Molecular Variants in the Anopheles Minimus

Anopheles minimus (Theobald) is one of the most important vectors of human malaria in Southeast Asia. Morphological studies now have revealed five sibling species as its complex, designated as species A to E. The present study investigated the genetic divergence among An. minimus populations from four countries (Japan, China, Thailand and Indonesia), based on the DNA sequences data of the D3 (the third domain of the 28S ribosomal gene) and ITS2 (the second internal transcribed spacer of the ribosomal gene) is reported. The D3 and ITS2 phylogenetic trees, and the electrophoretic profile of ITS1 (the first internal transcribed spacer of the ribosomal gene) indicated that our An. minimus populations are comprised of three groups: the Japanese population as group I, the population from Guangxi Province of China (GX population) as group II, and others, as group III. The results showed the morphological similarity of group III and GX with the species complex A and B, respectively. It is interesting that both two species A (YN population) and species B (GX) occur in China, and that both species, An. minimus species A (LB-95 population) and the closer population An. flavirostris (Ludlow) (LB-00 population) appeared to be present on the Lombok Island of Indonesia, although in far separated localities. Moreover, this molecular evidence confirms the previous suggestion that the population from the Ishigaki Island of Japan should be classified as a new genetic status species E. C from China (Subbarao, 1998) until now. It has been suggested that the prevalence of malaria infection could be associated with the morphological variations of the mosquitos inhabiting the infected areas (Yu and Li, 1984). Based on morphology of An. minimus, members of the complex are difficult to distinguish from closely related species, because they have overlapping morphological characters (Harrison, 1980), and An. minimus is a complex of at least two species, which are isomorphic and can only be distinguished using molecular markers. Variations in protein polymorphism were helpful for establishing a genetic identity between the morphological species A and C identified in Thailand (Sucharit et al, 1988; Green et al, 1990), Vietnam (Van Bortal et al, 1999), between species A and form B in China (Sawabe et al, 1996) and for species E in the Yaeyama Islands (Sawabe, unpublished). Green et al (1990) indicating that SOUTHEAST ASIAN J TROP MED PUBLIC HEALTH 772 Vol 34 No. 4 December 2003 the electrophoretic band patterns of the allele of Octanol dehydrogenase (Odh) can be used to distinguish between species A and C. In molecular phylogenetic studies using DNA sequences, Sharpe et al, (1999) developed two polymerase chain reaction (PCR)-based methods for identifying species A and C, and other closely related species at the 3 domain (D3) of the 28S gene of ribosomal DNA (rDNA) using PCR. Van Bortel et al (2000) reported distinguishing species at the second internal transcribed spacer (ITS2) of the ribosomal gene. In the previous study (Sawabe et al, 1996), the genetic differentiation between the two An. minimus complex species inhabiting the Yunnan and Guangxi Provinces of China corresponds to the species A and form B, respectively. We thus report here the molecular variations and confirm genetically the status between them, and also for populations from three other Southeast Asian countries based on D3 and ITS2 sequences, and ITS1 electrophoretic profiles. Although Somboon et al (2001) reported that the population from the Ishigaki Island of Japan should be classified as a new species E using the ISG population collected in 1999, we attempt to clarify their genetic background and also discuss the genetic identification of other ISG populations including the specimens used by Somboon et al (2001). MATERIALS AND METHODS Mosquitos used The details of all mosquitos used are given in Table 1. Ten An. minimus populations and one An. flavirostris were collected from four Southeast Asian countries. Morphological identification was performed on all individuals in the field for larvae and/or adults. Some populations were colonized and maintained at the laboratory after collection, and larvae at the end of the 4th-instar of the following generation were used for analysis. Based on the morphological characteristics and/or enzyme electrophoretic analysis, GX was confirmed as species B, ISG-93 and RKU as different from species A to D, and ISG-99 (namely ISG by Somboon et al, 2001) as species E. All other populations were confirmed as species A. Details of the ISG-99 and the CMU were also described in Somboon et al (2001). All specimens were stored at -80oC until used. For the DNA sequencing, male (CMU, LB00, LMP, ISG-99), female adults (fraviro-00) and larvae (GX, ISG-93, LB-95, PK, RKU and YN) were used. The sequences of species A (minimus A, AF114019 and AF230461 for the D3 and the ITS2, respectively), species C (minimus C, AF114017 and AF230462 for the D3 and the ITS2, respectively) and two Myzomyia Series, An. flavirostris (flavirostris, AF194483 for the D3) and An. aconitus (Dönitz) (aconitus, AF114015 and AF194491 for the D3 and the ITS2, respectively), were obtained from GenBank database as references. DNA extraction, amplification and sequencing Genomic DNA was extracted from individual mosquitos using an IsoQuick DNA extraction Kit (Tanehashi, Tokyo, Japan). The regions of the D3, ITS1 and ITS2 were amplified in a final volume of 25 μl containing RTG PCR Beads (Amersham Pharmacia Biotech, Uppsala, Sweden) and primers using PCR (Model 9600, PE Applied Biosystems, USA). The PCR cycling conditions were as follows: 30 seconds 95oC, 30 seconds 55oC and 1 minute 72oC for 35 cycles followed by a 4 minutes final extension at 72oC (for D3 primers); initial denaturation at 95oC for 5 minutes followed by 1 minute 95oC, 1 minute 39oC and 2 minutes 72oC for 45 cycles (for ITS1 and ITS2 primers). Primers used were 5′-GAC CCG TCT TGA AAC ACG GA-3′ and 5′-TCG GAA GGA ACC AGC TAC TA-3′ (Litvaitis et al, 1994) for the D3 region, 5′CCT TTG TAC ACA CGC CCC GT-3′ and 5′GTT CAT GTG TCC TGC AGT TCA C-3′ (Sharpe et al, 2000) for the ITS1 region, and 5′TGT GAA CTG CAG GAC ACA T-3′ and 5′TAT GCT TAA ATT CAG GGG GT-3′ (Beebe and Saul, 1995) for the ITS2 region, as a forward and reverse primer, respectively. A five-μl portion of the PCR product was electrophoresed on a 2% agarose gel (NU Sieve 3:1, FMS Bio Product, USA), and the remainder was concentrated using ethanol precipitation methods. An excising individual band was cut away from a 2% LMP agarose (SeaPlaque GTG MOLECULAR VARIANTS IN THE ANOPHELES MINIMUS Vol 34 No. 4 December 2003 773 agarose, USA) visualized by ethidium bromide, and cleaned using the methods for phenol-chloroform-ethanol purification. Each purified double-stranded PCR product was directly cyclesequenced from both ends using Dye Terminator Cycle Sequencing FS Ready Reaction Kit (PE Applied Biosystems) and the same primers used for PCR using the thermal profile 10 seconds 96oC, 5 seconds 55oC and 4 minutes 60oC for 25 cycles (Model 9600, PE Applied Biosystems), and ABI PRISM 310 Genetic Analyzer (PE Applied Biosystems). Phylogenetic analysis Alignment analyses were done using the program GENETYX-MAC ver. 10.01 (Software Development Co, Tokyo, Japan). Phylogenetic analysis was performed using distance and parsimony methods in MEGA (ver. 2.1; Kumar et al, 2001). Alignment gaps were treated as missing data. For the trees, a distance matrix was constructed using the Kimura 2-parameter model and trees were inferred using the neighbor-joining method. The parsimony trees also were constructed using the heuristic search of maximum parsimony method. Because of these two phylogenetic trees inferred by different algorithms mentioned above showed similar topology, the numbers of bootstrap replicates from parsimony trees were added but not to drown the trees in the text. As no great differences were found within a population, one or two (for LMP) typical alignments were used for the following analyses and shown in this text.