ISSN 0013-8738, Entomological Review, 2013, Vol. 93, No. 1, pp. 45–49. © Pleiades Publishing, Inc., 2013. Original Russian Text © N.V. Khrabrova, Yu.V. Andreeva, A.K. Sibataev, V.N. Stegniy, 2012, published in Parazitologiya, 2012, Vol. 46, No. 1, pp. 3–10.
Comparison of Ribosomal DNA Nucleotide Sequences of the Mosquito Genera Aedes and Ochlerotatus (Diptera: Culicidae: Aedini) N. V. Khrabrova, Yu. V. Andreeva, A. K. Sibataev, and V. N. Stegniy Research Institute of Biology and Biophysics, Tomsk State University, Tomsk, 634050 Russia e-mail:
[email protected] Received November 16, 2011
Abstract—Nucleotide rDNA sequences of 9 species of the mosquito genus Ochlerotatus and 2 species of the genus Aedes were obtained and compared. Variation both within and between these genera was characterized. The phylogenetic relationships among the studied species of Ochlerotatus generally correspond to the grouping of species based on morphological characters. DOI: 10.1134/S0013873813010077
In the Fauna of the USSR volume published in 1970, A.I. Gutsevich with co-authors classified the Palaearctic species of the subgenus Ochlerotatus into 4 groups for the sake of easier identification of adult mosquitoes; these groups were not given any taxonomic rank (Gutsevich et al., 1970). In the light of the new classification of European species recently proposed by Becker and co-authors (2010), it would be interesting to study these groups from the taxonomic viewpoint. Classification and identification of insect species provide an important basis for both fundamental and applied research. Morphological characters, especially quantitative ones, are not always convenient since they are often subject to individual, geographic, combinatorial, and modificational variation (Wilkerson et al., 1993). The complex approach using both morphological and molecular genetic data is now considered to be the most justified.
Mosquitoes of the family Culicidae are active blood-suckers and vectors of many serious diseases of human and animals. Of special interest is the complex taxonomic structure of the tribe Aedini which includes nearly half the species of blood-sucking mosquitoes in the world fauna and most of the species occurring in Russia. The genus Aedes Meigen, 1818 originally included species characterized by short maxillary palps. However, in view of the large number and variable morphology of its species, the researchers had to find additional characters, first of all those of the male genital apparatus; based on these characters, it was possible to split Aedes into smaller taxa. As a result, by the end of the XX century the genus Aedes included about 1000 species grouped into 44 subgenera (Khalin and Gornostaeva, 2008). Later, the taxonomic status of some representatives of the family was essentially reconsidered, new genera were established, some previously synonymized genera were restored, and some subgenera were promoted to genera. In particular, based on the study of many morphological characters, Reinert and co-authors (2000) elevated the subgenus Ochlerotatus Lynch Arribalzaga, 1891 to the status of a separate genus including about 500 species in 21 subgenera. As a result of the subsequent taxonomic changes proposed by Reinert and coauthors (2004, 2006), the genus Ochlerotatus presently includes 276 species grouped into the subgenera Ochlerotatus, Finlaya Theobald, 1903, and Rusticoidus Shevchenko et Prudkina, 1973 (Reinert et al., 2008).
The rDNA nucleotide sequences are used in taxonomic and phylogenetic studies. Each rDNA unit contains both conserved regions and variable ones with different rates of evolution; therefore these sequences can be used in phylogenetic analysis at different taxonomic levels (Hoy, 1994; Miller et al., 1996). The rDNA sequences include internal transcribed spacers (ITS1 and ITS2) which evolve at a high rate and therefore can reveal the changes taking place during speciation. In this communication we describe the results of sequencing and comparison of rDNA nucleotide sequences in some representatives of the genera Ochlerotatus and Aedes. 45
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Collection sites and dates and the number of mosquito larvae studied (Aedes and Ochlerotatus) Collection locality TP, Kireevsk TP, Takhtamyshevo-1 TP, petroleum storage depot TP, Takhtamyshevo-2 TP, Tegul’det
Coordinates longitude
latitude
56°24'50.63"N 56°22'29.16"N 56°26'37.43"N 56°22'38.97"N 57°19'18.68"N
84°4'38.09"E 84°51'43.33"E 84°55'35.96"E 84°51'47.86"E 88°11'46.78"E
Collection date
n
26.V.2008 20.V.2008 21.V.2008 V.VI.2009 26.VII.2009
38 93 3 121 36
Note: TP is Tomsk Province; n is the sample size.
MATERIALS AND METHODS IV instar larvae of mosquitoes of the genera Aedes and Ochlerotatus were collected in several districts of Tomsk Province. The collection localities are listed in table. In all, 291 larvae were studied. The species were identified using the published keys (Gutsevich et al., 1970; Gutsevich and Dubitskii, 1981). Identification was carried out using an MBS-10 binocular microscope. DNA was isolated from IV instar larvae with Invisorb® Spin DNA Extraction Kit (Invitek, Germany), following the manufacturer’s protocol. Amplification of rDNA was carried out using ISS2470F (5'-TTTAGAGGAAGTAAAAGT-3') and ISS228R (5'-GTTAGTTTCTTTTCCTCC-3') primers (Reno et al., 2000). The ITS2 regions were amplified using primers complementary to the conserved regions of 5.8S and 28S rDNA genes: 28S (5'-ATGCTTAAATTTAGGGGGTA-3') (Proft et al., 1999) and 5.8Sa (5'-ATCACTCGGCTCGTGGATCGAT-3') (Gordeev et al., 2004). The reaction mixture contained singlestrength PCR buffer (60 mM Tris-HCl, 25 mM KCl, 10 mM 2-mercaptoethanol, 0.1% Triton X-100), 1.5 mM MgCl2, 200 μM of each dNTP, 1 unit Taq DNA Polymerase (SibEnzyme, Novosibirsk), 5 pM of each primer, 10 ng genomic DNA, deionized water to 15 μl. DNA amplification was carried out in an MJ Mini™ Personal Thermal Cycler (Bio-Rad, US) under the following conditions: initial DNA denaturation step, 3 min at 94°C; 35 cycles with 3 stages: 30 s at 94°C, 30 s at 52°C, 1 min at 72°C; final elongation step, 10 min at 72°C. Amplification products were separated in 1.5% agarose gel with ×1 TAE buffer (0.04 M Tris-acetate, 0.002 M EDTA) at 100 V, stained with ethidium bromide (1 μg/ml), visualized in
UV light, and photographed. The DNA fragment size was estimated relative to a 100 bp + 1.5 kb DNA marker (SibEnzyme, Novosibirsk). The photographs were processed using Adobe® Photoshop® CS2 software. Sequencing was carried out in a 3130 Genetic Analyzer using a BigDye® Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems, US). The nucleotide sequences were edited in Sequencing Analysis 5.2 software (Applied Biosystems, US). Analysis, sequences alignment, and phylogenetic tree construction were performed using SeqMan™ II and MegAlign™ software (DNASTAR Inc.). RESULTS AND DISCUSSION Based on morphological characters, we identified 9 species of the genus Ochlerotatus and 2 species of the genus Aedes. Four larvae of the genus Ochlerotatus were tentatively referred to as Ochlerotatus species. These specimens were very similar in their morphology and very strongly resembled Och. caspius but differed from it in having longer gills. Although the gill length is known to be a highly variable character (Gutsevich and Dubitskii, 1981), we could not be completely certain about the species identity of the above specimens and used for them the name Ochlerotatus sp. The rDNA sequencing was performed for all these species. We sequenced not only the rDNA region limited by the primers ISS2470F and ISS228R (Reno et al., 2000) but also the ITS2 region located between the 5.8S and 28S genes (primers 28S (Proft et al., 1999) and 5.8Sa (Gordeev et al., 2004)). Both regions were sequenced for 4–6 specimens of each mosquito species. The studied DNA fragment included the internal transcribed spacers 1 and 2 (ITS1 and ITS2), and also the 5.8S gene. The primers were positioned in the conserved regions of the 5.8S, 18S, and 28S genes. The lengths of sequenced rDNA fragments were: 868 bp for Ae. cinereus, 889 bp for Ae. vexans, 803 bp for ENTOMOLOGICAL REVIEW Vol. 93 No. 1 2013
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Fig. 1. Aligned 5.8S gene sequences of 11 species of the genera Aedes and Ochlerotatus. Dots designate identical nucleotides; asterisks (*) mark the beginning and end of the 5.8S gene; dashes (–) designate deletions.
Och. cantans, 801 bp for Och. excrucians, 794 bp for Och. euedes, 777 bp for Och. cyprius, 758 bp for Och. diantaeus, 817 bp for Och. intrudens, 783 bp for Och. punctor, 748 bp for Och. dorsalis, and 767 bp for Ochlerotatus sp. On average, the rDNA fragments obtained for representatives of the genus Aedes were 90–100 bp longer than those of Ochlerotatus. Sequence alignment was carried out by the CLUSTAL method. In this method, sequences are clustered based on genetic distances between all the pairs of sequences. The clusters are first united in pairs, then in groups, and finally the total alignment system is produced. The sequences are characterized by numerous insertions and deletions as well as single nucleotide replacements. We observed 6 types of replacement which occurred with different frequencies: C/T—259; A/G—218; C/G—111; A/C—105; G/T— 81; A/T—65. It is noteworthy that interspecific differences were observed not only in the variable regions ENTOMOLOGICAL REVIEW Vol. 93 No. 1 2013
of the internal transcribed spacers but also in the conserved region, namely that of the 5.8S gene (Fig. 1). Among the four replacements in this gene, one replacement (C/A) constitutes the difference between Aedes and Ochlerotatus, one replacement (T/A) differentiates Ae. cinereus from the genus Ochlerotatus and from Ae. vexans, and two more replacements (A/C, T/G) are shared by Och. dorsalis, Ochlerotatus sp., and representatives of the genus Aedes. In addition, Och. dorsalis and Ochlerotatus sp. are characterized by two deletions: one is shared by the two forms and distinguishes them from the rest of the species studied, whereas the other occurs only in Och. dorsalis (Fig. 1). It should be noted, however, that the pattern of differences would probably change if more species were included in the analysis. The phylogeny was reconstructed by the NeighborJoining (NJ) method. The resulting tree is shown in Fig. 2. The genera Aedes and Ochlerotatus form two
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Fig. 2. Phylogenetic relations of 11 species of the genera Aedes and Ochlerotatus reconstructed by the NJ method.
separate branches, which is consistent with the treatment of Ochlerotatus as a distinct genus (Reinert, 2000). According to molecular genetic analysis, representatives of the cantans group (Gutsevich et al., 1970), namely Och. cantans, Och. excrucians, and Och. euedes, are characterized by high similarity (identity index 96.8–97.5%). At the same time, the fourth member of this group, Och. cyprius, is less similar to the others (77.9–81.0%), despite the fact that Och. cantans, Och. excrucians, Och. euedes, and Och. cyprius were included in one species group both by Gutsevich with co-authors (1970) (cantans group) and by Becker with co-authors (2010) (annulipes group). The differences between members of the communis group (Gutsevich et al., 1970) are greater (identity index only 73.7–79.0%), whereas Och. punctor is not clustered together with two other representatives of this group, Och. diantaeus and Och. intrudens. However, in the classification of Becker and co-authors (2010) Och. punctor is included in the punctor group, while Och. diantaeus and Och. intrudens belong to the intrudens group. Thus, our data support the latter classification variant; however, conclusive analysis will be possible only when the nucleotide sequences of all the other representatives of these groups become available for comparison. Ochlerotatus dorsalis and Ochlerotatus sp. are also clustered together, which may presume the position of Ochlerotatus sp. in the caspius group (Gutsevich et al., 1970; Becker et al., 2010). Analysis of morphological characteristics and comparison of the rDNA sequences of Ochlerotatus sp. with the database showed this form to be similar to Och. caspius.
It is interesting that similarity between Ae. cinereus and Ae. vexans is low, at the level of similarity between each of them and species of the genus Ochlerotatus. As mentioned above, the differences between Ae. cinereus and Ae. vexans were observed not only in the variable rDNA regions but also in the 5.8S gene. Aedes cinereus and Ae. vexans belong to different Aedes subgenera: Aedes and Aedimorphus, respectively. It should also be noted that Ae. vexans is the only representative of the subgenus Aedimorphus known in Europe (Becker et al., 2010). The essential differences in the rDNA sequences may be due to taxonomic position of these two species. Further complex studies involving a greater number of species will result in a better understanding of phylogenetic relationships both between and within the genera Ochlerotatus and Aedes. ACKNOWLEDGMENTS The work was financially supported by the Russian Foundation for Basic Research (grant no. 11-0400716). REFERENCES 1. Becker, N., Petric, D., Zgomba, M., et al., Mosquitoes and Their Control (Kluwer Acad. / Plenum Publ., New York, 2010). 2. Gordeev, M.I., Goryacheva, I.I., Zvantsov, A.B., et al., “Molecular Genetic Analysis of Central Asian MalariaTransmitting Mosquitoes,” Vestnik Tomsk. Gos. Univ. 10, 17–19 (2004). 3. Gutsevich, A.V. and Dubitskii, A.M., “New Species of Mosquitoes in the Fauna of the USSR,” Parazitol. Sbornik 30, 97–165 (1981). 4. Gutsevich, A.V., Monchadsky, A.S., and Stackelberg, A.A., “Mosquitoes of the Family Culicidae,” in Fauna ENTOMOLOGICAL REVIEW Vol. 93 No. 1 2013
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