Anopheles (Cellia) harrisoni Harbach & Manguin

Harbach, Ralph E., Garros, Claire, Manh, Nguyen Duc & Manguin, Sylvie, 2007, Formal taxonomy of species C of the Anopheles minimus sibling species complex (Diptera: Culicidae), Zootaxa 1654, pp. 41-54 : 44-51

Anopheles (Cellia) harrisoni Harbach & Manguin , sp. n.

Anopheles minimus species C of Green et al., 1990 (enzyme electrophoresis, morphology); Baimai et al., 1996 (mitotic karyotype); Sucharit & Komalamisra, 1997 (RAPD-PCR identification); Sharpe et al., 1999 (D3 rDNA, ASA and SSCP identification); Sharpe et al., 2000 (COII mtDNA, ITS2 rDNA, D3 rDNA, phylogenetic relationships); Van Bortel et al., 2000 (ITS2 rDNA, RFLP-PCR assay); Kengne et al., 2001 (RAPD-PCR, SCAR-PCR multiplex assay); Somboon et al., 2001 (D3 rDNA); Chen et al., 2002 (D3 rDNA, morphology, distribution); Choochote et al., 2002 (crossmating with An. minimus ); Rwegoshora et al., 2002 (adult bionomics); Zhou et al., 2002a (ITS2 rDNA, phylogenetic relationships); Zhou et al., 2002b (COII mtDNA, phylogenetic relationships); Chen et al., 2003 (COII mtDNA, D3 rDNA, phylogenetic relationships); Phuc et al., 2003 (ITS2 rDNA, multiplex assay); Van Bortel et al., 2003 (Odh locus, population genetics); Garros et al., 2004a (D3 rDNA, ITS2 rDNA, RFLP-PCR assay); Garros et al., 2004b (ITS2 rDNA, allele specific multiplex assay); Garros et al., 2005a (COII mtDNA, D3 rDNA, morphology, phylogenetic relationships); Garros et al., 2005b (COI mtDNA, ITS2 rDNA, D3 rDNA, phylogenetic relationships); Trung et al., 2004 (trophic behaviour); Van Bortel et al., 2004 (trophic behaviour); Kengluecha et al., 2005 (larval bionomics); Somboon et al., 2005a (crossmating with An. minimus E); Trung et al., 2005 (trophic behaviour); Harbach et al., 2006 (taxonomy); Garros et al., 2006 (general review); Potikasikorn et al., 2006 (insecticide resistance); Singh et al., 2006 (ITS2 rDNA, D2-D3 rDNA); Sungvornyothin et al., 2006a (morphology); Sungvornyothin et al., 2006b (bionomics).

Anopheles minimus form II of Van Bortel et al., 1999 (Odh locus).

Diagnosis. Sequences for the ITS2 and the D3 domain of the 28S rDNA and Cyt-b, COI and COII of mtDNA for An. harrisoni and other members of the Minimus Complex exhibit little intraspecific variation and sufficient interspecific variation to be diagnostic of the species (figs 2–6).

Comparative studies of the adult, larval and pupal stages of An. minimus and An. harrisoni (as An. minimus species A and C, respectively) from the type locality of the latter species (see Type series) were conducted using specimens from progeny broods identified by enzyme electrophoresis and PCR-RFLP (see Materials and methods). Attempts were made to find morphological characters in the various life stages that might differentiate the two species. Study of adult females included the use of scanning electron microscopy to examine the cibarial armature. Study of the larval and pupal stages involved systematic observations of all elements of chaetotaxy. The results of these studies indicated that An. minimus and An. harrisoni are essentially isomorphic in all life stages (also see Garros et al., 2005b). Since no morphological characters were found that would consistently and reliably distinguish these species, studies of their ecology and behaviour must relay on genetic and molecular methods of identification.

Molecular characterization. Three PCR-based assays have been developed that distinguish An. harrisoni from An. minimus and three related species ( An. aconitus Dönitz , An. pampanai Büttiker , An. varuna Iyengar ): SSCP-PCR of D3 for identifying four of the five species ( Sharpe et al., 1999); RFLP-PCR of ITS2 using the restriction endonuclease BsIZI ( Van Bortel et al., 2000); allele-specific PCR based on SCAR markers ( Kengne et al., 2001) and ITS2 nucleotide variations ( Phuc et al., 2003; Garros et al., 2004a).

Mean sequence divergence between An. minimus and An. harrisoni ranged from 2.3 to 3.3% in the Cyt-b region. Levels of variability of the D3 and COII sequences for these two species were reported previously ( Garros et al., 2005a, b).

Holotype female. ADULT: Exactly as the neotype series of An. minimus described by Harbach et al. (2006), except as follows. Head: Proboscis length about 1.6 mm. Maxillary palpus 1.5 mm long, apical pale band slightly longer than preapical dark and pale bands of equal length. Thorax: Pleura with 2 prespiracular, 2 prealar, 2 upper mesokatepisternal and 5 upper mesepimeral setae. Wing: Length 2.9 mm; costa with humeral pale spot in addition to presector pale, sector pale, subcostal and preapical pale spots, sector and accessory sector pale spots of R fused; distal 0.25 of 1A pale-scaled; pale fringe spots fused at apices of R3 and R4+5 (total of 6 pale fringe spots). PUPAL EXUVIAE: Habitus and chaetotaxy as described and illustrated for An. minimus by Harbach et al. (2006), except as follows; number of branches of setae in table 1. Cephalothorax: Seta 4-CT with 7/5 branches (1–3 in An. minimus ) [ Harrison (1980) observed 7-10 branches in specimens from Thailand that may have included An. harrisoni ]1; 8-CT forked on one side (single in An. minimus ). Trumpet: Length 0.35 mm, meatus 0.10 mm, pinna 0.35 mm. Abdomen: Length 2.33 mm. Seta 5-I with 1/2 branches (2–4 in An. minimus ); 7-I with 6/7 branches (4–6 in An. minimus ) [3–7 branches in Thai specimens]; 9-I with 6/8 branches (2–5 in An. minimus ); 0-IV with 5/4 branches (2–4 in An. minimus ) [1–6 branches in Thai specimens]; 4-IV with 3/5 branches (2,3 in An. minimus ) [1–6 branches in Thai specimens]; 8,10,11-II absent. Genital lobe: Longer, length 0.20 mm. Paddle: Length 0.67 mm, width 0.43 mm, index 1.56; marginal serrations begin 0.15 from base and end 0.47 from base; refractile index 0.31. LARVAL EXUVIAE (fourthinstar): Habitus and chaetotaxy as described and illustrated for An. minimus by Harbach et al. (2006), except as follows; number of branches of setae in table 2. Head: Width 0.55 mm, length 0.59 mm. Seta 7-C with 19/

1. From this point onward, information in square brackets refers to data of Harrison (1980).

15 branches (14–19 in An. minimus ) [15–20 in Thai specimens]; 8-C with 8/9 branches (4–7 in An. minimus ) [5–10 in Thai specimens]; 8-P with 35/? branches (31–34 in An. minimus ) [27–38 in Thai specimens]. Antenna: Length 0.23 mm. Thorax: Mesothorax without pair of submedian notal plates. Seta 8-P with 35/? branches (31–34 in An. minimus ) [27–38 in Thai specimens]; Abdomen: Segments I–VII with distinct submedian accessory tergal plates. Seta 0-III with 3/2 branches (1,2 in An. minimus ) [1–3 in Thai specimens]; 1- III,V with 23/22 and 22/20 branches, respectively (16–22 and 16–21 in An. minimus ) [17–25 and 17–22 in Thai specimens]; 4-I,II with 9/9 and 7/9 branches, respectively (both 4–7 in An. minimus ) [4–8 and 5–9 in Thai specimens]; 5-VI,VIII with 8/9 and 7/7 branches, respectively (9–13 and 4–6 in An. minimus ) [9–12 and 5–7 in Thai specimens]; 6,7-I both with?/34 branches (23–31 and 22–33 in An. minimus ) [26–34 and 26–33 in Thai specimens]; 7-II with 36/39 branches (22–33 in An. minimus ) [24–36 in Thai specimens]; 7-IV with 10/7 branches (5–7 in An. minimus ) [4–8 in Thai specimens]. Pecten plate with 13/12 spines. Saddle length 0.23 mm. Seta 4-X with 4–14 branches (4–13 in An. minimus ).

nc = not counted.

Systematics. Anopheles harrisoni , like An. minimus , is very similar to three other species of the Myzomyia Series that occur within its range of distribution in the Oriental Region, i.e. An. aconitus , An. fluviatilis James and An. varuna (see e.g. Van Bortel et al., 2001). As pointed out by Harrison (1980), no morphological characters are completely reliable for distinguishing the adults of these species. Furthermore, the adults of An. pampanai are also often misidentified as An. minimus , and hence An. harrisoni , because the distinguishing features of the wings are not easily discerned. Consequently, adults of An. harrisoni (as well as those of other members of the Minimus Complex), cannot be distinguished from the adults of these species with certainty without associated larval and pupal exuviae. The morphological characters in the identification keys of Harrison (1980) and Rattanarithikul et al. (2006) that distinguish An. minimus from the closely related species also distinguish An. harrisoni from those species. However, because of the uncertainties associated with morphological differentiation, the various types of molecular assays developed by Sharpe et al. (1999), Va n B o rt el et al. (2000), Kengne et al. (2001), Phuc et al. (2003) and Garros et al. (2004a, b) should be used for the unequivocal identification of An. harrisoni (= their An. minimus species C).

*Range of branches for individual setae (9 pairs).

Bionomics. Little specific bionomical information is available for An. harrisoni because this species was not distinguished from An. minimus during the course of ecological and epidemiological studies conducted before, and even after, the advent of molecular methods of identification. The trophic behaviour and seasonality of An. harrisoni (as An. minimus C) has been examined in northern Vietnam and western Thailand ( Rwegoshora et al., 2002; Van Bortel et al., 2004; Trung et al., 2005, Sungvornyothin et al., 2006b) where this species occurs in sympatry with An. minimus , but its larval ecology and biology are still unknown. Both species may inhabit the same larval habitats (Garros et al., 2006). In northern Vietnam (Hoa Binh Province), adults of An. harrisoni were particularly abundant in October during the dry season ( Van Bortel, 2002; Garros et al., 2006). In western Thailand, a two-year survey showed that populations of the species peaked in April– June and November–December, which corresponds with the beginning and the end of the rainy season ( Sungvornyothin et al., 2006b). In contrast, an earlier one-year study showed that populations in the same area of Thailand exhibited peak biting density at the end of the rainy season in October–November and a second, smaller peak during the latter part of the dry season in January–March ( Rwegoshora et al., 2002). The density of An. harrisoni was exceptionally high at sites in western Thailand (Ban Phu Toei, Sai Yok District, Kanchanaburi Province) and central Vietnam (Lang Nhot Village, Khanh Phu Commune, Khanh Vinh District, Khanh Hoa Province) ( Rwegoshora et al., 2002; Garros et al., 2005c; Kengluecha et al., 2005; Sungvornyothin et al., 2006b). Two biting peaks were observed indoors in Thailand, one around 1900 h and another after midnight (around 0 100 h) ( Sungvornyothin et al., 2006b). Only the later peak was recorded outdoors. The relative risk of being bitten before 2200 h was higher for An. harrisoni than for An. minimus , which exhibited peak feeding activity after 2200 h in Vietnam ( Trung et al., 2005).

Studies of trophic behaviour have shown that Anopheles harrisoni is more zoophilic than anthropophilic, and is exophagic and exophilic in both northern Vietnam and western Thailand ( Rwegoshora et al., 2002; Van Bortel et al., 2004; Trung et al., 2005; Sungvornyothin et al., 2006b). It will not be surprising if future studies find that the feeding behaviour of An. harrisoni is as highly variable as that of An. minimus , which is known to be an opportunist feeder (Van Bortel et al., 2004; Trung et al., 2005). The vectorial status of An. harrisoni has not been determined, but evidence suggests that it is a major vector of malaria in southern China (Chen et al., 2002).

Species Localities Latitude/longitude Accession no.

28S/D3 ITS2 COI COII Cyt-b (341 bp) (470 bp) (524 bp) (631 bp) (711 bp) Distribution. Current data show that An. minimus is the predominant species of the Minimus Complex in the Oriental Region. It is recorded from northeastern India to eastern China ( Taiwan) and southward from Sichuan Province of China through Laos, Thailand, Vietnam and Cambodia ( Subbarao, 1998; Van Bortel et al., 1999, 2000; Kengne et al., 2001; Chen et al., 2002; Somboon et al., 2005b). In comparison, available records indicate that An. harrisoni has a smaller, patchy distribution in Southeast Asia. Populations have been documented in south-central China (Chen et al., 2002), central Myanmar ( Singh et al., 2006), northern and central Vietnam ( Van Bortel et al., 1999, 2000; Kengne et al., 2001; Garros et al., 2005c) and northwestern Thailand along the Thai-Myanmar border ( Green et al., 1990; Sharpe et al., 1999; Rattanarithikul et al., 2006; Singh et al., 2006; Sungvornyothin et al., 2006a, b). Whether An. harrisoni occurs in areas between these disjunctive localities, i.e. Laos, central and eastern Thailand, and Cambodia, is unknown.

Etymology. This species is named in honor of Dr. Bruce A. Harrison (Public Health Pest Management, North Carolina Department of Environment and Natural Resources, Winston-Salem, North Carolina) for his many important contributions to our knowledge of Anopheles mosquitoes in the Oriental Region, especially his taxonomic investigations of the Myzomyia Series ( Harrison, 1980) that provided the foundation for integrated morphological and molecular studies of this medically important group of insects.

Type series. One hundred and sixty-five specimens from 2 progeny broods (18 females [Ψ], 17 males [ɗ], 65 larval exuviae [Le], 65 pupal exuviae [Pe]). Holotype, Ψ (HB4-14), with LePe on microscope slide, VIET- NAM: Hoa Binh Province, Tan Lac District, Phu Cuong Commune, village of Khoi, 10.ix.1999 ( NIMPE staff) ( BMNH). Paratypes, 17ɗLePe (HB4-11, -13, -15, -17 through -20; HB5-5, -8, -9, -11 through -14, -17 through -19), 17ΨLePe (HB4-2, -4 [head and cibarium on SEM stub], -5, -7, -9, -10, -12 [head and cibarium on SEM stub]; HB5-1 through -4, -6, -7, -10, -15 [head and cibarium on SEM stub], -16, -20 [head and cibarium on SEM stub]), 65LePe (HB4-3, -21 through -41; HB5-21, -24 through -30), same data as holotype ( BMNH).