Sequence polymorphism in spacer ITS2 of Anopheles (Cellia ... · ITS2 SEQUENCE POLYMORPHISM 125 IN...

Sequence polymorphism in spacer ITS2 of Anopheles (Cellia) sub-pictus Grassi (Diptera: Culicidae)

Taruna Kaura, Monika Sharma, Sudarshan Chaudhry and Asha Chaudhry*

Mosquito Cytogenetics Unit, Department of Zoology, Panjab University, Chandigarh-160014, India.

Abstract — Mosquito cytogenetic studies have revealed that the majority of disease vector species exist as cryptic species complexes. In relevance to this, the present results include the sequence analysis of r-DNA in-ternal transcribed spacer 2 (ITS2) of two populations of Anopheles subpictus Grassi collected from Chandigarh (Pop.A) and Hoshiarpur, Punjab, India (Pop.B). These sequences were compared with fi ve other populations C, D, E, F and G worked out earlier. ITS2 sequence in Pop. A was 681 bp compared to 491 bp in Pop. B. All the sequences were G:C rich. In populations A-E, which form the Indian component of An. subpictus, the ts/tv frequencies ranged from 0.25 to 0.77. Populations F and G, which represent the Srilankan fauna, were much higher at 0.83 to 0.90 in inland and coastal populations respectively. The SRF revealed various common interspersed repeats in the form of dimers, trimers, tetramers, pentamers and polymers. The TG repeat motif, which was repeated 50 to 60 times, was common in Indian and Srilankan populations of An. subpictus. Further, there were as many as seven different types of polymers/repeats in populations A-E of Indian region but none was shared by F and G of Srilanka. Phylogeny analysis of ITS2 sequence had well supported India and Srilanka clades in accordance with their allopatric status.

Key words: Anopheles subpictus populations, ITS2, sequence analysis.

*Corresponding author: phone: 0172-2534205 (O), 0172-2541942 (PP), 0172-4416614, mo: 9876581480; e-mail: [email protected]

CARYOLOGIA Vol. 63, no. 2: 124-133, 2010

INTRODUCTION

During the last four decades, the work on mosquito cytogenetics has grown to such an ex-tent that it has helped to illuminate their basic biology, evolution, speciation and genetics of vectorial capacity and insecticide resistance. It has also been realized that majority of the epi-demiologically important species of mosquitoes exist in the form of complexes of two or more cryptic species with similar, overlapping or con-fusing morphotaxonomic characters. This makes their identifi cation diffi cult (SUBBARAO 1996; SUBBARAO and SHARMA 1997; CHAUDHRY 1999). Subspecifi c variants were detected by using a multidisciplinary approach which included the

analysis of polytene chromosomes, amino acid and enzyme profi les (DUFFY 1964; ADAK et al. 1988). This approach has resulted in a variety of molecular markers for establishing the exact tax-onomic and phylogenetic status of species and their populations (NARANG et al. 1993a; b; MUN-STERMANN 1995; REINERT et al. 1997; SEVERSON et al. 2001; WHO 2003; CHAUDHRY et al. 2006). In addition to the use of nuclear and mitochondrial genes, the internal transcribed spacers 1 and 2 (ITS1, 2) with hypervariable sequence composi-tion have also been found to be ideal molecular markers (FRITZ et al. 1994; MILLER et al. 1996; CORNEL et al. 1996; MARRELLI et al. 2005; BANER-JEE et al. 2007).

Ribosomal DNA has been used extensively and very successfully for phylogenetic analy-sis of both closely and distantly related organ-isms. The internal transcribed spacer 2 (ITS2) sequence, which like the internal transcribed spacer 1 (ITS1), evolves faster than coding se-quence, has been used for discrimination of closely related anopheline mosquitoes in the An.

ITS2 SEQUENCE POLYMORPHISM IN ANOPHELES SUBPICTUS 125

gambiae Giles complex (PASKEWITZ and COLLINS 1990), An. funestus Giles complex (HACKETT et al. 2000) and An. dirus complex (XU et al. 1998). These sequences, therefore, are good candidates for the analysis of phylogenetic relationships be-tween members of a species complex.

The identifi cation of repetitive DNA se-quences and variations in the spacer length have practical utility in the application of RFLP-PCR based genomic studies (MATHIOPOULOS et al. 1995; KOEKEMOER et al. 1998). Repetitive sequences are presumed to be important in a number of regulatory functions and are princi-ple causes of genomic instability (ZHANG 1998; ZHANG and HEWITT 2003). When repeat se-quences get mutated excessively both tandem and dispersed repeats become diffi cult to locate and one of the major challenges in repeat detec-tion is the location of such hidden or latent re-peats. The repetitive sequences having high level of polymorphism can also infl uence the role of functional DNA (TAUTZ et al. 1986; KASHI et al. 1997). In this, the technique of polymerase chain reaction (PCR) has actually proved to be an as-set in the genomic diagnostics of cryptic species and their populations, as it allows the selective amplifi cation of DNA fragments upto 10 kbs in size from a complex genome of an organism.

The present investigations were aimed at the comparative study of sequence variations in ITS2 of seven allopatric populations (5- Indian, 2- Srilankan) of Anopheles subpictus. This spe-cies is considered an emerging vector of malaria in some parts of the Indian subcontinent (PAN-ICKER et al. 1981; AMERSINGHE et al. 1992). The sequences obtained from these two populations were compared with those of fi ve other popula-tions worked out earlier from India and Srilanka so as to generate data of signifi cance variations in this taxon of epidemiological signifi cance (AB-HYAWARDANA et al. 1996a; b; CHHILAR 2008).

MATERIALS AND METHODS

For the present research work, the blood fed female specimens of Anopheles subpictus Grassi were collected from the places near Panjab Uni-versity campus, Chandigarh (Pop.A) and from a village Khani near Hoshiarpur, Punjab (Pop.B). It is a widely distributed species in the entire In-dian regions where its breeding is associated with clear rain water pools (RAO 1984). The identifi -cation of the species from its morphotaxonomic characters was also confi rmed from a unique

polytene chromosome marker in the form of species-specifi c X-chromosome banding pattern and a large nucleolar heterochromatin mass as-sociated with its centomeric end (WATTAL and KALRA 1967; RAO 1984; CHAUDHRY 1986; CHAUD-HRY et al. 2005). The blood fed gravid females were collected from their resting sites in the early morning hours from the cattle sheds and human dwellings, after which they were allowed to lay eggs in distilled water fi lled petridishes placed in the breeding cages. Alternatively, two to fi ve specimens were held in test tubes where the eggs were received on a strip of damp fi lter paper. A larval colony was raised from these eggs in a BOD incubator by feeding the stocks with a diet consisting of a mixture of fi nely powdered dog biscuits and yeast tablets (SINGH et al. 1975; CLEMENTS 1992). Freshly hatched adults were stored in separate eppendorf tubes at –20°C for DNA extraction.

Extraction and PCR amplifi cation of DNA - The DNA was extracted by following the proto-col of AUSUBEL et al. (1999) and the one stand-ardized in this laboratory (NEETU and CHAUDHRY 2005; CHAUDHRY and SHARMA 2006) according to which 10 specimens (one at a time) of freshly hatched unfed females, each of the two popula-tions, were homogenized and a PCR master mix was prepared by mixing 10X PCR buffer, dNTP mix (100mM each), MgCl2, Taq polymerase (3 units/μl), double distilled water and template DNA. The specifi c forward and reverse prim-ers (FP, RP) used in the process consisted of 19 base oligomers having a sequence of 5’-TGT-GAACTGCAGGACACACAT-3’ (Code 46JB) and 5’-TGTGCTTAAATTCAGGGGGT-3’ (Code 47JB) (SHARPE et al. 2000; SHOUCHE and PATOLE 2000). The amplifi cation reactions were performed as per the scheme of WILLIAMS et al. (1990) and CHAUDHRY and KOHLI (2007) ac-cording to which, each of the 25 µl of reaction mixture was loaded in a thermocycler which was programmed for the initial one cycle for dena-turation of DNA at 94°C for 10 mins. This was followed by 35 cycles each of denaturation, an-nealing of primer and extension of DNA at 94°C for 1min, 56°C for 1 min and 72°C for 1 min re-spectively. This was followed by fi nal extension of 5 mins. In all such amplifi cations, a negative control consisting of all the components of reac-tion mixture except the DNA, was also carried out so as to rule out the experimental errors. The PCR products and standard DNA ladder were electrophorased in 2% agarose gel. Fi-nally the sequences were also submitted to Gen-

KAURA, SHARMA, CHAUDHRY and CHAUDHRY126

Bank under accession numbers EU847232 and EF192277 for populations A and B respectively while the sequences taken for comparisons were as follows (Table 1).

RESULTS AND DISCUSSION

In fi gures 1 and 2, lane M carries the bands of standard DNA ladder while lanes A and B have the amplifi ed products from ITS2 of populations A and B of Anopheles subpictus with 491 and 657bp respectively. The DNA of these bands were sequenced and aligned with Clustal W multiple sequence alignment algorithm (www.ebi.ac.uk/clustalw/). These sequences from all the populations were found to be G:C rich.

In fi gure 3 the loci marked with asterisks (*) indicate those regions where nucleotide se-quences are identical in both populations A and B. Dashes (-) were used to indicate the insertions and/ or deletions (indels). In addition to the places marked as * and -, some regions marked with arrows provide examples of the differences in the complementary bases in the sequences arising due to substitutions in the form of tran-sitions and transversions (ts/tv). The transition/transversion (ts/tv) ratios of all the seven popu-lations (A- G) under consideration were 0.315, 0.776, 0.545, 0.25, 0.40, 0.90 and 0.833 respec-tively. In populations A- E, the ts/tv frequencies ranged from a minimum of 0.25 to maximum of 0.77 while in populations F and G the ts/tv fre-quencies were 0.83 and 0.90 in inland and coast-

TABLE 1 — Details of the populations covered under ITS2 sequence analysis

S.No. Population Locality Country Accession number Reference 1 A Chandigarh India EF192277 Present 2 B Hoshiapur “ EU847232 “ 3 C Sangrur “ EF601870 Chhilar, 2008 4 D Bhiwani “ EF601869 “ 5 E Sonipat “ EF601868 “ 6 F Inland Srilanka AF406616 Abhayawardana et al. 7 G Coastal “ AY049004 1996a, b

Fig. 1 — PCR amplifi ed ITS2 sequence of population A. Lane N- negative control, lane M- DNA ladder, lane A- DNA band from pop A.

Fig. 2 — PCR amplifi ed ITS2 sequence of population B. Lane N- negative control, lane M- DNA ladder, lane B- DNA band from pop B.


Fig. 3 — ITS2 sequence multiple sequence alignment of populations A,B, C, D, E F and G of Anopheles subpictus.


Fig. 3 — Contd.


al populations respectively (ABHYAWARDANA et al. 1996a; b). This indicates that the base replace-ment process is essential for rapid and adaptive changes for two different types of breeding habi-tats comprising fresh and brackish water. These differences are also in agreement with the ob-servations of COLUZZI and SABATINI (1967) and CHAUDHRY and SONI (2000) in which the salivary polytene X- chromosome of fresh and salt water populations of An. gambiae and An. subpictus respectively had distinct banding pattern differ-ences leading to two types of genotypes desig-nated as cryptic species A and B. The present

sequence was also subjected to Spectral Repeat Finder (SRF) and Tandem Repeat Occurrence Locator (TROLL) programmes (SHARMA et al. 2004; BENSON 1999) for identifying the presence of interspersed and tandem repeats respectively. The SRF revealed various common interspersed repeats which were further categorized as dim-ers, trimers, tetramers, pentamers and polymers. In the present sequence of An. subpictus dimer TG repeat had the highest copy number as it was 50 to 60 times more common in Indian and Srilankan populations (Table 2, Fig. 4). Tetramer GTGC and pentamer GGTGC were also found

Fig. 3 — Contd.

Fig. 4 — Frequency of dimer TG in the ITS2 sequence of populations C, D, E and G.

Fig. 5 — Frequency of tetramer GTGC in the ITS2 se-quence of populations C and E.


to be common among one of the Srilankan pop-ulations (G) and two populations (C and E) of the Indian region (Figs. 5,6). There were seven different types of polymers in populations A-E of Indian region none of which were shared by Srilankan F and G (Fig. 7). It was also noticed that in the spacers of similar length, the number of insertion/deletions (indel bias) was higher in those spacers which had the lower number of re-peats, a condition which was also encountered in human genome (VINOGRADOV 2002).

ITS2 sequence analysis revealed that the inte-gration of repeats did not show positive correla-tion with spacer length but the number of in-dels were consistent with its length. The present

authors applied TROLL (BENSON 1999) to the ITS2 sequences of Anopheles subpictus, Anophe-les stephensi, Culex quinquefasciatus and Armig-eres subalbatus, in which, unlike the other three species, An. subpictus did not have tandem re-peats. A phylogram was generated with the help of Gene Bee Service programme (YUSHMANOV and CHUMAKOV 1988) (Fig. 8). Populations C and E from Sangrur and Sonipat and F and G from Srilanka 1 and Srilanka 2 formed the fi rst and second clades in accordance with their al-lopatric status. The polytene chromosome data was supplemented with the RAPD-PCR based genomic data from two populations of this spe-cies. SHARMA (2003) isolated several DNA bands

Fig. 6 — Frequency of pentamer GGTGC and CGGCG in the ITS2 sequence of populations C, D, E and G.

Fig. 7 — Frequency of polymers CTCGGCGTG, GACGTG and TGTGCG in the ITS2 sequence of pop-ulations B, D,E, F and G.

TABLE 2 — Spectral repeat fi nder (SRF) based ITS2 sequence characteristics of An.subpictus populations (● common repeats).

Population Base Dimer Trimer Tetramer Pentamer Polymer Pair Sequence Copy Sequence Copy Sequence Copy Sequence Copy Sequence Copy Length No. No. No. No. No. A 491 AC 36 – – CCTA 15 GAGTG 7 ACCTTGAG 5 GCAT 17B 681 – – GTG 21 CGTG 44 – – CTCGGCGTG● 6C 687 TG● 45 – – GTGC● 42 GGTGC● 26 CCGGCGT 5 CGGCG● 11 GTGCCGGCG 7D 687 TG● 64 – – – – GGTG 26 CTCGGCGTG● 6 CGGCG● 10E 670 TG● 46 – – GTGC● 45 GGTGC● 28 CGGCGC 11 CTCGGCGTG● 6F 668 CG 54 – – TGCA 47 TGGAC 17 GACGTG● 11 TGTGCG● 11 GGACGC● 11G 618 TG● 50 – – GTGC 43 GTGCA 15 GACGTG● 11 GCGT 43 GGTGC● 15 TGTGCG● 11


of variable size in which intraspecifi c differences were detected in the form of presence-absence, population specifi c and species specifi c bands. CHHILAR (2008) analysed morphotaxonomic characters and sequence variations in spacer ITS2 and CO II gene of three different popu-lations of this species. He was able to establish the exact status of cryptic species in this taxon. CHHILAR’S evidence suggested the presence of only two cryptic species, A and B (inland and coastal) instead of A, B, C and D (unpublished). Sequence repeats are well known in the genome of different animal groups where they are more common at centromeric and telomeric regions of the chromosomes. Thus the sequence repeats observed here should be a valuable addition to the knowledge on sequence characteristics of mosquitoes of public health importance espe-cially for RFLP-PCR based studies (NARANG et al. 1993 a). For example, the tetramer TGCA has a restriction site for a restriction enzyme Hpy-CH4IV (FANELLO et al. 2002; JUAN et al. 2002). The present studies suggest that the distribution of sequence polymorphism throughout the pop-ulations of a species is a type of intragenomic mechanism which promotes genotypic varia-tions due to constantly changing sequences.

Acknowledgements — The authors are thankful to Chairperson, Department of Zoology, Panjab Uni-versity, Chandigarh for providing the necessary fa-

cilities under Centre of Advance Studies Programme of U.G.C, New Delhi India to carry out the present research work.

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Received May 15th 2009; accepted November 6th 2009

Sequence polymorphism in spacer ITS2 of Anopheles (Cellia ... · ITS2 SEQUENCE POLYMORPHISM 125 IN...

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