Nyx pholeocola Linton & Harbach

Harbach, Ralph E., Kitching, Ian J., Culverwell, C. Lorna, Howard, Theresa M. & Linton, Yvonne-Marie, 2013, Nyx pholeocola, a new genus and cavernicolous species of tribe Aedini (Diptera: Culicidae) from southern Thailand based on morphological and molecular data, Zootaxa 3683 (2), pp. 159-177 : 162-164

Nyx pholeocola Linton & Harbach , sp. n.

Female ( Figs 2 , 3 ). As described for genus, bearing the following features and specific characters listed in the Appendix. Dark scaling light to dark brown, pale scaling white. Head: Dorsum with large semi-erect pale falcate scales and few broad spatulate scales laterally on one or both sides, relatively few pale erect scales that become darker (brown) laterally. Antenna length about 2.0 mm; pedicel with small pale spatulate scales and flagellomere 1 with few small dark spatulate scales on mesal surfaces. Proboscis (except labella) and maxillary palpus ( Fig. 2 D) dark-scaled; proboscis length about 1.8 mm, slightly longer than forefemur; maxillary palpus bare beneath, with relatively few setae on dorsal and lateral surfaces, length 0.3 mm. Thorax: Integument light brown, darker dorsally. Scutum ( Fig. 2 B) with pattern of coarser pale scales on background of finer dark scales, pale scaling as follows: acrostichal line bifurcating into lateral prescutellar lines, lateral patch on scutal fossa continuous with anterior dorsocentral area and posterior fossal line that joins posterior dorsocentral line and narrow line on margin of supraalar area; scutellum with pale falcate scales on mid and lateral lobes; brown setae on antepronotum and posteriorly on postpronotum, golden to golden-brown setae on upper proepisternum, postspiracular area, upper and lower mesokatepisternal areas, prealar area, upper mesepisternal area and 1 or 2 (usually 2) on lower anterior area of mesepimeron; pale falcate scales on postpronotum and few scattered pale falcate scales on postpronotum, pale spatulate scales on upper proepisternum, upper and lower posterior mesokatepisternum and upper anterior mesepimeron anteroventral to upper mesepimeral setae. Wing: Length about 3.0 mm, entirely dark-scaled. Halter : Integument pale, capitellum darker dorsally with faint pale scales. Legs: Anterolateral surface of forecoxa with pale spatulate scales and prominent golden-brown setae, midcoxa with pale spatulate scales on anterior side of midlateral row of prominent setae, hindcoxa without scales, with posterolateral row of prominent setae; ventral surface of trochanters with pale scales and setae distally; femora with distinct apical pale rings (knee spots) and subdued posteroventral pale stripes extending to or near apex; forefemur length about 1.7 mm. Abdomen: Terga II–VIII with relatively narrow straight basal pale bands, often narrower medially on terga VII and VIII and occasionally obsolescent medially on tergum VIII; sterna dark-scaled from segment II posteriorly. Genitalia ( Fig. 3 ): Segment VIII fully retracted into segment VII ( Fig. 2 F); tergum VIII broad anteriorly, index about 0.7, relatively long stout setae on caudal margin, sparse short setae anterior to these, basolateral seta present, broad scales intermixed with setae; sternum VIII broadened laterally at midlength, index about 0.6, median posterior emargination separating broad lateral lobes, setae on caudal 0.67 progressively longer toward margin of lobes, setae 1–3-S in posterolaterally directed lateral line, basolateral seta present; tergum IX index about 0.6, caudal margin with broad median concavity separating setose lobes; upper and lower vaginal lips relatively broad, moderately pigmented; postgenital lobe (PGL) somewhat heart-shaped, middle of posterior margin slightly concave and lateral margins broadly convex, ventral length about 0.06 mm, width at midlength about 0.08 mm; cercus with more or less straight mesal margin and curved outer margin, broad in proximal half and progressively narrower to blunt apex, distal area of dorsal and lateral surfaces with scattered setae, length about 0.16 mm, greatest width about 0.08 mm, index about 2.0, cercus/dorsal PGL index about 3.2; spermathecal capsules with few small pores near orifice.

Male. Generally smaller but otherwise similar to the female excepting obvious sexual differences. Head: Antenna with strongly developed whorls of numerous long setae projecting dorsally and ventrally, 2 terminal flagellomeres disproportionately long compared to other flagellomeres; length about 1.7 mm. Proboscis with indication of false joint 0.6 from base. Maxillary palpus ( Fig. 2 C, E) about 0.4 length of proboscis, slender, darkscaled, palpomeres 1–3 without scales ventrally, apex of palpomere 3 with few short setae, palpomere 4 bud-like. Wing: Generally paler, veins with fewer scales, tertiary fringe scales absent; length about 2.4 mm. Abdomen: Tergum and sternum VIII with straight posterior margins; sternum VIII (post-rotation dorsal position) mainly palescaled with incomplete posteromedian dark band; tergum VIII mainly dark-scaled. Genitalia ( Fig. 3 ): Tergum IX lobes weakly produced, narrowly joined medially by membrane, small setae extend laterally along posterior margin from apex of each lobe; sternum IX about as long as broad, with 1–3 small centrally located setae; gonocoxite elongate, more or less cylindrical, mesal membrane developed to apex, basal dorsomesal lobe slightly produced with dense cluster of relatively long setae, dorsal surface with short setae, lateral and ventral surfaces with long setae and spatulate scales, apicodorsal lobe weakly developed with patch of setae; gonostylus long, about 0.75 length of gonocoxite, narrow proximally, dorsoventrally flattened and laterally expanded in distal 0.67, lateral margins of expanded portion lined with relatively long setae to apex; gonostylar claw relatively long, slightly longer than 0.1 length of gonostylus, dorsoventrally flattened and slightly bent ventrad; claspette relatively slender and slightly compressed, with 1 stout apical seta and a small dorsomesal seta at its base; proctiger about 0.25 length of gonocoxite, paraproct distally tapered and bent tergally, without apical teeth; cercal sclerite poorly defined, more or less membranous, fused with paraproct; aedeagus with lateral plates bent tergally, joined distally, tergolateral margin of each lined with short teeth to apex; paramere nearly as long as aedeagus, articulated with apex of basal piece near midlength.

Pupa ( Fig. 4A, B ). As described for genus, bearing the following features and specific characters listed in the Appendix; character and positions of setae as illustrated. Cephalothorax: Moderately but unevenly pigmented, scutum darker posteriorly. Setae 1–3,6,11-CT single; setae 4,5,7–9,10,12-CT branched. Trumpet: Moderately and evenly pigmented; length about 0.5 mm, tracheoid weakly developed on one side of trumpet, length about 0.08 mm, pinna about 0.13 mm, width at midlength about 0. 0 8 mm, index about 6.3. Abdomen: Moderately pigmented, posterior area of tergum I and anterior fold lines of sterna II–VIII darker; length about 3.2 mm. Seta 1-I broom-like, with short thick stem and numerous (approximately 70) thin simple branches; setae 3,6-I long, single; seta 11-I usually present, single; alveolus of seta 13-I usually present; seta 1-II multiple branched, 1-III–VII with fewer branches (usually 2) on succeeding posterior terga; seta 6-II–VI relatively long, usually single, sometimes double on segments II and III; seta 10-II usually absent; seta 9-VIII with 2–4 long aciculate branches, nearly half length of paddle. Genital lobe: Moderately pigmented; long in male, length about 0.3 mm, about 0.8 length of paddle; about 0.1 mm in female. Paddle: Lightly and evenly pigmented, midrib darker, midrib distinct to distal area before seta 1- Pa; length about 0.8 mm, width about 0.6 mm, index 1.3; seta 1-Pa long, single.

Larva, fourth-instar ( Fig. 5 ). As described for genus; bearing the following features and specific characters listed in the Appendix; character and positions of setae as illustrated (larva reconstructed from larval exuviae, relative positions of setae may differ slightly from those shown). Head: Nearly round in dorsal view, length and width about 0.8 mm; moderately pigmented, collar darker. Dorsomentum with 7–9 teeth on either side of slightly larger median tooth, teeth nearest median tooth abutted, lateral 2 or 3 teeth separated. Setae 5,6,8–10-C single. Antenna: Moderately pigmented, bent mesad, length about 0.3 mm; seta 1-A inserted at mid-length, setae 2,5-A very long, 2-A nearly twice length of 5-A ( Fig. 5 B). Thorax: Integument hyaline, smooth. Seta 1-P double or triple; setae 3,8-P single or double; setae 14-P,M and 13-T more strongly developed (longer) than in Isoaedes, 14-P double, 14-M double or triple, 13-T with 4 or 5 branches; setae 13-M and 8-T double or triple. Abdomen: Integument hyaline, smooth. Seta 3-I–VII normally single, moderately long, all longer than seta 4; seta 6-I–VI with long, stout, aciculate branches, 6-VI noticeably shorter than 6-I–V; seta 2-I,II,VII anterolateral to seta 1, 2-III–VI more or less directly anterior to seta 1; seta 8-II–VI normally single; seta 10-II–VI approaches length of following segment; seta 13-III,IV single or double, longer than following segment. Segment VIII: Comb comprised of 6–9 spine-like scales in single row, with minute basolateral denticles. Siphon: Moderately pigmented, slightly swollen at level of seta 1-S, surface smooth; length about 0.9–1.0 mm, width at base about 0.3 mm, index 3.0–3.3; acus present, very small, detached from siphon; pecten with 10–12 spines with few denticles basally on ventral margin, distal 1 or 2 spines more widely spaced; seta 1-S branched, inserted more or less at level of distal pecten spine. Segment X: Saddle moderately pigmented; dorsal length about 0.3–0.4 mm; siphon/saddle index 2.3–3.3. Seta 1-X single, simple, slightly shorter than saddle; setae of ventral brush with 2 or 3 branches, mostly 3-branched, precratal seta single, simple. Dorsal and ventral anal papillae same length, short and relatively stout, slightly longer than saddle.

Egg. Unknown.

DNA sequence. ITS2 and COI fragments were amplified from two specimens (SS74-19 and SS74-40, see type series below). The sequences of the ITS2 fragments were identical. These fragments (primers removed), which also include a short portion of the flanking 5.8S and 28S genes, were 287 bases long and 50.87% AT rich (65A, 81T, 74C, 67G). The sequence shares greatest similarity (94%) with that of Borichinda cavernicola Rattanarithikul & Harbach (GenBank accession EF370410 ), reflecting the cladistic relationship noted above. The COI barcode fragments (658 bp) exhibited no intraspecific variation and the haplotype was 68.38% AT rich (184A, 266T, 115C, 93G). Despite the presence of COI sequence for Bc. cavernicola in GenBank ( Harbach et al., 2007), the COI gene of Nx. pholeocola proved to be most similar (89%) to that of Stegomyia albopicta (Skuse) ( HQ398902 ), which emphasizes the need for further phylogenetic investigations before the true affinities of Nyx are resolved. Future studies should also include DNA sequences for Isoaedes cavaticus (Reinert).

Etymology. The name pholeocola is a Latin noun (masculine or feminine) derived from pholeos, meaning hole, cave or den, and - cola, meaning dweller or inhabitant. It was chosen because the species was discovered in and is only known to inhabit the cave at Wat Tham Phanturat in Khlong Sok, near Khao Sok National Park in southern Thailand (see below). The specific name is feminine in agreement with the gender of Nyx .

Bionomics. Larvae and pupae of Nx. pholeocola were collected from a rimstone pool (20 x 50 cm) in Wat Tham Phanturat cave, Khlong Sok, near Khao Sok National Park in Surat Thani Province in southern Thailand. The water in the pool was clear and cold, 10 cm deep and devoid of vegetation and plant matter. The cave harbours an enormous number of bats that are likely to be the primary source of food for adult females. Females, however, avidly attacked collectors inside the cave; thus, Nx. pholeocola may have the potential to opportunistically transmit zoonotic pathogens between bats and humans.

Distribution. Nyx pholeocola is only known from the cave at Wat Tham Phanturat Khlong Sok, near the Khao Sok National Park in the Surat Thani Province of southern Thailand ( Fig. 4 C).

Type series. Eighty-six specimens: 21 Ƥ, 2 Ƥ genitalia, 11 3, 4 3 genitalia, 16 fourth-instar larval exuviae [Le] and 35 pupal exuviae [Pe]. Holotype, 3 (SS74-24), with LePe on microscope slide, THAILAND: Surat Thani Province, Khlong Sok (near Khao Sok National Park), Wat Tham Phanturat cave (08º 53' 59.6" N, 98º 31' 31.2" E), shallow rimstone pool (20 x 50 cm), 23.i.2008 (Y.-M. Linton et al.). Paratypes (same data as holotype except specimens of collection SS75 captured landing on humans in cave; specimens with dissected genitalia on microscope slides are indicated with an asterisk*), 3Ƥ (SS75-1, -3, -4); 7 ƤPe (SS74-2, -4, -5 [dissected head on microscope slide], -6*, -8, -10, -13*); 11 ƤLePe (SS74-23, -25 -35, -36, -37, -38, -39, -40 [Ƥ used for DNA extraction; COI ( KC860482 , body; KC860484 , leg only), ITS2 ( KC860486 )] -41, -42, -46 [LePe of -35 to -46 retained in 80% ethanol]); 73Pe (SS74-3*, -7*, -17, -19 [3 used for DNA extraction: COI ( KC860483 ), ITS2 ( KC860485 )], -20*, -22*, -31); 3 3LePe (SS74-9 [dissected head on microscope slide], -26, -43 [LePe retained in 80% ethanol]); 7 Pe (SS74-11, -14, -15, -27, -28, -29, -30). All specimens are deposited in the Natural History Museum, London.

Figures

FIGURE 2. Anatomical elements of adult females and males of Nyx pholeocola. A, Thorax (left side) of a female. B, Mesonotum (dorsal) of a female. C, Head (left side) of a male showing the short maxillary palpi. D, Maxillary palpi (dorsal) of a female. E, Left maxillary palpus (dorsal) of a male. F, Terminal abdominal segments (dorsal) of a female (segment VIII is fully retracted into segment VII). Ce = cerci; Pl = palpifer; MPlp = maxillary palpus; 1 – 3 = palpomeres; V – VII = abdominal segments.

FIGURE 3. Genital structures of Nyx pholeocola (aspects as indicated). A – I, Male genitalia: A, with right gonocoxopodite removed; B, C, gonocoxopodite; D, tergum IX; E, F, proctiger; G, aedeagus with parameres; H, claspettes; I, sternum IX. J, K, Female genitalia: J, principal parts; K, tergum IX. Ae = aedeagus; BP = basal piece; Ce = cercus; Cl = claspette; Gc = gonocoxite; GC – gonostylar claw; Gs = gonostylus; I = insula; Par = paramere; PGL = postgenital lobe; Ppr = paraproct; X-Te = tergum X. Scales bars = 0.1 mm (A – C, J) and 0.05 mm (D – I, K).

FIGURE 5. Fourth-instar larva of Nyx pholeocola (reconstructed from larval exuviae; positions of some setae may differ slightly from those illustrated). A, Head, dorsal (left) and ventral (right) aspects of left side. B, Apex of antenna with setae 2 – 6 - A. C, Thorax and abdominal segments I – VI, dorsal (left) and ventral (right) aspects of left side. D, Abdominal segments VII – X, left side. A = antenna; C = cranium; CS = comb scale; Dm = dorsomentum; M = mesothorax; P = prothorax; PS = pecten spine; S = siphon; Sa = saddle; T = metathorax; I – VIII, X = abdominal segments I – VIII and X; 0 – 15, 18 = setal numbers for specified areas, e. g. seta 5 - C.

FIGURE 4. A, B, Pupa of Nyx pholeocola. A, Left side of cephalothorax, dorsal to right. B, Dorsal (left) and ventral (right) aspects of metathorax and abdomen. C, Outline map of Thailand and Surat Thani Province showing the location of the cave at Wat Tham Panthurat. CT = cephalothorax; GL = genital lobe; Pa = paddle; T = trumpet; I – VIII, X = abdominal segments I – VIII and X; 0 – 11, 14 = setal numbers for specified areas, e. g. seta 1 – II.