Variation in Number and Formation of Repeat Sequences in the rDNA ITS2 Region of Five Sibling Species in theAnopheles barbirostrisComplex in Thailand

Abstract

Repeat sequences of approximately 100 base pairs in length were found in the rDNA ITS2 region of Anopheles barbirostris van der Wulp (Diptera: Culicidae) species A1, A2, A3, A4, and An. campestris-like in the An. barbirostris complex. Variation in the number of repeats was observed among the five sibling species. Specifically, 10 repeats were observed in A1, eight in A2, A4, and campestris-like, and three in A3. Based on similarities in the sequences of the repeats, related repeats were classified into nine groups. Although A2, A4, and the campestris-like species had the same number of repeats, the ITS2 region of the three species contained different groups of repeats. Excluding the repeat sequences facilitated good alignment of the ITS2 region in the five sibling species. Phylogenetic analyses of the 95 isolines were compared with results obtained from mitochondrial genes (COI and COII). The results revealed marked differences among the five sibling species, particularly regarding the ITS2 region of A3, which was more distinct from the other four species than COI and COIL Repeat sequences in the ITS2 region of other Anopheles species retrieved from GenBank also were analyzed. New repeat sequences were found in An. beklemishevi Stegnii and Kabanova, An. crucians Wiedemann and An. funestus Giles, suggesting that the occurrence of repeat sequences in the ITS2 region are not rare in anopheline mosquitoes.

Systematics of Anopheles barbirostris van der Wulp and a sibling species of the Barbirostris Complex (Diptera: Culicidae) in eastern Java, Indonesia

“…Within the Barbirostris Subgroup, the ITS2 amplicon is exceptionally large (>1.5 kb), larger than in any other Anopheles sp., due to a complex series of internal repeats (Paredes‐Esquivel et al , 2009; Otsuka, 2011). Consequently, sequencing this region involved amplification followed by cloning, as described in Paredes‐Esquivel et al (2009).…”

“…barbirostris . Variation of repeat sequences in the ITS2 region of the five purported species of the Barbirostris Complex in Thailand (Saeung et al , 2008; Suwannamit et al , 2009) was investigated by Otsuka (2011).…”

Systematics of theAnopheles barbirostrisspecies complex (Diptera: Culicidae: Anophelinae) in Thailand

“…wejchoochotei was referred to informally as An. campestris ‐like in recent chromosomal, cross‐mating, and molecular studies (Saeung et al ., ; Suwannamit et al ., ; Otsuka, ). Although An.…”

“…nov. These polymorphisms are mainly the result of differences of the number of repeats in the ITS2 sequences (Otsuka, ). Sequence data are not currently available for specimens definitively identified as An.…”

“…Anopheles barbirostris species A1 of Saeung et al ., (Thailand, mitotic karyotype, cross‐matings, COI and COII mtDNA, ITS2 rDNA); Suwannamit et al ., (Thailand, cross‐matings, metaphase karyotype, COI and COII mtDNA, ITS2 rDNA); Thongsahuan et al ., (Thailand, Plasmodium susceptibility); Otsuka, (Thailand, ITS2 rDNA).…”