WEJCHOOCHOTEI, Taai & Harbach, 2015

Taai, Kritsana & Harbach, Ralph E., 2015, Systematics of the Anopheles barbirostris species complex (Diptera: Culicidae: Anophelinae) in Thailand, Zoological Journal of the Linnean Society 174 (2), pp. 244-264 : 259-261

ANOPHELES ( ANOPHELES ) WEJCHOOCHOTEI SP. NOV.

Anopheles (Anopheles) barbirostris ( campestris -like) in part (?) of Harrison & Scanlon, 1975 (northern Thailand, ♀ * ♂ * L* P*, taxonomy) .

Anopheles barbirostris in part (?) of Harrison et al., 1988 ( Thailand, A L P morphology).

Anopheles barbirostris form B in part of Baimai et al., 1995 ( Thailand, metaphase karyotype).

Anopheles campestris -like Forms B and E of Saeung et al., 2007 ( Thailand, mitotic karyotype, crossmatings, COI and COII mtDNA, ITS2 rDNA).

Anopheles barbirostris Clade V ( An. campestris ) of Paredes-Esquivel et al., 2009 ( Thailand, COI mtDNA, ITS2 rDNA).

Anopheles campestris -like Form E of Suwannamit et al., 2009 ( Thailand, cross-matings, metaphase karyotype, COI and COII mtDNA, ITS2 rDNA).

Anopheles campestris -like Forms B, E, and F of Thongsahuan et al., 2009 ( Thailand, cross-matings, mitotic karyotypes, COI and COII mtDNA, ITS2 rDNA). Thongsahuan et al., 2011 ( Thailand, Plasmodium susceptibility).

Anopheles campestris -like of Otsuka, 2011 ( Thailand, ITS2 rDNA).

Anopheles campestris of Paredes-Esquivel & Townson, 2014 ( Thailand, ITS2 rDNA).

Diagnosis

Anopheles wejchoochotei is morphologically similar to An. campestris but ITS2 sequence data (not available for An. campestris ) indicate that it is the sister species of An. barbirostris ( Fig. 4 ). Anopheles wejchoochotei and An. barbirostris are distinguished and identified by differences in their COI and ITS2 sequences (see below). Some potentially differential morphological characters are denoted below, but the two species are essentially isomorphic.

Description

Female

Anatomical features compared with other species of the Barbirostris Complex in Table 1, generally smaller than An. barbirostris , lengths of proboscis, maxillary palpus, antennal flagellum, wing, vein R 4+5, and forefemur statistically significantly shorter ( Table S2); similar to the female of An. campestris ; apical pale band of hindtarsomere 3 0.06–0.10 mm (mean 0.09 mm), not extended across joint onto base of hindtarsomere 4 in 10% of females examined.

Male

Similar to the male of An. campestris ; aedeagus with three to five pairs of leaflets; other characters of genitalia listed in Table 2, none statistically significantly different from those of An. barbirostris ( Table S3).

Pupa

As described for An. campestris ; setal branching in Table S12; branching of seta 2 compared with other species of the complex in Table 3; differences include seta 1-II with three to seven (four) branches; sum of branches of pair of seta 1-II = 7–13 (10), pair of seta 3-III = 8–13 (8).

Larva, fourth instar

As described for An. campestris ; setal branching in Table S13; differences include seta 14-P with four to five (five) branches; seta 8-M with four to 11 (nine) branches; seta 2-T single; seta 5-V with three to five (three) branches; seta 13-II with four to 17 (12) branches; sum of branches of pair of seta 13-C = 11–16, pair of seta 7-P = 41–47, pair of seta 8-M = 15–19, pair of seta 1-II = 28–38, pair of seta 2-VIII = 10–17(10), pair of seta 5-IV = 6–8.

Mitotic karyotype

Three types of X chromosome (X 1, X 2, X 3) and three types of Y chromosome (Y 2, Y 5, Y 6) comprising three karyotypic forms (X 2 Y 2, X 1 X 2 X 3 Y 5, X 2 X 3 Y 6) have been identified in the early fourth-instar larval brains of An. wejchoochotei ( Thongsahuan et al., 2009) .

Cross-matings

Cross-matings of An. wejchoochotei (as An. campestris - like Form E) with An. campestris -like Forms B, E, and F produced fully fertile offspring, yielding high percentages of emergence. Backcrosses between the F 1 offspring and the respective parental strains yielded fertile F 2 progeny. However, crosses between An. campestris -like Form E and An. barbirostris Forms A (= An. barbirostris , An. dissidens and An. saeungae ) and B (= An. dissidens and An. saeungae ) failed to yield offspring ( Saeung et al., 2007; Suwannamit et al., 2009).

DNA sequence

Specimens identified as An. wejchoochotei are shown in Table S1, together with GenBank accession numbers for ITS2 and COI sequences. The ITS2 subunit for An. wejchoochotei yields a dominant product of 1612 bp. The three interspecifically variable sites at bases 202, 316, and 556 of the COI gene that are unique for this species are shown in Figure 4 . The results of Bayesian analyses of ITS2 and COI sequences of An. wejchoochotei are shown in Figures 5 and 6 , respectively. Both trees show that An. wejchoochotei is well separated from the other species of the Barbirostris Complex. Our ITS2 sequences for An. wejchoochotei (HCE) fall within a strongly supported clade ( Fig. 5 , BPP 100%) with two sequences (bsk34 and csk10) of An. campestris (Clade V) of Paredes-Esquivel et al. (2009).

Bionomics

Suwannamit et al. (2009) found larvae of An. wejchoochotei (as An. campestris -like form E) in rice fields at 310 m above sea level in San Sai District of Chiang Mai Province. Adult females are known to attack and bite humans (the mothers of the broods that comprise the type series of this species were collected in human-baited traps). Limrat et al. (2001) and Apiwathnasorn et al. (2002) reported that either An. barbirostris or An. campestris is a probable vector of malaria in Sa Kaeo Province in eastern Thailand where high numbers of females were captured landing on humans both indoors and outdoors; however, no sporozoites of P. vivax developed in An. wejchoochotei females (as An. campestris -like Form E) from this province during experimental infection studies conducted by Thongsahuan et al. (2011). It is interesting to note, however, that 66.67 and 64.29% of females of Forms B and E, respectively, of this species from Chiang Mai Province did develop sporozoites of P. vivax .

Distribution

Based on COI, COII, and ITS2 sequences, An. wejchoochotei is currently only definitely known to occur in Thailand (Ayuttaya, Chanthaburi, Chiang Mai, Chiyaphum, Chumphon, Kamphaeng Phet, Khon Kaen, Maha Sarakham, Mukdahan, Prachuap Khiri Khan, Sa Kaeo, and Udon Thani Provinces; Thongsahuan et al., 2009, as An. campestris -like; present study). However, because it is so widely distributed in Thailand, it is likely to occur in neighbouring countries.

Etymology

This species is named in honour of the late Prof. Dr Wej Choochote ( Department of Parasitology , Faculty of Medicine, Chiang Mai University, Chiang Mai, Thailand) for his many contributions to our knowledge of mosquitoes in Southeast Asia, especially his studies of the Barbirostris Group, which provided the taxonomic foundation for further studies of this medically important group of insects .

Type series

Two-hundred and fifty-two specimens (63 ♀, 41 ♂, 59 Le, 65 Pe, 24 L) derived from six molecularly

identified progeny broods: HCE1 (1) , HCE2 (1) , HCE3 (1) , HCE4 (1) , HCE5 (1) , and HCE7 (1) . Holotype, ♀ [ HCE1 (1)- 15], with Le and Pe on microscope slide, offspring of female collected as follows: THAILAND, Chiang Mai Province, San Sai District , Ban Nong Chom, humanbaited trap, 2.xi.2013, coll. Choochote et al. Paratypes, same data as holotype: 30 ♀ LePe [ HCE1 (1)-1, -5, -9, -10, -12, -13, -16 to -20; HCE2 (1)-9 to -13, -16 to -20; HCE3 (1)-3, -6, -9, -16 to -20, -23]; 1 ♀ Le [ HCE1 (1)- 14]; 5 ♀ Pe [ HCE3 (1)-2, -21, -22, -25, -26]; 26 ♀ [ HCE4 (1)-2 to -8, -10 to -16; HCE5 (1)-1, -2, -5 to -8, -10; HCE7 (1)-3, -5 to -7, -9]; 27 ♂ LePe [ HCE1 (1)-2 to -4, -6 to -8, -11; HCE2 (1)-1 to -8, 14, -15; HCE3 (1)-4, -5, -7, -8, -10 to -12, -14, -15, -24]; 2 ♂ Pe [ HCE3 (1)- 1, -13]; 12 ♂ [ HCE4 (1)-1, -9; HCE5 (1)-3, -4, -9, -11, -12; HCE7 (1)-1, -2, -4, -8, -10]; 24 L [ HCE1 (1)-A, -B -C, -D, -E, -F; HCE2 (1)-A, -B -C, -D, -E, -F; HCE3 (1)- A, -B -C, -D, -E, -F; HCE4 (1)-A, -B; HCE5 (1)-A, -B; HCE7 (1)-A, -B]. The type series is deposited in BMNH.

Figures

Figure 4. Alignment of the polymorphic sites along a 658-bp fragment of the mitochondrial cytochrome c oxidase subunit I gene in Anopheles barbirostris (one female), Anopheles dissidens sp. nov. (13 females), Anopheles saeungae sp. nov. (nine females), Anopheles wejchoochotei sp. nov. (six females), and An. barbirostris species A3 (three females) (Saeung et al., 2008). Codes for the specimens are shown in Table S1. Bases enclosed in boxes represent species-specific differences among the five species.

Figure 5. Bayesian consensus tree for internal transcribed spacer 2 sequences. The tree is rooted on Anopheles (Cellia) dirus. Only Bayesian posterior probabilities greater than 50% are shown on the branches. The bar represents 0.1 substitutions per site.

Figure 6. Bayesian consensus tree for cytochrome c oxidase subunit I sequences. Only Bayesian posterior probabilities greater than 50% are shown on the branches. The bar represents 0.01 substitutions per site.