Evidence to Support Karyotypic Variation of the Mosquito, Anopheles peditaeniatus in Thailand

Eight isoline colonies of Anopheles peditaeniatus Leicester (Diptera: Culicidae) were established from wild-caught females collected from buffalo-baited traps at 8 localities in Thailand. They showed 2 types of X (X2, X3) and 4 types of Y (Y2, Y3, Y4, Y5) chromosomes based on the number and amount of major block(s) of heterochromatin present in the heterochromatic arm, and were tentatively designated as Forms B (X2, X3, Y2), C (X3, Y3), D (X3, Y4) and E (X2, X3, Y5). Form B was found in Nan, Ratchaburi, and Chumphon provinces; Form C was obtained in Chon Buri province; Form D was recovered in Kamphaeng Phet province; and Form E was acquired in Chiang Mai, Udon Thani, and Ubon Ratchathani provinces. Crossing studies among the 8 isoline colonies, which were representative of 4 karyotypic forms of An. peditaeniatus, revealed genetic compatibility in providing viable progenies and synaptic salivary gland polytene chromosomes through F2-generations, thus suggesting the conspecific nature of these karyotypic forms. These results were supported by the very low intraspecific sequence variations (0.0 – 1.1%) of the nucleotide sequences in ribosomal DNA (ITS2) and mitochondrial DNA (COI and COII) of the 4 forms.


Introduction
The Hyrcanus group of the Myzorhynchus series of the subgenus Anopheles (Diptera: Culicidae) comprises a large number of species that occur widely in Asia. At least 8 species of this group, i.e. Anopheles argyropus Swellengrebel, An. crawfordi Reid, An. nigerrimus Gilles, An. nitidus Harrison, Scanlon and Reid, An. paraliae Sandosham, An. peditaeniatus Leicester, An. pursati Laveran, and An. sinensis Wiedemann are recorded in Thailand (Harrison and Scanlon 1975;Rattanarithikul et al. 2006). Among these, An. nigerrimus, An. peditaeniatus, and An. sinensis are suspected as vectors of Plasmodium vivax Grassi and Feletti in Thailand (Harrison and Scanlon 1975;Rattanarithikul et al. 1996), while An. sinensis has been incriminated as a natural vector of P. vivax in Korea (Chai 1999;Ree et al. 2001) and An. peditaeniatus as a secondary vector of Japanese encephalitis virus in China and India (Mourya et al. 1989;Zhang 1990; Kanojia et al. 2003). Although An. peditaeniatus has been found abundantly and widely distributed throughout Thailand, its status as a vector of the Japanese encephalitis virus remains a crucial question that needs to be clarified more thoroughly. Additionally, this species was also considered an economic pest of cattle because of its vicious biting-behavior and ability to transmit cervid filariae of the genus Setaria (Reid 1968;Harrison and Scanlon 1975).
Regarding the cytogenetic investigations of An. peditaeniatus, the results indicated that at least 3 types of X (X 1 , X 2 , X 3 ) and 5 types of Y (Y 1 , Y 2 , Y 3 , Y 4 , Y 5 ) chromosomes were found in both sympatric and/or allopatric populations in Chanthaburi, Chiang Mai and Phrae provinces (Baimai et al. 1993).
Chromosomes X 1 , X 2 and X 3 differ from each other in the number and amount of major block(s) of heterochromatin present in the heterochromatic arm, making them appear as metacentric X 1 , small submetacentric X 2 , and large submetacentric X 3 chromosomes. Likewise, the evolution of Y chromosome types, i.e. very small telocentric Y 1 , medium telocentric Y 2 , large telocentric Y 3 , very large telocentric Y 4 , and submetacentric Y 5 could have arisen via the process of gain, rather than loss, of major block(s) of heterochromatin (Baimai et al. 1993;Baimai 1998). Although marked genetic variation at the chromosomal level of An. peditaeniatus has obviously been illustrated, little is known about its genetic proximities. Accordingly, the chromosomal variant and/or distinction might be manifested as an important role in generating post-mating barrier and DNA sequence variation of some specific genomic regions. Thus, this paper presents the results of crossing experiments and comparative DNA sequencing of the ribosomal DNA (ITS2) and mitochondrial DNA (COI and COII) regions of 4 karyotypic forms of An. peditaeniatus strains from 8 localities in Thailand.

Field collections and the establishment of isoline colonies
Wild, fully engorged female An. peditaeniatus were collected from buffalo-baited traps from November 2007 to September 2008 at 8 localities in Thailand ( Figure 1; Table 1). Eight isoline colonies were successfully established and maintained in an insectary using the techniques described by Kim et al. (2003). These isoline colonies were used for studies on metaphase karyotypes, crossing experiments, and molecular analyses.

Metaphase chromosome preparation
Metaphase chromosome preparation was the technique used for chromosome preparation in adult mosquitoes, as described by Choochote et al. (2001). Briefly, newly emerged adult males of laboratory-raised An. peditaeniatus (aged about 6-12 hr) were intra-thoracically inoculated with 0.30 μl of 1% ethanol-extracted Gloriosa superba L. (Liliales: Colchicaceae) solution and held in an insectary at 27 ± 2 o C, with 70-80% relative humidity for 3 hr. The excised testes were incubated in 1% hypotonic sodium citrate solution, fixed in Carnoy's fixative, stained with 10% Giemsa in phosphate buffer pH 7.2, mounted in Permount (Fisher, www.fishersci.com), and examined under a compound microscope. Identification of types of sex chromosomes followed the cytotaxonomic key of Baimai et al. (1993).

DNA extraction, amplification, sequencing and analysis
One individual F 1 -progeny adult female from each isoline colony of An. peditaeniatus forms was used for DNA extraction and amplification. Genomic DNA was extracted from individual adult mosquitoes using a RED Extract-N-Amp TM Tissue PCR Kit (Sigma-Aldrich). The ribosomal DNA (rDNA) internal transcribed spacer 2 (ITS2), and mitochondrial cytochrome c oxidase subunit I (COI) and subunit II (COII) were amplified using the primers described by Park et al. (2003), with minor modifications: 5.8S + 35 (5'-ACG CAT ATT GCA CGT CGT GG-3') and 28S -20 (5 -GGG TTG TCA CAC ATA ACT TGA GGC-3 ) for ITS2; LCO1490 (5 -GGT CAA CAA ATC ATA AAG ATA TTG G-3 ) and HCO2198 (5 -TAA ACT TCA GGG TGA CCA AAA AAT CA-3 ) for COI; AnoCO2+1 (5 -GAT TAG TGC AAT GAA TTT AAG C-3 ) and AnoCO2END (5 -GAG ATC ATT ACT TGC TTT CAG TC-3 ) for COII. The PCR condition, cloning, and sequencing followed the techniques previously reported by Park et al. (2008). The PCR products were purified using the QIAquick ® Gel Extraction Kit (Qiagen, www.qiagen.com), and directly sequenced with an ABI PRISMH ® 3700 DNA Analyzer (Applied Biosystems, www.appliedbiosystems.com) using a Dye Terminator Cycle Sequencing Ready Reaction Kit (Applied Biosystems). Both strands were sequenced and aligned using the ClustalX multiple alignment programs (Thompson et al. 1997). The nucleotide sequence data reported are in the DDBJ/EMBL/GenBank nucleotide sequence databases with the accession numbers AB539056-AB539079. Geographical type of specimens and their sequence accession numbers within GenBank are denoted in Table 1.
For crossing experiments, details of hatchability, pupation, emergence, and adult sex-ratio of parental, reciprocal, and F 1 -hybrid crosses among the 8 isoline colonies of An. peditaeniatus Forms B (X 2 , X 3 , Y 2 ), C (X 3 , Y 3 ), D (X 3 , Y 4 ), and E (X 2 , X 3 , Y 5 ) are shown in Table 2. All crosses yielded viable progenies through F 2 -generations. No evidence of genetic incompatibility and/or post-mating reproductive isolation was observed among these crosses. The salivary gland polytene chromosomes of the 4 th stage larvae from all crosses showed complete synapsis along the whole length of all autosomes and the X chromosome (Figure 3).
In the DNA sequence analysis, DNA sequences were determined and analyzed for the ITS2, COI, and COII regions from 8 isoline colonies representative of 4 karyotypic forms of An. Peditaeniatus. In these, all sequences of the ITS2 region were found to be completely identical with a length of 463 bp, but in comparison with An. lesteri they had a very high interspecific sequence variation of 35.4%. The results of comparative sequences of COI and COII regions revealed 548 bp for COI with 0.0 -1.1% intraspecific sequence variations, and 672 bp for COII with 0.0 -0.8% intraspecific sequence variations, and seven variable sites were observed from both ( Figure 4). Interspecific sequence variations between An. peditaeniatus and An. lesteri in COI and COII were 3.6 -4.0% and 3.1 -3.5%, respectively.

Discussion
Karyotypic variation, due to the addition of an extra block of herterochromatin on sex chromosome (X, Y), is an important mechanism in the speciation process of anopheline mosquitoes and/or other dipteran insects. It could be used as a primary marker for further investigations of sibling species or subspecies status in natural populations of mosquitoes, particularly in those that have heteromorphic sex chromosomes as anophelines (Baimai 1998;Subbarao 1998). Nonetheless, limitation in use should be kept in mind since either markedly different or identical metaphase karyotypes could be cytological characteristics of sibling species or subspecies (cytological races). For example, An. minimus Theobald (minimus species A) has uniquely submetacentric X 1 , medium submetacentric X 2 , and submetacentric Y 1 chromosomes. Anopheles harrisoni Harbach and Manguin (minimus species C) has unique large submetacentric X 3 and large submetacentric Y 2 chromosomes (Baimai et al. 1996). Anopheles barbirostris Van der Wulp species A1, A2, A3, and A4 share common characteristics of medium submetacentric X 2 and subtelocentric Y 1 chromosomes, whereas submetacentric X 1 , large submetacentric X 3 , submetacentric Y 2 , and large submetacentric Y 3 chromosomes were common phenomena of the karyotypic variation of An. barbirostris species A1 ).
Journal of Insect Science | www.insectscience.org 7 Crossing experiments for determining hybrid non-viability, sterility, or breakdown are still a useful tool used in the recognition of anopheline species complexes. Detailed genetic incompatibility, including lack of insemination, embryonation, hatchability, larval survival, pupation, emergence, adult sex distortion, abnormal morphology, and reproductive system are useful criteria for elucidating sibling species or subspecies status (Baimai et al. 1987(Baimai et al. , 1988Sawadipanich et al. 1990;Subbarao 1998). However, a point worth noting is that an isoline colony established from the combinative characters of morphological and/or cytological markers has to be seriously considered. A laboratoryraised colony established from a naturally mixed population should be omitted, since it may be a mixture of cryptic species or sibling species. Several intra-taxa of the anopheline species that were primarily detected with cytological differences and/or variations that  (Baimai et al. 1987(Baimai et al. , 1988Sawadipanich et al.1990), An. maculatus Theobald complex (Chabpunnarat 1988, Thongwat et al. 2008, and An. minimus complex (Choochote et al. 2002b;Somboon et al. 2005); and subspecies (cytological races), e.g. An. sinensis Wiedemann Forms A and B (Park et al. 2008), An. vagus Doenitz Forms A and B (Choochote et al. 2002a), An. pullus Yamada Forms A and B ), An. aconitus Doenitz Forms B and C (Junkum et al. 2005), and An. campestris-like Form B, E, and F (Thongsahuan et al. 2009).
In this study, 4 tentative karyotypic forms of An. peditaeniatus, i.e. Form B (X 2 , X 3 , Y 2 ), C (X 3 , Y 3 ), D (X 3 , Y 4 ), and E (X 2 , X 3 , Y 5 ) were obtained from natural populations in Thailand. It is interesting to note that the ancestral Form A (X 2 , Y 1 ), reported by Baimai et al. (1993), was not detected in any isoline colonies, as only a few samples appeared to be used in the current study. Even though Form A (X 2 , Y 1 ) was not detected in the present investigation markedly distinct characteristics, particularly the Y chromosomes among the 4 karyotypic forms, were enough to perform their genetic proximity thoroughly. Accordingly, the crossing experiments were carried out among the 4 karyotypic forms in order to determine the degree of genetic proximity. In addition, their comparative DNA sequences of ITS2, COI, and COII were included in this study. The results of no post-mating reproductive isolation among the 4 karyotypic forms, by yielding viable progenies and synaptic salivary gland polytene chromosomes through F 2 -generations, suggested their conspecific nature. The very low intraspecific sequence variations (0.0 -1.1%) of the nucleotide sequences of ribosomal DNA (ITS2) and mitochondrial DNA (COI and COII) of the 4 karyotypic forms were strong supportive evidence. Additionally, the length (463 bp) and sequences of ITS2 regions of An. peditaeniatus forms obtained in this study were identical to that of a previous report (Ma and Xu 2005