assay on 185 specimens collected throughout Thailand showed the wide distribution of An. wejchoochotei, previously presented as An. campestris-like species in a published distribution map [5]. This species is often found in sympatry with An. saeungae and An. dissidens based on collections from 13 and 11 provinces, respectively. Anopheles barbirostris, collected in one site

Conclusions
This study provides a simple, rapid, specific and efficient multiplex PCR assay for identifying the six described species members of the Barbirostris Complex. This assay has been validated on five species present in Thailand. Specimens of An. vanderwulpi from Indonesia and An. barbirostris species A3 from Thailand should now be tested using this AS-PCR in order to validate the primers. This multiplex PCR is a reliable identification tool for allowing a wide range of studies on the known species of the Barbirostris Complex. The assay also provides a tool with the possibility of recognition of new cryptic species populations throughout the broader geographical range of this complex.