Anopheles campestris Plasmodium Anopheles campestris Systematics of the Anopheles barbirostris species complex (Diptera: Culicidae: Anophelinae) in Thailand Taai, Kritsana Harbach, Ralph E. Zoological Journal of the Linnean Society 2015 2015-05-14 174 2 244 264 9CJDC Meigen 1818 [867,1002,518,539] Insecta Culicidae Anopheles Animalia Diptera 15 259 Arthropoda genus    Anopheles barbirostrisin part (?) of Harrison et al., 1988( Thailand, A L P morphology).   Anopheles barbirostrisform 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, COIand COIImtDNA, ITS2rDNA).   Anopheles barbirostrisClade V (  An. campestris) of Paredes-Esquivel et al., 2009( Thailand, COImtDNA, ITS2rDNA).   Anopheles campestris-like Form E of Suwannamit et al., 2009( Thailand, cross-matings, metaphase karyotype, COIand COIImtDNA, ITS2rDNA).    Anopheles campestris-like Forms B, E, and F of Thongsahuan et al., 2009( Thailand, cross-matings, mitotic karyotypes, COIand COIImtDNA, ITS2rDNA). Thongsahuan et al., 2011( Thailand,  Plasmodiumsusceptibility).   Anopheles campestris-like of Otsuka, 2011( Thailand, ITS2rDNA).    Anopheles campestrisof Paredes-Esquivel & Townson, 2014( Thailand, ITS2rDNA).  Diagnosis   Anopheles wejchoochoteiis morphologically similar to  An. campestrisbut ITS2sequence data (not available for  An. campestris) indicate that it is the sister species of  An. barbirostris( Fig. 4).  Anopheles wejchoochoteiand  An. barbirostrisare distinguished and identified by differences in their COIand ITS2sequences (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 in10% 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 typesof X chromosome (X 1, X 2, X 3) and three typesof Y chromosome (Y 2, Y 5, Y 6) comprising three karyotypic forms (X 2Y 2, X 1X 2X 3Y 5, X 2X 3Y 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 1offspring and the respective parental strains yielded fertile F 2progeny. However, crosses between  An. campestris-like Form E and  An. barbirostrisForms A(=  An. barbirostris,  An. dissidensand  An. saeungae) and B (=  An. dissidensand  An. saeungae) failed to yield offspring ( Saeung et al., 2007; Suwannamit et al., 2009).  DNA sequence Specimens identified as  An. wejchoochoteiare shown in Table S1, together with GenBank accession numbers for ITS2and COIsequences. The ITS2subunit for  An. wejchoochoteiyields a dominant product of 1612 bp. The three interspecifically variable sites at bases 202, 316, and 556 of the COIgene that are unique for this species are shown in Figure 4. The results of Bayesian analyses of ITS2and COIsequences of  An. wejchoochoteiare shown in Figures 5and 6, respectively. Both trees show that  An. wejchoochoteiis well separated from the other species of the Barbirostris Complex. Our ITS2sequences 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 mabove 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 typeseries of this species were collected in human-baited traps). Limrat et al. (2001)and Apiwathnasorn et al. (2002)reported that either  An. barbirostrisor  An. campestrisis a probable vector of malaria in Sa Kaeo Provincein eastern Thailandwhere high numbers of females were captured landing on humans both indoors and outdoors; however, no sporozoites of  P. vivaxdeveloped in  An. wejchoochoteifemales (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 Provincedid develop sporozoites of  P. vivax.  Distribution Based on COI, COII, and ITS2sequences,  An. wejchoochoteiis 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 ThaniProvinces; 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 MaiUniversity, 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.   Typeseries 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 inBMNH. Thailand Thailand 15 259 2 1 1 Thailand Department of Parasitology Faculty of Medicine 16 260 1 Chiang Mai [435,534,198,220] HCE 17 261 HCE1 1 [545,644,198,220] HCE 17 261 HCE2 1 [656,754,198,220] HCE 17 261 HCE3 1 [144,241,229,251] HCE 17 261 HCE4 1 [251,349,228,250] HCE 17 261 HCE5 1 [407,505,229,251] HCE 17 261 HCE7 1 2013-11-02 HCE Choochote Thailand San Sai District 17 261 HCE1, HCE2, HCE3, HCE4, HCE5, HCE7 104 63 41 Chiang Mai holotype