Anopheles, Meigen, 1818

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 : 245-253

ANOPHELES ( ANOPHELES ) BARBIROSTRIS VAN DER WULP

Anopheles barbirostris van der Wulp, 1884 (♀). Holotype ♀: Mount Ardjoeno , Java, Indonesia (Nationaal Natuurhistorisch Museum, Leiden, Netherlands).

Anopheles martini Laveran, 1902 (♀). Type locality: near Pursat, Cambodia (Institut Pasteur, Paris). It is not possible to know for certain whether or not this nominal species is conspecific with An. barbirostris or another member of the Barbirostris Complex.

Anopheles barbirostris innominata Stoker & Waktoedi Koesoemawinangoen, 1949 (♀ ♂). Type locality: Sulawesi, Indonesia (type non-extent). This nominal form may be a distinct member of the Barbirostris Complex.

Anopheles (Anopheles) barbirostris in part of Bonne-Wepster & Swellengrebel, 1953 ( Indonesia, ♀ * ♂ * L*); Reid, 1962 ( Indonesia, Malaysia, Thailand, ♀ * ♂ E* L P*, taxonomy); Reid, 1968 ( Malaysia, Borneo, ♀ * ♂ * E L* P*, taxonomy, biology); Harrison & Scanlon, 1975 ( Thailand, ♀ * ♂ * L* P*, taxonomy); Reid, Harrison & Atmosoedjono, 1979 ( Indonesia, Malaysia, Thailand, ♀ * L P* morphology, taxonomy).

Anopheles barbirostris in part (?) of Harrison, Rattanarithikul & Mongkolpanya, 1988 ( Thailand, A L P morphology); Ndoen et al., 2010, 2011 (West Timor, Java, ecology, bionomics).

Anopheles barbirostris form A in part of Baimai, Rattanarithikul & Kijchalao, 1995 ( Thailand, metaphase karyotype).

Anopheles barbirostris Clade I of Paredes-Esquivel et al., 2009 (Bornean Indonesia, Thailand, Vietnam, COI mtDNA, ITS2 rDNA); Paredes-Esquivel & Townson, 2014 (Bornean Indonesia, Thailand, ITS2 rDNA).

Anopheles barbirostris species A4 of Suwannamit et al., 2009 ( Thailand, cross-matings, metaphase karyotype, COI and COII mtDNA, ITS2 rDNA); Thongsahuan et al., 2011 ( Thailand, Plasmodium susceptibility); Otsuka, 2011 ( Thailand, ITS2 rDNA).

Anopheles (Anopheles) barbirostris of Townson et al., 2013 ( Javan Indonesia, ♀ * ♂ * L* P*, COI mtDNA and ITS2 rDNA, taxonomy, bionomics, distribution) .

Diagnosis

Females of An. barbirostris resemble other members of the Barbirostris Group in having entirely darkscaled maxillary palpi with palpomeres 1 and 2 particularly shaggy and a tuft of black scales on abdominal sternum VII. The other sterna usually have a few scattered pale scales in both males and females. The thoracic pleura generally have pale scales associated with setae on the upper proepisternum, mesokatepisternum, and mesepimeron. The wing usually has a pale fringe spot at the apex of the cubitus and a few scattered pale scales on the proximal half of the costa. Larvae have bushy lateral palatal brushes and thoracic seta 1-P with long branches from near the base.

Anopheles barbirostris is very similar to other members of the Barbirostris Complex. Females of the complex are usually recognizable by the absence of a pale fringe spot at the apex of vein R 2, the presence of a pale fringe spot at the apex of vein R 4+5, extensive pale scaling on the cubitus, a few pale scales on the abdominal sterna, and narrow apical pale bands on tarsomeres of the foreleg. Pupae have a secondary cleft on the trumpet, setae 1,5-III–VII with numerous branches, seta 2-VII with few branches, and seta 9-II–VIII yellow to pale brown. Larval seta 3-C normally has more than 40 stiff, broom-like branches, palmate seta 1-II is well developed and pigmented, and the spiracular apparatus lacks an anterior median process. Sequences for the COI region of mtDNA distinguish An. barbirostris from other species of the Barbirostris Complex (see below).

Description

Female

Large, brownish-black mosquito with finely dappled wings and narrowly banded tarsi ( Fig. 1 ). Measure- ments and statistical analysis of selected anatomical features compared with other species of the complex in Tables 1 and S 2, respectively.

Head: Vertex dark-scaled with patch of pale scales before interocular space; interocular space with mostly dark setae and narrow pale scales. Antenna about twothirds length of proboscis; pedicel with scales on dorsolateral surface; flagellomere 1 with patch of scales, other flagellomeres without scales. Proboscis length about 2.45 mm, entirely dark-scaled, noticeably shaggy in proximal 0.5–0.7, labella also dark; forefemur/ proboscis ratio 0.90–0.98 (mean 0.93). Maxillary palpus same length as proboscis, entirely dark-scaled, palpomeres 1 and 2 particularly shaggy.

Thorax: Scutum with longitudinal bare areas between rather broad lines of golden piliform scales on acrostichal, dorsocentral, and marginal areas; anteri- or promontory with whitish piliform scales medially; posterior segments of acrostichal and dorsocentral areas and entire prescutellar area with covering of golden piliform scales, scutal setae slightly darker and longer than scales; scutellum with golden piliform scales along bases of large golden to golden-brown setae in transverse posterior row. Mesopostnotum and postpronotum bare. Antepronotum with prominent patch of dark scales on dorsoanterior surface and rather sparse fine piliform scales posteriorly. Pleura with dark setae on upper proepisternum and golden to golden-brown setae on prespiracular area, prealar knob, upper and lower mesokatepisternal areas, and upper and middle mesepimeron; with four or five upper proepisternal setae, eight or nine prealar setae, five or six upper and four to six lower mesokatepisternal setae, and six to 15 upper and zero to two lower mesepimeral setae; scales usually accompany setae on upper proepisternum, mesokatepisternum, and mesepimeron; scales and setae often indistinct on middle of mesepimeron.

Wing: Pattern as illustrated, costa often with small humeral pale spot (at least on posterior margin of vein) and usually scattered pale scales between humeral crossvein and small subcostal pale spot, remainder of costa dark to preapical pale spot (sometimes absent); remigium usually with median pale scales; humeral crossvein with dark scales; vein R with scattered pale scales extending to sector pale spot; vein R 1 with apical pale spot adjoining preapical pale spot of costa and usually a line of interspersed pale scales at subcostal area; vein R 2 with subapical pale spot adjoining apical pale spot of R 1; vein R 3 usually with distinct postbasal and preapical pale spots; vein R 4+5 with variable density of interspersed pale scales on median 0.80, length of pale-scaled area 1.64–2.00 mm (mean = 1.82 mm); vein M 1 with postbasal and preapical pale spots, sometimes with variable pale scaling in between; vein M 2 largely pale-scaled except distally; vein M 3+4 with mostly pale scaling between basal and apical dark scaling; mediocubital crossvein with medial pale spot; vein CuA mainly pale-scaled with distinct short postbasal and apical dark spots, apex often with a few pale scales, postbasal dark spot usually separated by more than its length from medial dark spot on vein 1A; vein 1A largely pale-scaled with distinct medial and apical dark spots; narrow pale fringe spots at apices of veins R 4+5 and CuA.

Halter : Integument of scabellum pale; integument of pedicel and capitellum dark and dark-scaled.

Legs: Mainly dark-scaled; coxae with small patches of pale scales; forefemur swollen towards base; all femora with a few pale scales at base; all tibiae narrowly palescaled at apex; foretarsomeres 1 and 2 with narrow apical pale marks or bands, midtarsus entirely dark-scaled, hindtarsomeres 1–4 with narrow apical pale bands or patches that sometimes cross the joints, apical band of hindtarsomere 3 0.06–0.10 mm (mean 0.08 mm), not extended across joint onto base of hindtarsomere 4.

Abdomen: Integument brownish black, sterna paler, especially basally on either side of midline; sterna II– VII with a median patch of pale scales, usually a few pale scales forming row along lateral margins and infrequently with scattered pale scales in between lateral rows and median patch, sternum VII with prominent posteromedian tuft of black scales, often a few dark scales in same position on sternum VI.

value)

- P 0.000 0.000 0.001 0.000 0.000 0.001 0.000 0.000 0.220 0.000 0.208 0.103 0.109 0.001 0.323

parentheses in modes / Anopheles wejchoochotei sp nov.. 2.08 () – 1.92 2.24 2.15 – 1.84 2.40 ()) 1.48 1.31 – 1.12 (3.36 – 3.72) (4.08 –) (0.88 0.96 1.04 – (0.80 0.96) 0.88) (1.64 – 1.42 1.54 (1.68) 2.00 – 1.83) (0.96 0.88 – 0.81 –) 5 3 (4 6 0) 8 (– (4 4) – 2 3 4) – 6 (8 16 (– 5) 3) (– 0 0 means (

)))))))) Thailand (2.27 2.40 2.60 (2.39 1.56 (1.43 4.03 (4.24 1.22 2.04 ((1.80 1.67 (2.12 2.07 0.91 0.95 (in Anopheles campestris 2.12 – 2.08 – 1.28 – – 3.76 0.96 – 1.08 – 1.58 1.96 – – 0.88 (4,) 3 3 5 – 4 (7) – 6 0 2 5 3) (– 9 14 – 0) 6 0 (– Complex Barbirostris the Anopheles saeungae nov.. sp) 2.12 1.84 2.02 – (1.80 (2.00 2.12) – 1.46) 1.36– 1.60 (3.60 4.00) 3.84 – (0.89 0.80 0.96 () – 0.90 (0.98 – 0.82) –) 1.51 1.34 (1.62 1.76 1.91) – 2.04 () (– 1.00 0.87 0.94 2 (1 2,) (0 – 9 0)) 1 – 6 (2) – 5 2 (1 5 10 () – 7) 0 0 3 – (of species) 1.73) 1.78 1.34) 3.49) 0.79)) 0.82 1.38)) 1.63) 0.94 five females of Anopheles dissidens . sp. nov 2.04 – (1.60 (1.64 2.20 – 1.92 1.16 (– 3.20 – (3.84 0.72 0.84 – ((– 0.92 0.70 1.24 – 1.56 ((1.40 1.76 – 1.05 (0.90 – – 6 2 3) ((–) 2 2 9 –) (0 6 2 4 3) – (0 (5 5) 7 – 0 in observed 2.45) () 2.54 (() 1.70 (4.58) 1.04) 1.12 ((1.10)) (1.82 () 2.27 0.98 0.93 () features Anopheles barbirostris 2.60 2.16 – – 2.64 2.44 1.80 1.56 – 4.24 4.80 – 0.96 – 1.04 1.12 – 2.00 – 1.64 2.12 2.36 – 0.90 – 4) 4 (5, 0) 0 9 (– (, 6 5 5) 4 –6 (4) 15 – 6) 2 (0 – 0 anatomical setae setae of palpus ratio setae. 1 Table Comparison Morphological feature of proboscis Length Length maxillary of flagellum Length of Length of wings wings Width of R Length vein of 2 R of Length vein 5 4 + Length of forefemur / proboscis Forefemur Upper proepisternal Prealar setae mesokatepisternal Upper mesokatepisternal Lower Upper setae mesepimeral Lower setae mesepimeral

Male

Like female except as follows.

Head: Maxillary palpus usually completely darkscaled, sometimes with indistinct apicolateral pale bands on palpomeres 4 and 5.

Wing: Scaling of veins posterior to radius not as dense as in female; small pale fringe spot often between apices of veins R 2 and R 4+5.

Abdomen: Tergum VIII with median patch of dark scales, sometimes with a few pale scales proximal to dark scales.

Genitalia: ( Fig. 2C ; Tables 2, S 3) Lateral surface of gonocoxite with dark scales that become longer, more prominent, and black toward apex, proximal scaling less distinct with a few pale scales sometimes present at base; two parabasal setae borne on small protuberance at base of dorsomesal surface, lateral parabasal about twice as long as mesal one; inner seta on middle of ventromesal surface similar to lateral parabasal seta; ventral lobe of claspette small and ill-defined, with two simple setae borne on ventrolateral margin, the more ventromesal one longer than the other; dorsal lobe of claspette with club formed of four or five contiguous and distally fused setae; aedeagus with five or six pairs of leaflets, longest leaflet about half as long as aedeagus, bearing a blunt tooth at base and fine serration mainly on distal two-thirds of both edges, other large leaflets with serration on one or both edges.

Pupa

Lightly to moderately pigmented; positions and character of setae as figured ( Fig. 2A, B ); setae usually with ring of darker cuticle at base; numbers of branches in Table S4.

Cephalothorax: Trumpet darkly pigmented, with thin uniform rim and deep secondary cleft; wings with blurred, lattice-like pattern of darker spots; antenna with incomplete dark rings at joints and dark apex; proboscis and fore- and midfemora and -tibiae with blurred rings of darker cuticle; sum of branches of pair of seta 12-CT = 2−5 (3), pair of seta 3-III = 11–16 (11).

Abdomen: Lightly to moderately pigmented. Branching of serially homologous seta 2 compared with other species of the Barbirostris Complex in Table 3. Seta 1-II with three to eight (five) branches, sum of branches of pair of seta 1-II = 6–13 (7); seta 2-II–VII well developed; sum of branches of pair of seta 3-III = 11–16 (11); seta 5-II with three to seven (four) branches; setae 1,5-III–VII strongly developed with numerous fine branches (fewer than actual number of branches shown in Fig. 2B ), central branch usually distinctly longer than other branches; setae 1,5,9-IV–VII more darkly pigmented than integument; seta 8-II present or absent.

Paddle: With darkly pigmented base and spot of darker cuticle around insertions of setae 1,2-Pa; seta 1-Pa

usually single with apex split into one to three elements.

Larva, fourth instar

Generally darkly pigmented; positions and character of setae as illustrated ( Fig. 3 ); numbers of branches in Table S5.

Head: About as wide as long; unevenly pigmented, with mottled pattern of darker cuticle, collar and dorsomentum darkly pigmented. Seta 2-C simple; seta 3-C broom-like; seta 4-C small, with one to three (two) branches; setae 6,7-C often with eight to 14 (ten, 13) branches; setae 8,9-C usually with five to 11 (eight, nine) branches.

Antenna: Moderately pigmented; mesal and ventral surfaces strongly spiculate; length about 0.28 times length of head.

Thorax: Integument hyaline, smooth. Seta 1-P often with six to eight (seven) branches from near base; seta 11-P with one or two simple branches arising from short basal stem; sum of branches of pair of seta 13-P = 7– 13 (11); seta 13-M with four to 11 (six) branches; seta 14-M commonly with eight to 14 (11) branches; seta 3-T palmate with pale lanceolate leaflets; seta 8-T normally with 13–26 (21) branches.

Abdomen: Integument smooth except for fine spicules on midventral areas of segments II–VIII. Seta 1-I similar to seta 3-T; seta 1-II–VII fully palmate, leaflets with blades darkly pigmented proximal to steplike margins of tapered terminus; seta 13-I often with five to 14 (nine) branches; seta 2-II generally with three to seven (five) branches; seta 5-III frequently with five to nine (seven) branches; sum of branches of pair of seta 10-III = 3–5 (3); seta 13-II with six to ten (eight) branches, seta 13-IV usually with three to seven (three) branches; seta 9-VII often with two to six (five) branches; pecten plate darkly pigmented, usually with eight or nine longer spines; seta 8-S with one to three branches, most often three; membrane posterior to saddle with numerous relatively long spicules; seta 1-X about as long saddle.

Mitotic karyotype

Two types of X chromosome (X 1, X 2) and one type of Y chromosome (Y 1) comprising a single karyotypic form (X 1 X 2 Y 1) have been identified in the early fourthinstar larval brains of An. barbirostris ( Baimai et al., 1995; Suwannamit et al., 2009).

Cross-matings

Reciprocal cross-matings between An. barbirostris , Anopheles dissidens sp. nov., Anopheles saeungae sp. nov., and Anopheles wejchoochotei sp. nov. (as species A4, A1, A2, and campestris -like, respectively) conduct- ed by Suwannamit et al. (2009) revealed strong reproductive isolation. The crosses yielded a few viable eggs that gave rise to F 1 larvae with asynaptic polytene chromosomes and F 1 adults with abnormally developed reproductive systems. Females had abnormal ovarian follicles and the accessory glands and testes of males were atrophied.

DNA sequence

Specimens identified as An. barbirostris are shown in Table S1, together with GenBank accession numbers for ITS2 and COI sequences. The ITS2 subunit for An. barbirostris yields a dominant product of 1637 bp. The seven interspecifically variable sites at bases 55, 100, 266, 389, 562, 565, and 607 that are unique for the COI gene of An. barbirostris are shown in Figure 4 . The results of Bayesian analyses of ITS2 and COI sequences are shown in Figures 5 and 6 , respectively. Both trees show that An. barbirostris is well separated from the other species. Our ITS2 sequence for An. barbirostris (BACm6) falls within a strongly supported Clade, Bayesian posterior probability (BPP) 100%, with two sequences (th1.3 and k2) previously report- ed by Paredes-Esquivel et al. (2009, as Clade I = An. barbirostris s.s. of Townson et al., 2013) ( Fig. 5 ).

Bionomics

In general, An. barbirostris is a foothill mosquito. In Thailand, immature stages have been collected at elevations from sea level to 500 m a.s.l. and adults have been captured biting humans at elevations between 750 and 1400 m ( Scanlon & Esah, 1965). Habitats of the immature stages include river and stream margins, river and stream pools, ditches with flowing and stagnant water, moats, lakes, permanent and temporary ground pools, flood pools, rice fields, wells, canals, marshes and swamps, ponds, rock pools, seepage springs, and buffalo and elephant footprints. Most habitats contain vegetation and are located in open sunny and partially shaded areas. Females are generally zoophilic but bite humans in the absence of their usual hosts. Adults have been found resting in animal shelters and inside and outside houses.

Both Plasmodium falciparum and Plasmodium vivax infections have been detected in mosquitoes identified morphologically as An. barbirostris s.l. in Sumatra and Sulawesi in the Greater Sundas ( Bangs & Rusmiarto, 2007; Syafruddin et al., 2007), Flores, Adonara Island, and Timor-Leste in the Lesser Sundas ( Bangs & Rusmiarto, 2007; Cooper et al., 2010), Sri Lanka ( Amerasinghe et al., 1999), and Bangladesh ( Alam et al., 2010). However, no oocysts or sporozoites of either P. falciparum or P. vivax were found in females of An. barbirostris s.s. (as An. barbirostris species A4) during experimental infection studies of Thai populations conducted by Thongsahuan et al. (2011). Future vector incrimination studies in other areas of the reported range of this species should include DNA applications to retrospectively unequivocally identify malaria infective females.

Distribution

Anopheles barbirostris is the most widespread member of the Barbirostris Complex. It is abundant and widely distributed in Thailand, but is not found at higher elevations or in heavily shaded forest. It is uncommon in the rice plains north of Bangkok where An. campestris also occurs. Literature records suggest that this species occurs widely in the Oriental Region from China southward to Indonesia and from Vietnam westward to India and Sri Lanka, but molecular data have only documented its presence in Indonesia (Java, South Kalimantan), Thailand, and Vietnam ( Suwannamit et al., 2009; Paredes-Esquivel et al., 2009; Townson et al., 2013; present study).

Material examined

Fifty-seven specimens (19 ♀, 9 ♂, 13 Le, 16 Pe ) derived from four progeny broods: BACm6(1), JV52, JV58, and JV73 (the last three from the study of Townson et al., 2013). THAILAND, Chiang Mai Province, Chiang Dao District , Ban Wang Jom , buffalo-baited trap, offspring of female collected 23.xi.2013 (coll. Choochote et al.): 2 ♀ LePe [BACm6(1)-2, -5]; 2 ♀ Pe [BACm6(1)- 100, -101], 3 ♂ LePe [BACm6(1)-1, -3, -4], 1 ♂ Pe [BACm6-102]. INDONESIA, Java, Malang Regency , East Java Province, Karangploso Subdistrict , Donowarin Village , Karang Hamlet (07°52 ′ 55 ′′ S, 112°35 ′ 02 ′′ E), 2280 ft, offspring of females collected 12.xi.2007 (coll. McAlister et al.): 6 ♀ LePe [JV52-3, -4, -6, -8 to -10], 8 ♀ [JV52-14 to -17, JV58-1, -2, -4, -5], 2 ♂ LePe [JV52- 5, -7], 3 ♂ [JV52-11 to -13]; Semerang Regency, Central Java Province, Jambu Subdistrict, Kelurahan Village , Krajan Hamlet (07°17 ′ 03 ′′ S, 112°21 ′ 53 ′′ E), 1715 ft, offspring of female collected 14.xi.2007 (coll. McAlister et al.): 1 ♀ [JV73-4]. The specimens are deposited in the Natural History Museum, London ( BMNH) .

Figures

Figure 1. Female of Anopheles barbirostris van der Wulp (dorsal), modified from Townson et al. (2013).

Figure 2. Pupa and male genitalia of Anopheles barbirostris van der Wulp (modified from Townson et al., 2013). A, pupa, left side of cephalothorax, dorsal to right. B, pupa, dorsal (left) and ventral (right) aspects of metathorax and abdomen. C, male genitalia, dorsal (tergal) aspect. Abbreviations: Ae, aedeagus; c, club on dorsal lobe of claspette; CT, cephalothorax; Gc, gonocoxite; Gs, gonostylus; InS, internal seta; LAe, leaflets of aedeagus; Pa, paddle; PBS, parabasal setae; Pr, proctiger; I–IX = abdominal segments I–IX; 1–14 = setal numbers for specified areas, e.g. seta 3-I.

Figure 3. Fourth-instar larva of Anopheles barbirostris van der Wulp (modified from Townson et al., 2013). A, head, dorsal (left) and ventral (right) aspects of left side. B, thorax and abdominal segments I–VI, dorsal (left) and ventral (right) aspects of left side. C, abdominal segments VII, VIII, and X, left side. Abbreviations: A, antenna; C, cranium; P, prothorax; M, mesothorax; S, spiracular lobe; T, metathorax; I–VIII,X = abdominal segments I–VIII and X; 1–15 = setal numbers for specified areas, e.g. seta 5-C.

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.