Zootaxa, Description of a New Species

Description of a new species, Anopheles pseudosundaicus (Diptera: Culicidae) from Kerala, India

B.K. TYAGI1, J. HIRIYAN, S.C. TEWARI, K. AYANAR, P. PHILIP SAMUEL, N. ARUNACHALAM, R. PARAMASIVAN, R. KRISHNAMOORTHY, K.J. DHANANJEYAN, S. VICTORJERALD LEO, & R. RAJENDRAN Centre for Research in Medical Entomology (ICMR), 4-Sarojini Street, Chinna Chokkikulam Madurai – 625002, TN, India 1Corresponding author. E-mail: [email protected]; [email protected]

Abstract

Specimens of Anopheles pseudosundaicus, new species, belonging to the Pyretophorus Series of Anopheles (Cellia), were collected from the coastal areas of Alleppey and Kollam districts in Krerala, southwest India. Female (holotype), male (allotype), pupa and fourth-stage larva of this species are described and illustrated to distinguish it from An. subpictus Grassi and An. sundaicus. (Rodenwaldt). It differs from An. sundaicus by the absence of speckling on legs and from An. subpictus in having a complete prehumeral dark spot on costa in wings. A partial sequence of Cytochrome oxidase C subunit 1 (CO1) of mt DNA places An. pseudosundaicus phylogenetically distant from An. sundaicus and closer to An. subpictus.

Key Words: Anopheles pseudosundaicus, Culicidae, Kerala, India

Introduction

Fifty eight species of Anopheles have so far been recorded in India (Knight & Stone 1977; Walter Reed Biosystematics Unit 2001) and as many as ten species are considered primary or secondary vectors of malaria (Roy et al. 1978, Nagpal & Sharma 1995, Tyagi & Yadav, 2001, Tyagi 2002, 2004). The last endemic species, An. pinjaurensis Barraud was discovered from India more than seven decades ago (Christophers 1933). In India only a few species, e.g., An. subpictus Grassi and An. sundaicus (Rodenwaldt), have been reported breeding in the fresh and/or brackish water of coastal areas (Covel 1944, Reid 1968). The coastline of the Indian peninsular region is a rich, yet little explored habitat for mosquitoes. Studies initiated after the tsunami on December 26, 2004 revealed a new mosquito species, Anopheles pseudosundaicus, belonging to the Pyretophorus Series of Anopheles (Harbach 2004). This species was encountered in Kerala State, southwestern India. Although, the new taxon seems to be close to An. subpictus and An. sundaicus, nevertheless, it differs distinctly from either of them both in morphological (adult and immature) and molecular characteristics. In this paper we describe this new species, morphologically and molecularly using mtDNA sequence, to distinguish it from An. subpictus and An. sundaicus. The terminology used as well as abbreviations of generic and subgeneric names are followed from various references (Reinert 1975, Harbach & Knight 1980, 1982, Bickley & Ward 1989).

Accepted by G. Courtney: 17 Jul. 2009; published: 3 Sep. 2009 49 Materials and methods

Larval specimens were collected from the tsunami-hit coastal localities of Kuzhithura, Kollam District and Valiazheekal, Alleppey District, Kerala State in southwestern India, from January to July, 2005. Samples were individually reared to adult stage for species identification using keys by Christophers (1933) and Reid (1968). Associated larval and pupal exuviae were examined for chaetotaxy analysis for which abbreviations used in text are Le = larval exuviae; Pe = pupal exuviae. To plot the genetic variation, individual adult mosquito specimens of An. subpictus and An. pseudosundaicus (CRME Museum Ref #A605) were used for isolating mtDNA, while comparative molecular characteristics for An. sundaicus were obtained from GenBank. DNA was isolated using “DNA Isolation Solution” kit (Genei, Bangalore, India) following manufacturer’s instructions, with slight modification. A polymerase chain reaction (PCR) was set to amplify the subunit 1 of cytochrome oxidase gene of the mtDNA using GeNeiTM PCR Master Mix (Genei, Bangalore, India) following manufacturer’s instruction. The universal CO1 primers C1-J-1718 (5’ – GGA GGA TTT GGA AAT TGA TTA GTT C – 3’) and C1-N-2191 (5’ – CCC GGT AAA ATT AAA ATA TAA ACT TC – 3’) were used following the thermal cycling of initial denaturation at 94oC for 10 min., followed by 40 cycles of denaturation at 94oC for 30 sec., annealing at 50oC for 30 sec., and elongation at 72oC for 1 min. followed by final elongation at 72oC for 10 min. The thermal cycling was carried out in Master cycler (Eppendorf, Germany). The amplicons were visualized in a 1.2% agarose gel stained with ethidium bromide. The PCR amplicon was purified by ultrafiltration (Millipore, India). The purified PCR product was sent for custom sequencing service (MWG Germany, India Office, Bangalore city, India). DNA sequencing was performed using Big Dye terminator reaction Kit in ABI, Foster City, CA, USA. To determine the sequence identity, the sequences were subjected to BLAST analysis (Altschul et al., 1990) CLUSTAL W analysis (Thompson et al., 1994; Higgins et al., 1996) was used for multiple sequence alignment. The evolutionary history was inferred using the Neighbor-Joining method (Saitou & Nei 1987). The bootstrap consensus tree inferred from 1000 replicates is taken to represent the evolutionary history of the taxa analyzed (Felsenstein 1985). The tree is drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree. The evolutionary distances were computed using the Kimura 2-parameter method (Kimura 1980) and are in the units of the number of base substitutions per site. All positions containing gaps and missing data were eliminated from the dataset (complete deletion option). There were a total of 363 positions in the final dataset. Phylogenetic analyses were conducted in MEGA4 (Tamura et al. 2007).

Anopheles (Cellia) pseudosundaicus Tyagi et al.

Description. Female (Figs.1 a, b) Head: antenna dark brown about 0.7 length of proboscis (mean 0.7; range 0.58–0.75; n=13); proboscis dark brown, about 0.9 the length of maxillary palpi (range 0.75–0.99; n=13); palpus with three pale bands; apical pale band little shorter than the subapical dark band; subapical pale band very narrow, about 1/5 the length of subapical dark band. Vertex covered with broad dark brown scales and erect white scales, frontal setae white, large and bifurcate. Thorax: antepronotum and postpronotum without scales; propleuron with 2 or 3 setae; pleura with broad pale scale patches on mesepimeron and mesokatepisternum; coxae with a patch of broad pale scales. Abdomen: abdominal segments covered with numerous golden setae, the last two segments intermixed with few narrow yellow scales also; terga I–VIII each covered with broad pale scales and few dark brown ones posteriorly; sterna I–VIII each covered with broad dark brown scales and pale scaled patch basally. Legs: femora I, II mainly covered with dark brown scales with few scattered pale scales; femur III largely covered with pale scales anteriorly; tibae I–III dark brown scaled with a narrow stripe of pale scales; tarsi I–III mainly with dark brown scales; fore tarsi with tarsomere 1 with a narrow apical pale band, tarsomeres 2–4 with both basal and apical pale bands, tarsomere 5

50 · Zootaxa 2219 © 2009 Magnolia Press TYAGI ET AL. FIGURE 1. A. Adult Female palpus (left), and proboscis (right); B. Wing; C. Male genitalia, phallosome; D. Male genitalia, claspette. completely dark; mid-tarsi with tarsomeres 1–4 with narrow basal and apical pale bands; tarsomere 5 dark; hind tarsi with tarsomeres 1–4 with narrow apical bands, tarsomere 5 completely dark. Wings: mainly dark scaled, about 3mm in length; prehumeral usually completely dark scaled, occasionally interrupted with few

AN. PSEUDOSUNDAICUS SPEC. NOV. FROM INDIA Zootaxa 2219 © 2009 Magnolia Press · 51 pale scales, costa with the following pale spots: humeral, presector, sector, subcostal, and preapical; dark spots other than humeral and preapical are large; pale fringe spots at the end of all veins and rarely a feebly pale fringe spot between 1A and base of wing.

FIGURE 2. A. Pupal abdomen; B. Pupal trumpet.

Male (Figs. 1 c, d). General morphological characteristics of body including wings and legs are similar to those of the female. Genitalia (n=12): phallosome with 6 pairs of leaflets, smaller ones spiny form, the longest ones blade-shaped; claspette with a long apical spine (mean 0.27mm; range 0.18–0.31) which is more than

52 · Zootaxa 2219 © 2009 Magnolia Press TYAGI ET AL. twice the length of the club (mean 0.12mm; range 0.09–0.14), 2 small spines one about 1/2 (mean 0.05mm; range 0.03–0.06) and another about 1/3 (mean 0.04mm; range 0.03–0.05) length of the club, arising between the apical spine and the club.

FIGURE 3. A. Fourth-instar larva, head; B. Fourth-instar larva, thorax; C. Fourth-instar larva, pecten plate

Pupa (Figs. 2 a, b; Table 1). Cephalothorax: moderately pigmented; Trumpet: heavily pigmented with scattered spicules, index 2.34–2.83, mean 2.68 (sd ± 0.23), meatus about 0.40 of the trumpet length. Abdomen; Seta 7-I 2-5(3) branched, shorter than 6,9-I Paddle: width about 3/4 of its length, index 0.7–0.8; refractile border about 4/5 of paddle length (index 0.8) with small clearly defined teeth, fringed hairs on posterior border with hooked tips; 1-P long and hooked; 2-P bifid.

AN. PSEUDOSUNDAICUS SPEC. NOV. FROM INDIA Zootaxa 2219 © 2009 Magnolia Press · 53 Larva (Fig. 3 a, b, c; Table 2). Head: setae 1-4-C slender and single, 2-C about twice the length of 3-C; 4- C about equal length of 3-C. Thorax: setae 1-P, 7-15 branched; setae 9-12-P all single; seta 1-M with 25–30 branches, 4-M single, rarely bifid ; setae 9-12-M all single; seta 3-T poorly developed with 3-5 branches. Abdomen: seta 1-I (palmate hair) weakly developed with 4–9 leaflets, seta 1-II-VII (palmate hair) well developed, evenly pigmented, filaments sharply differentiated, about twice the length of the blade; tergal plates small to moderate sized, smallest on abdominal segment II; seta 1-VIII single to 7 branched; pecten plate with 12–15 (mean 12) uneven teeth.

54 · Zootaxa 2219 © 2009 Magnolia Press TYAGI ET AL. Type material. Holotype female (coded A #538) with associated larval (Le #254) and pupal (Pe #256) exuviae mounted on slides with following collection data:- India: Kerala State, Kollam District, Kuzhithura, 22 January 2005, collected as larva from brackish water pool, G. Baskaran collector, deposited in the Museum of Centre for Research in Medical Entomology (MCRME), Indian Council of Medical Research, Madurai, India. The allotype male (A #543) with associated larval (Le #259) and pupal (Pe #261) exuviae with collection data same as holotype also deposited in MCRME, Madurai. Paratypes (36 males, 72 females, pinned and 78 Le and 78 Pe mounted on slides) with the same collection data as holotype are also deposited in MCRME. Distribution: Based on collections from two coastal localities in Kerala, south India, a total of 302 specimens of An. pseudosundaicus were examined: Kuzhithura (Kollam district), brackish waterpool, 8 males, 16 females, 11 Le and Pe , 22 Jan. 2005; Valiazheekal (Alleppey District), brackish waterpool, 28 males, 58 females, 5 male genitalia, 69 Pe 27 Le, 23 Jan. 2005 and 21 Jul 2005. Bionomics: Larvae of An. pseudosundaicus were collected in brackish water pools associated with floating algae, from coastal localities of Kollam and Alleppey Districts, Kerala State, southwest India. Culex (Culex) sitiens Wiedemann was the only mosquito species found co-breeding with An. pseudosundaicus.

FIGURE 4. The gel picture showing amplification of ~565bp of CO1 region from An. pseudosundaicus (#A605) and An. subpictus. L1—Molecular weight marker; L2—CO1 amplification from An. pseudosundaicus (#A605); L3—CO1 amplification from An. subpictus.

AN. PSEUDOSUNDAICUS SPEC. NOV. FROM INDIA Zootaxa 2219 © 2009 Magnolia Press · 55 FIGURE 5. Phylogenetic analysis of An. pseudosundaicus (#A605) species based on partial sequence of Cytochrome oxidase C subunit 1 gene of mt DNA.

Diagnosis. Adult Anopheles pseudosundaicus differ distinctly from those of An. sundaicus due to the absence of leg speckling. Anopheles pseudosundaicus differs from An. subpictus in wing markings; the former

56 · Zootaxa 2219 © 2009 Magnolia Press TYAGI ET AL. having a complete prehumeral dark spot whereas in An. subpictus this dark spot often is divided by pale scales (Tyagi 1984). The mid tarsus in An. pseudosundaicus bears narrow apical and basal bands whereas that of An. subpictus has only apical bands. The male genitalia of An. pseudosundaicus has two spines near the base of club of the claspette compared to the single spine in An. subpictus and 1–3 spines in An. sundaicus. The larva of An. pseudosundaicus differs from that of An. subpictus in having 4-M single compared to bifid in the latter species. The pupae of An. pseudosundaicus and An. subpictus can be distinguished by seta 7-I: 2-5(3) branched and shorter than 6,9-I in An. pseudosundaicus vs. simple and as long as 6,9-I in An. subpictus (Table 3). Etymology. This species is named based on its similarities to An. sundaicus particularly its morphological characteristics and breeding habitat in brackish waters. Remarks. The specimens used in this study comprised field collected larva and pupa as well as adults reared from immatures from tsunami waters in coastal Kerala, India.

Discussion

With the inclusion of An. pseudosundaicus, there are a total of 24 described species in the Anopheles (Cellia) Pyretophorus Series (Harbach 2004). The series is characterized by a set of characters which include: (i) Adult—maxillary palpus with three pale bands, propleuron with 2,3 setae, pronotal lobes without scales, banded tarsi, tarsomere 5 all dark; Pupa—seta 1-V-VII long, seta 9-V-VII long, curved and tapering, seta 1-P long and hooked; Larva—setae 1,2-C simple, setae 1,2,3-P with separate roots, seta 1-I (palmate hairs) with sharply defined filaments, tergal plates medium sized. Anopheles pseudosundaicus possesses Pyrotophorus Series’s characteristics, but is distinguished from its nearest allies, An. subpictus and An. sundaicus, by specific characteristics of the larva, pupa and adult. In Southeast Asia, particularly in Sulawesi and the south coast of Java, An. subpictus, is known to have four cytotypes A,B,C and D (Baimai et al. 1996) and is a coastal species of minor malariological importance (Ramachandra Rao 1984; Sundararaman et al. 1957; Suguna et al. 1994). All four cytotypes occur in India, but only An. subpictus B breeds in brackish water and transmits malaria as a secondary vector (Panicker et al. 1981; Suguna 1982; Reuben & Suguna 1983). In the neighbouring island country, Sri Lanka, An. subpictus B is restricted to coastal localities and plays a major role in the transmission of malaria (Abhayawardana et al. 1966). It is well documented that members of the Pyretophorus Series are chiefly open pool breeders with a tendency to produce forms adapted to salt water which obfuscates species identification (Reid 1968). Interestingly, An. subpictus has also been demonstrated to transmit Japanese encephalitis virus as a bridge vector in south India (Thenmozhi et al. 2005). Contrary to the presence of An. subpictus on the mainland India, An. sundaicus is endemic to Andaman & Nicobar islands where it is the only major vector of malaria (Ramachandra Rao 1984; Balaraman et al. 2005). Anopheles pseudosundaicus is generally dark coloured having wing veins largely dark scaled. Unlike An. sundaicus, it has large prehumeral and preapical dark spots on the costa, lacks a pale fringe spot between veins CuA and 1A, and lacks leg speckles. The diagnostic features of An. pseudosundaicus are: (1) the apical pale band on female palpi shorter than the subapical dark band, (2) longer prehumeral and preapical dark spots on costa, (3) absence of pale fringe spot between wing veins CuA and 1A, (4) venter of abdominal segments with pale scale patches, (5) mid-tarsi with narrow apical and basal bands, (6) male claspette with two spines arising between the long apical spine and the club, (7) phallosome with 6 pairs of leaflets, (8) larva with seta 4-M usually single, but rarely bifid, and (9) pupa with seta 7-I usually 3 branched and shorter than setae 6, 9-I (Table 3).

AN. PSEUDOSUNDAICUS SPEC. NOV. FROM INDIA Zootaxa 2219 © 2009 Magnolia Press · 57 Molecular characteristics of An. pseudosundaicus were compared with those of An. subpictus and An. sundaicus. The PCR amplification produced a fragment of ~565 bp (Fig. 4) approximating to the values of both other species. The BLAST (Altschul et al. 1990) search revealed that the CO1 sequences were very close to those of An. subpictus and An. sundaicus. The phylogenetic analysis of An. pseudosundaicus (#A605) showed that this taxon was clustered with An. subpictus, suggesting a rather recent divergence of the former species (Fig. 5). Ironically, An. pseudosundaicus appears morphologically closer to An. sundaicus than An. subpictus, indicating missing links between the new taxon and An. sundaicus. Additional studies are needed to test this hypothesis.

Acknowledgement

We thank the Director General, Indian Council of Medical Research, New Delhi, for facilities and encouragement; and Shriyuts S.P. Kandasamy, K. Pazhaninathan, G. Baskaran, K. Venkatasubramani, A. Venkatesh and K. Moorthi for their field and laboratory assistance including illustration work.

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