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Genetic compatibility between Anopheles lesteri from Korea and Anopheles paraliae from Thailand

Abstract

To assess differentiation and relationships between Anopheles lesteri and Anopheles paraliae we established three and five iso-female lines of An. lesteri from Korea and An. paraliae from Thailand, respectively. These isolines were used to investigate the genetic relationships between the two taxa by crossing experiments and by comparing DNA sequences of ribosomal DNA second internal transcribed spacer (ITS2) and mitochondrial DNA cytochrome c oxidase subunit I (COI) and subunit II (COII). Results of reciprocal and F1-hybrid crosses between An. lesteri and An. paraliae indicated that they were compatible genetically producing viable progenies and complete synaptic salivary gland polytene chromosomes without inversion loops in all chromosome arms. The pairwise genetic distances of ITS2, COI and COII between these morphological species were 0.040, 0.007-0.017 and 0.008-0.011, respectively. The specific species status of An. paraliae in Thailand and/or other parts of the continent are discussed.

Anopheles lesteri; Anopheles paraliae; crossing experiments; second internal transcribed spacer; cytochrome c oxidase subunit I; cytochrome c oxidase subunit II


The Anopheles hyrcanus Group has a wide range of distributions extending from Iberia in Europe to East and Southeast Asia, including some of the off-lying islands of the Indian and Pacific Oceans. Up until now, at least 27 species have been reported within this group (Harrison & Scanlon 1975, Harbach 2010). It is well known that some species of the Hyrcanus Group are involved in transmission of human diseases, particularly in the Oriental and contiguous parts of the eastern Palaearctic regions. For example, human malaria Plasmodium vivax was detected in Anopheles sinensis, Anopheles lesteri, Anopheles kleini, Anopheles pullus and Anopheles belenrae (Harrison 1973, Ree et al. 2001, Whang et al. 2002, Ma & Xu 2005, Lee et al. 2007, Joshi et al. 2009, 2011, Rueda et al. 2010). Moreover, Brugia malayi was found in An. sinensis and An. lesteri (Sasa 1976) while Anopheles peditaeniatus was infected with Japanese encephalitis virus (Zhang 1990, Kanojia et al. 2003). In addition, some members of the Hyrcanus Group have also been considered as economic pests of cattle because of their vicious biting-behaviour and ability to transmit cervid filariae of the genus Setaria (Reid et al. 1962, Reid 1968, Harrison & Scanlon 1975).

An. lesteri has been found in the Philippines (type locality) and the Palaearctic regions (China, Korea and Japan) whereas Anopheles paraliae has been detected in the coastal areas of Peninsular Malaysia, Sabah, Sarawak, Brunei, Vietnam and Thailand. However, the taxonomic ambiguity of An. paraliae was raised as early as 1959. Morphologically, An. paraliae has a narrower apical fringe spot on the wing compared with that of An. lesteri, but their immature stages can not be distinguished from each other. Consequently, An. paraliae was considered to be a subspecies, An. lesteri paraliae, by earlier authors (Sandosham 1959, Reid 1963, 1968, Harrison & Scanlon 1975). Nevertheless, this subspecies was elevated subsequently to species status, i.e., An. paraliae, based on distinct characteristics of the adult wings and immature habitats (brackish and/or peaty water) (Harrison et al. 1991). Yet there is no evidence of genetic differences between An. lesteri and An. paraliae. This article presents the results of crossing experiment and cytogenetic study of these two species and comparative DNA sequence analyses of the second internal transcribed spacer (ITS2) of ribosomal DNA (rDNA), the cytochrome c oxidase subunit I (COI) and subunit II (COII) of mitochondrial DNA (mtDNA).

MATERIALS AND METHODS

Collection sites - Samples of An. lesteri from Korea were caught in a vinyl tent in a rice paddy field of the district of So Rae, Incheon City, northern of the province of Gyeonggi. The An. paraliae specimens from Thailand were obtained by using cow-baited traps in three localities, i.e., district of Damnoen Saduak, province of Ratchaburi, district of Pak Panang, province of Nakhon Si Thammarat and district of Hat Yai, province of Songkhla (Table I). Species identification using F1-progeny of each iso-female line followed the keys of Rueda et al. (2005) and Rattanarithikul et al. (2006). The distinctive characteristics of wings to separate An. lesteri from An. paraliae are illustrated in Fig. 1.

TABLE I
Locations, code of iso-female lines of Anopheles lesteri and Anopheles paraliae and their GenBank accessions

Fig. 1A:
wing of Anopheles lesteri from Korea showing wide pale fringe spot extending from tip of vein R1 to R4+5 and two dark spots on anal vein (1A); B-D: wings of Anopheles paraliae from Thailand showing very narrow pale fringe spot at tip of vein R2, and two dark spots on 1A similar to that of An. lesteri (B), narrow fringe spot at tip of vein R2 and two dark spots on 1A (C) and moderated fringe spot extending from tip of vein R1-3 and one dark spot on 1A (D).

Establishment of iso-female lines - Three and five iso-female lines of An. lesteri (ilG1, ilG2, ilG3) and An. paraliae (ipR1, ipR2, ipN1, ipS1, ipS2), respectively, were established successfully using the methods of Choochote et al. (1983) and Kim et al. (2003). They have been maintained in colonies for more than five consecutive generations in our laboratory and they were used for crossing experiments and comparative DNA sequence analyses.

Crossing experiments - One iso-female line (ilG1) of An. lesteri and three iso-female lines (ipR1, ipN1, ipS1) of An. paraliae were arbitrarily selected for crossing experiments to determine post-mating reproductive isolation by employing the techniques previously reported by Saeung et al. (2007). Study on salivary gland polytene chromosomes of 4th instar larvae of F1-hybrids from the crosses followed the techniques of White et al. (1975) and Kanda (1979).

DNA extraction and amplification - Individual F1-progeny adult females of each iso-female line of An. lesteri (ilG1, ilG2, ilG3) and An. paraliae (ipR1, ipR2, ipN1, ipS1, ipS2) were used for DNA extraction and amplification. Molecular analysis of ITS2, COI, COII was performed to determine intraspecific sequence variation in An. lesteri and An. paraliae. Genomic DNA was extracted from adult mosquitoes using the DNeasy(r) Blood and Tissue Kit (Qiagen). Primers for amplification of ITS2, COI and COII regions followed the methods of Saeung et al. (2007). Polymerase chain reaction (PCR) reaction was performed in total 20 µL volume 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. For ITS2, the conditions for amplification consisted of initial denaturation at 94ºC for 1 min, 30 cycles at 94ºC for 30 sec, 55ºC for 30 sec and 72ºC for 1 min and a final extension at 72ºC for 5 min. The amplification profile of COI and COII comprised initial denaturation at 94ºC for 1 min, 30 cycles at 94ºC for 30 sec, 50ºC for 30 sec and 72ºC for 1 min and a final extension at 72ºC for 5 min. The amplified products were subjected to electrophoresis in a 1.5% agarose gel and stained with ethidium bromide. Finally, the PCR products were purified using the QIAquick(r) PCR Purification Kit (Qiagen) and their sequences directly determined using the BigDye(r) Terminator Cycle Sequencing Kit and 3130 genetic analyzer (Applied Biosystems). The sequence data of this pa-per have been deposited in the DDBJ/EMBL/GenBank nucleotide sequence database under accessions AB733020-AB733043. The ITS2, COI and COII sequences obtained from this study were also compared with deposited sequences available through GenBank (Table I).

Sequencing alignment and phylogenetic analysis - Sequences of ITS2, COI and COII were aligned using the CLUSTALW multiple alignment program (Thompson et al. 1994). Gap sites were excluded from the following analysis. The Kimura two-parameter method was used to calculate genetic distances (Kimura 1980). Construction of neighbour-joining (NJ) trees (Saitou & Nei 1987) and the bootstrap test with 1,000 replications were performed 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, GTR+I, which was selected by MrModeltest version 2.3 (Evolutionary Biology Centre, Uppsala University, 2004) as the best-fit model for ITS2, COI and COII. Bayesian posterior probabilities were calculated from the consensus tree after excluding the first 25% trees as burnin.

RESULTS

Crossing experiments - Details of hatchability, pupa-tion, emergence and adult sex-ratio of parental, reciprocal and F1-hybrid crosses between An. lesteri from Korea and An. paraliae from Thailand are shown in Table II. All crosses yielded viable progenies through the F2-generations. No evidence of genetic incompatibility and/or post-mating reproductive isolation was observed among these crosses (repeated twice: experiments 2 and 3, data are not shown). The salivary gland polytene chromosomes of F1-hybrid larvae from all crosses showed complete synapsis without inversion loops in all chromosome arms (Fig. 2).

TABLE II
Crossing experiments among the four iso-female lines of Anopheles lesteri and Anopheles paraliae

Fig. 2
complete synapsis in all arms of salivary gland polytene chromosome of F1-hybrid larvae of crosses between Anopheles lesteri and Anopheles paraliae. A: ilG1 female x ipR1 male; B: ipR1 female x ilG1 male; C: ilG1 female x ipN1 male; D: ipN1 female x ilG1 male; E: ilG1 female x ipS1 male; F: ipS1 female x ilG1 male.

Sequence analysis of ITS2, COI and COII regions - The level of genetic distance and number of base substitutions between sequences of the three regions are presented in Tables III-V. Analysis of the ITS2 sequence revealed no intraspecific sequence variation among the three and five iso-female lines of An. lesteri and An. paraliae, respectively. Comparison of ITS2 sequences indicated that An. lesteri differed from An. paraliae by 16 base substitutions (pairwise distance = 0.040). In addition, three iso-female lines of An. lesteri from Korea were identical with An. lesteri from China (= Anophe-les anthropophagus) (AY803732, AY375467), Japan (AB159606) and Korea (EU789791), but they differed from those of the Philippines (AY375469) by three base substitutions (pairwise distance = 0.007). The average percentages of base composition for the ITS2 sequence of the eight iso-female lines (3 of An. lesteri from Korea and 5 of An. paraliae from Thailand) were A: 29.9% (29.2-30.5%), T: 24.2% (23.6-24.9%), G: 25.2% (25-25.4%) and C: 20.8% (20.6-20.9%). Percentage of GC content was 46% in An. lesteri (448 bp) and 45% in An. paraliae (448 bp). All eight sequences differed markedly from An. sinensis (pairwise distance = 0.321-0.338) and An. peditaeniatus (pairwise distance = 0.550-0.566) (Table III). The analysis of COI (658 bp) among the eight iso-female lines revealed four-nine base substitutions (pairwise distance = 0.007-0.017). On the contrary, An. lesteri and An. paraliae showed significant differences from An. sinensis (pairwise distance = 0.034-0.042) and An. peditaeniatus (pairwise distance = 0.037-0.041) (Table IV). The analysis of COII (685 bp) among the eight iso-female lines revealed five-seven base substitutions (pairwise distance = 0.008-0.011). These two species also showed significant differences from An. sinensis (pairwise distance = 0.039) and An. peditaeniatus (pairwise distance = 0.031-0.036) (Table V).

TABLE III
Genetic distance and number of nucleotide substitutions in second internal transcribed spacer sequences among Anopheles lesteri, Anopheles paraliae, Anopheles sinensis and Anopheles peditaeniatus
TABLE IV
Genetic distance and number of nucleotide substitutions in cytochrome c oxidase subunit I sequences among Anopheles lesteri, Anopheles paraliae, Anopheles sinensis and Anopheles peditaeniatus
TABLE V
Genetic distance and number of nucleotide substitutions in cytochrome c oxidase subunit II sequences among Anopheles lesteri, Anopheles paraliae, Anopheles sinensis and Anopheles peditaeniatus

Phylogenetic analysis - The NJ and Bayesian trees of An. lesteri, An. paraliae, An. sinensis and An. peditaeniatus were constructed based on the ITS2, COI and COII sequences (Fig. 3). For ITS2, An. lesteri (n = 8) and An. paraliae (n = 5) were clustered in each monophyletic and well separated from An. sinensis and An. peditaeniatus with high bootstrap values (93-100%) in both NJ and Bayesian trees. The trees indicated that An. lesteri was more closely related to An. paraliae (average genetic distances = 0.038) than to the other species. Further, lower sequence divergences (0.000-0.002) were found within the population of each species. For COI and COII, the trees showed that An. lesteri was more closely related to An. paraliae than to the other species with low level of average genetic distances (0.008-0.011) for both regions, while very low genetic distances (0.003-0.005) were obtained within the population of each species.

Fig. 3:
neighbour-joining (NJ) trees inferred from sequences of three loci. A: second internal transcribed spacer; B: cytochrome c oxidase subunit I (COI); C: COII of Anopheles paraliae, Anopheles lesteri, Anopheles sinensis and Anopheles peditaeniatus. Numbers on branches are bootstrap values (%) of NJ analysis and Bayesian posterior probabilities (%). A hyphen (-) shows that the branch did not appear in majority rule (50%) consensus trees of Bayesian analysis. Branch lengths are proportional to genetic distance (scale bar).

DISCUSSION

Crossing experiments using iso-female lines of closely related species of the Oriental Anopheles have proven to be a robust systematic procedure for clarifying species status, for example, Anopheles minimus and Anopheles aconitus (Harrison 1980, Sucharit & Choochote 1982), Anopheles annularis and Anopheles philippinensis (Choochote et al. 1984), Anopheles nivipes and An. philippinensis (Klein et al. 1984) and An. minimus and Anopheles flavirostris (Somboon et al. 2000). These methods are useful for solving taxonomic problems of some sibling species complexes, e.g., Anopheles dirus (Baimai et al. 1987), Anopheles maculatus (Thongwat et al. 2008), An. minimus (Somboon et al. 2001, 2005, Choochote et al. 2002) and Anopheles barbirostris (Saeung et al. 2007, 2008, Suwannamit et al. 2009). Likewise, the status of subspecies or cytological races of Anopheles can be elucidated by the same approach of cytogenetic study as exemplified in An. pullus (= Anopheles yatsushiroensis) (Park et al. 2003), Anopheles vagus (Choochote et al. 2002), An. aconitus (Junkum et al. 2005), An. sinensis (Choochote et al. 1998, Min et al. 2002, Park et al. 2008), An. barbirostris species A1 (Saeung et al. 2007, Suwannamit et al. 2009), Anopheles campestris-like taxon (Thongsahuan et al. 2009) and An. peditaeniatus (Choochote 2011). Our findings in this study showed no post-mating reproductive isolation between An. lesteri from Korea and An. paraliae from Thailand. These results were clearly supported by cytological evidence and DNA analysis. Thus, complete synapsis of salivary gland polytene chromosomes without inversion loops along the entire lengths of all chromosome arms was observed in the F1-hybrid larvae between An. lesteri and An. paraliae which strongly indicated genetic compatibility between them.

Analysis of ITS2 sequences of An. lesteri from Korea (ilG1, ilG2, ilG3) revealed identical sequences to An. lesteri from China (= An. anthropophagus), Japan and Korea (genetic distance = 0.000), although they showed little difference from those of the Philippines (genetic distance = 0.007) (Wilkerson et al. 2003, Ma & Yang 2005, Park et al. 2008, K Sawabe et al., unpublished observations). Our results were in agreement with those previously reported by Ma and Xu (2005). Moreover, the low level of pairwise distance (0.040) detected between An. lesteri from Korea and An. paraliae from Thailand, based on ITS2 sequences, was in accordance with previous reports of different groups of Anopheles, e.g., the Anopheles gambiae complex (0.4-1.6%) (Paskewitz et al. 1993), Anophe-les dunhami and Anopheles nuneztovari (mean genetic distance = 0.025) (Ruiz et al. 2010), Anopheles fluviatilis S and An. minimus C (pairwise distance = 0.036) (Singh et al. 2006), Anopheles kunmingensis and Anopheles liangshanensis (pairwise distance = 0.0381) and An. pullus (= An. yatsushiroensis) and Anopheles junlianensis (pairwise distance = 0.03081) (Hwang 2007). Currently, Calado et al. (2008) showed that An. nuneztovari A is not conspecific with An. nuneztovari B/C based on COI sequences (genetic distance = 0.00818-0.02071) and An. dunhami has been reported as new record in the Brazilian Amazon by comparing sequences with those of An. nuneztovari A (genetic distance = 0.01436-0.03343). Similarly, comparative sequences for COI and COII between An. lesteri and An. paraliae revealed low average genetic distance between them (0.008-0.011). Despite such low genetic distances, phylogenetic trees seem to indicate that An. lesteri and An. paraliae were well separated from each other with NJ and Bayesian analyses for three regions, except for the Bayesian tree of COI. Although these two species were distinguished apparently by DNA sequence analysis, they obviously showed genetic compatibility by crossing experiments. Controversy over taxonomic problems with respect to full-fledged species, sibling species and subspecies within a taxon of Anopheles has occurred when only data of comparative DNA sequence analyses of certain specific genomic regions were used as first hand criteria for separating them. For example, An. fluviatilis S was considered a synonym of An. minimus C based on comparison of the D3 domains of 28S (28S-D3) (Harbach 2004, Garros et al. 2005, Chen et al. 2006). However, Singh et al. (2006) carried out molecular analysis on ITS2 and D2-D3 domains of 28S rDNA regions of An. fluviatilis S and An. minimus C. The authors suggested that these Anopheles species did not deserve synonymous status. Hence, crossing experiments between An. fluviatilis S and An. minimus C using iso-female lines are essential prior to a definite conclusion as to their conspecificity. Our studies using crossing experiments between An. lesteri from Korea and An. paraliae from Thailand together with data on species distributions, morphological variants, cytology and comparative DNA sequence analyses have clearly indicated that they are conspecific within the taxon An. lesteri. Additionally, the population genetic structure will be studied further in order to evaluate the gene flow among An. lesteri and An. paraliae populations before definitely concluding that An. lesteri is a synonym of An. paraliae.

ACKNOWLEDGEMENTS

To Dr Niwes Nantachit, Dean of the Faculty of Medicine, Chiang Mai University, for his interest in this research.

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Publication Dates

  • Publication in this collection
    May 2013

History

  • Received
    24 Aug 2012
  • Accepted
    26 Nov 2012
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