Additional data on Spinitectus petterae (Nematoda: Rhabditida) from Clarias gariepinus (Siluriformes: Clariidae) in the Vaal River system: conserved morphology or high intraspecific genetic variability? Author Austin, Lucinda Author Santos, Quinton Marco Dos Author Avenant-Oldewage, Annemariè text Folia Parasitologica 2023 002 2023-01-10 70 1 12 http://dx.doi.org/10.14411/fp.2023.002 journal article 10.14411/fp.2023.002 1803-6465 8143713 Spinitectus petterae Boomker, 1993 Figs. 2–5 Material examined: ten females , ten males and four immature females using LM; five females , six males , 12 sets of spicules and two immature females using SEM. Fig. 5. Scanning electron micrographs of immature female of Spinitectus petterae Boomker, 1993 collected from Clarias gariepinus (Burchell) . A – apical view of cephalic region; B – vulva; C – conical tail end; D – conical tail. Abbrevations : A – anus; CA – caudal papilla; L – labium; MT – mucron tip; PL – pseudolabium. Description (for measurements: see Supplementary Tables S2, S3 and S4). Adult . Specimens small to medium-sized (length 7–12 mm ). Intestine brown, remainder of body white and opaque. Gravid females (9.4–12.0 mm) larger than adult males (7.0– 9.5 mm ). Blunt and rounded cephalic area ( Fig. 2A,C,D ), elongated body, conical tail at posterior end ( Figs. 2H , 3A ). Cuticle surface bears annular spines separated into half-circles by lateral line ( Fig. 2A,B ). First five rings largest, with first two rings closer together. Subsequent spines diminish in size and organisation towards posterior ( Fig. 2G,H ). Fe- males with spines throughout whole body, males only with spines to their midbody ( Fig. 3A ). Elongated dorsoventral oral aperture on cephalic area, flanked by two large, flat la- bia, ventrally and dorsally ( Fig. 2C–E ). Each labium with broad base, smaller, simple, slightly sclerotised and slender sublabium bordering oral opening. Lateral to oral opening, large, broad, pseudolabia project medially, fold into oral opening. Two elongated submedian cephalic papillae interspersed by two lateral amphids at base of each pseudolabium ( Fig. 2E ). No deirids observed. Most specimens display thinwalled, distinctly widened vestibule with posterior prostom. Some specimens (three of nine) showed collapsed vestibule. Telostom present, both relaxed and contracted states observed. Alternate states of vestibule and telostom impacted other morphometric characters such as distance of nerve ring to vestibule and anterior extremity. Oesophagus divided into anterior muscular section (more slender and shorter than wide) and long glandular section. Nerve ring location variable, mostly encircling muscular oesophagus. Excretory pore located at fourth annular ring ( Fig. 2F ). Male. Ten randomly selected males measured (Supplementary Table S3). Total body length 7.0– 9.5 mm , maximum width 100–189. First ring of spines 147–182 from anterior. Number of spines: 39–50 in first, 40–47 in second, 36–48 in third ring. Length of spines 13–16 in first, 15–18 in second, 14–16 in third ring. Two individuals with extra ring of 4 spines between second and third rings. At postoesophageal region, cuticular spines become smaller, sparser, begin to lose annular pattern, absent at posterior ( Fig. 3A ). Vestibule plus prostom length 47–64, uncollapsed vestibule 11–20 long and 14–23 wide, prostom 30–48 long and 6–9 wide. Relaxed telostom 10–17 long and 7–15. Contracted telostom length 5–12 and width 14–28. Relaxed telostom generally longer, thinner than in contracted state. Muscular oesophagus 408–563 long, glandular oesophagus 1.5– 2.7 mm long, ratio of muscular to glandular oesophagus lengths 1: 3.1–5.1. Vestibule plus total oesophagus length 2.0– 3.3 mm , percentage of vestibule plus oesophagus of total body length 24–41%. Nerve ring 117–285 from anterior extremity, 59–339 from vestibule. Excretory pore on fourth ring, 277–379 from anterior extremity, 248–329 from vestibule. Caudal alae absent. Tail 144–191 long, mucron tip terminal, 2–5 long. Rugosa ridges present on ventral surface, 10 rows ( Figs. 3A,B , 4A,C ). Pedunculated preanal papillae in four pairs, postanal papillae in three pairs, distal group of three pairs ( Figs. 3A,C , 4A,C ). Left spicule delicate, with internal supporting rings 0.7–1.0 mm long, shaft length 280–553, left spicule shaft percentage of total spicule 40–53 %. Left spicule covered by spicule pouch; pouch attached to spicular muscle ( Fig. 4A ). Manubrium at base of spicule, vas deferens connected to manubrium ( Figs. 3K , 4A ). Lumen of spicule cytoplasmic core opens at anterior third of spicule, and terminates distally ( Figs. 3E–H , 4A ). Proximal cytoplasmic core opening observed in scanning electron microscopy, resembles arch in LM. Blade of left spicule with V-shaped vellum, twisting around blade, distal cytoplasmic core opening ( Figs. 3G–I , 4B ). V-shaped vellum resembles spine-like structure in LM. Right spicule small, robust 124–162 long, including distended fleshy membrane 131–170 long ( Fig. 3J ). Right spicule shallow, boat-like structure with fleshy tip in undigested SEM spec- imens. Once digested, right spicule structure appears deep- 813447 Spinitectus petterae Tanzania 291616 Spinitectus petterae Egypt OP800410 Spinitectus petterae 1FB96Cg9 OP800407 Spinitectus petterae 2FVD96Cg3 OP800411 Spinitectus petterae 2FVB96Cg9 OP800404 Spinitectus petterae N1G Vaal River system OP800406 Spinitectus petterae 1FVD96Cg7 OP800409 Spinitectus petterae 1MVD96Cg11 OP800408 Spinitectus petterae 1LVD96Cg8 OP800405 Spinitectus petterae N2G OP800412 Spinitectus petterae SPC 1 Crocodile OP800413 Spinitectus petterae SPC 3 River DQ503464 Spinitectus carolini JF803922 Spinitectus tabascoensis MH754719 Spinitectus tabascoensis MG594296 Spinitectus macrospinosus Americas MG594297 Spinitectus gracilis MK341639 Spinitectus mexicanus MK353469 Spinitectus mexicanus Rhabdochona mazeedi MK353468 Rhabdochona xiphophori 0.004 Fig. 6. Phylogenetic relationships of Spinitectus spp. based on available 18S rDNA, based on the Bayesian (BI) method, with Rhabdochona xiphophori Caspeta-Mandujano, Moravec et Salgado-Maldonado, 2001 and Rhabdochona mazeedi Prasad et Sahay, 1965 serving as outgroups. Posterior probability (BI) and 1,000 bootstrap replicate (maximum likelihood (ML)) support indicated (BI/ML), nodes with less than 0.5 (50 %) support not annotated. Data shaded in colour indicate geographical locality or river system. er with sharp ( Fig. 3K,L ), fleshy tip, and hollow cytoplas- mic core opening visible in LM. Female . (10 randomly selected gravid females measured – Supplementary Table S2). Total body length 9.4– 12.0 mm, maximum width 222–292. First ring of spines 170–192 from anterior extremity. Number of spines: 40–50 in first, 39–52 in second, 37–49 in third ring. Length of spines (measured from spine tip to connection with collar – Fig. 2A,B ): 14.0– 16.3 in first, 15.5–18.6 in second, 15.5–17.8 in third ring. Three individuals with extra spine ring with 1–9 spines between second and third rings. In post-oesophageal region, cuticular spines become smaller, sparser, begin to lose annular pattern, dissipating towards tail ( Fig. 2A,G,H ). Vestibule plus prostom 51–80 long, vestibule 15–20 long and 4–22 wide, prostom 36–49 long and 6–9 wide. Relaxed telostom length 11–17 and width 12–22. Contracted telostom length 8–20 and width 16–22. Relaxed telostom generally longer, thinner than contracted one. Muscular oesophagus length 482–648, glandular oesophagus length 1.4–2.2 mm , ratio of muscular to glandular oesophagus lengths 1: 2.5–4.7. Vestibule plus total oesophagus length 2 mm , percentage of vestibule plus oesophagus to body length 19–29 %. Nerve ring 73–176 from anterior, 14–115 from vestibule. Excretory pore 363–399 from anterior and 296–338 from vestibule. Vulva not protruding ( Fig. 2H,I ), in posterior half of body, 9.0– 11.4 mm from anterior body, 280–532 from anus, 353–632 from tail tip. Vulva 94–96% from anterior end. Reproductive structure mono-opisthodelphic, ovary with two oviducts posterior to uterus and muscular vagina, all directed anteriorly ( Fig. 2I ). Fully developed eggs present in uterus of gravid females. Eggs oval, smooth, lacking accessories, thick walled, larvated; 33–36 long and 21–25 wide. Tail 71–99 long, with small spines, terminal mucron tip 1.1–6.0 long ( Fig. 2J,K ). Immature female. ( four immature females measured Supplementary Table S4). Total body length 8.3–9.1 mm , maximum width 210–257. Cephalic structures underdeveloped ( Fig. 5A ). First ring of spines 56–70 from anterior. Number of spines generally lower than in adult females: 41–43 in first, 42–47 in second, 36–48 in third ring. Length of spines; 12–13 in first, 15–17 in second, 13–15 in third ring. In postoesophageal region, cuticular spines become smaller, sparse, begin to lose annular pattern. Prostom plus 0.02 Fig. 7. Phylogenetic relationships of Spinitectus spp. based on available cox 1 mtDNA for Spinitectus based on Bayesian inference (BI)), with Rhabdochona xiphophori Caspeta-Mandujano, Moravec et Salgado-Maldonado, 2001 as the designated outgroup. Posterior probability (BI) and 1,000 bootstrap replicate (maximum likelihood (ML)) support indicated (BI/ML), nodes with less than 0.5 (50 %) support not annotated. Data shaded in colour from indicated geographical locality or river system, and three haplotypes recorded from the Vaal River system indicated (VRS1–VRS3). vestibule 54–64 long, prostom 47–50 long, vestibule 8–15 long. Muscular oesophagus length 304–428, glandular oesophagus 0.7–1.8 mm long, ratio of muscular to glandular 1: 2.1–5.0. Vestibule plus whole oesophagus length 2.0– 2.9 mm ; ratio of oesophagus plus vestibule length to total body length 20–30%. Nerve ring not observed. Excretory pore situated between row 1, 3, 4 and 5, 9–214 from anterior extremity. Vulva not protruding, located in posterior-half of body ( Fig. 5B ), 7.8–8.8 mm from anterior end, 277–429 from anus. Muscular vagina connected to uterus anteriorly. Oval, smooth, unfertilised eggs in uterus, 33–37 long and 20–22 wide. Tail 74–88 long; small, dispersed spines anterior to mucron, mucron length 2–6 ( Fig. 5C,D ). Two caudal papillae lateral to mucron.