Camallanidae Railliet and Henry, 1915

Nofal, Ashlee P., Dos Santos, Quinton M., Jirsa, Franz & Avenant-Oldewage, Annemari`e, 2022, Camallanid nematodes from Clarias gariepinus (Burchell, 1822) in the Crocodile River, Gauteng, South Africa: Exploring diversity and divergence in an acid-mine drainage impacted environment, International Journal for Parasitology: Parasites and Wildlife 19, pp. 196-210 : 199-203

Family Camallanidae Railliet and Henry, 1915 Genus Procamallanus Baylis, 1923

Procamallanus (Procamallanus) pseudolaeviconchus Moravec and van As, 2015

Type host: African sharptooth catfish, Clarias gariepinus (Burchell, 1822) ; Siluriformes ; Clariidae .

Other hosts: According to Moravec (2019), the following are considered definitive hosts: Clarias spp. including Clarias agboyiensis Sydenham, 1980 , Clarias alluaudi Boulenger, 1906 , Clarias anguillaris (Linnaeus, 1758) , Clarias buthupogon Sauvage, 1879 , Clarias ebriensis

Pellegrin, 1920, Clarias liocephalus Boulenger, 1898 , Clarias macromystax Günther, 1864 , Clarias stappersi Boulenger, 1915 , Clarias theodorae Weber, 1897 and Clarias werneri Boulenger, 1906 .

Locality: Lake Heritage (25 ◦ 57 ′ 31.7 ′′ S, 27 ◦ 51 ′ 20.2 ′′ E), Crocodile River, Muldersdrift, South Africa .

Site of infection: Oesophagus and stomach.

Prevalence: 78.4% (CI = 67.1–89.7%); 40 out of 51 infected.

Intensity: 1–26 (8.8 ± 1).

Abundance: 0–26 (6.9).

Specimens deposited: A total of 15 voucher specimens were deposited: 5 (2 females and 3 males) in each of the 3 museums; Institute of Parasitology , Czech Academy of Sciences, Ceske ˇ´Budˇejovice, Czech Republic ( IPCAS N-1068 ) , the Natural History Museum , London ( NHMUK 2022.10 .13.1–13.5), and Iziko Museum of South Africa ( SAMC-A094714–18 ) .

Genetic data: All genetic material produced deposited to GenBank, eight 18S rDNA sequences (OP602987 to OP602994) and ten CO1 mtDNA sequences (OP602960 to OP602969).

General description. Medium-sized nematodes, sexually dimorphic, males distinctively smaller. Body long and narrow, maximum width equatorially. Cuticle thick and continuous, transverse striations along entire length of body. Anterior end with terminal, circular oral opening, characterized by unlobed or smooth peribuccal flange ( Fig. 2A and B ). Eight submedian cephalic papillae encompass oral opening, arranged in inner and outer rings of four papillae each ( Fig. 2B ). Two amphids, each lateral to oral opening ( Fig. 2B ). Buccal capsule elongated, sclerotized, yellow-orange, barrel-shaped, six marginal, crescent-shaped elevations anteriorly ( Fig. 2D ). Interior of buccal capsule smooth, narrow posterior ring followed by basal ring located at point of oesophageal attachment ( Fig. 2E and F ). Oesophageal cup posterior to

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buccal capsule, relatively small and poorly sclerotized. Muscular oesophagus triradial and claviform, preequatorially encircled by nerve ring. Glandular oesophagus cylindrical and claviform, longer than muscular oesophagus. Excretory pore posterior to nerve ring, anterior to widened part of muscular oesophagus base ( Fig. 2G, G (i)). Small, simple, short, single, blunt deirids lateral on opposing sides, at level of nerve ring ( Fig. 2H and I ). Narrow intestine; light-brown in adults. Tail tapered, ending in rounded tip.

Female ( Fig. 3A–C ; supplementary file A). Morphometrics based on 14 adult specimens for LM; ten whole specimens studied using SEM. Body length 3.25–8.26 (6.77) mm; maximum width 111–212 (162). Entire buccal capsule (including basal ring) 49–77 (73) long, 39–64 (55) wide. Basal ring 6–37 (15) long, 7–37 (28) wide. Oesophageal cup 5–12 (8) long, 9–20 (13) wide. Muscular oesophagus 378–544 (430) long, 5–12 (7) % of body length; 19–46 (36), 31–62 (49) and 44–99 (67) wide at anterior, mid-length and posterior region, respectively. Glandular oesophagus 586–1044 (811) long, 10–18 (12) % of body length; 38–64 (48), 40–99 (60), 42–103 (68) wide at anterior, mid-length and posterior region, respectively. Length of entire oesophagus and buccal capsule 17–32 (20) % of body length. Nerve ring 168–276 (212) from anterior end. Excretory pore 202–493 (280) from anterior end. Deirids 180–244 (213) from anterior end. Vagina directed posteriorly, causing lateral body wall protrusion near vulva. Vulva situated postequatorially on body, directed laterally with distinct lips ( Fig. 3A and B ), 1.77–4.78 (3.68) mm from anterior end 49–60 (54) % of body length. First-stage larva observed exiting vulva ( Fig. 3C ). Tail 85–220 (122) long with three digit-like processes.

Male ( Fig. 3D–G ; supplementary file B). Morphometrics based on 16 adult specimens for LM; ten whole specimens and 25 specimens for spicule isolation studied using SEM. Body length 2.98–6.14 (4.10) mm; maximum width 90–162 (114). Entire buccal capsule (including basal ring) 52–67 (58) long, 37–53 (42) wide. Basal ring 5–33 (10) long, 6–51 (24) wide. Oesophageal cup 4–12 (7) long, 6–18 (11) wide. Muscular oesophagus 219–495 (368) long, 5–15 (9) % of body length; 21–46 (31), 28–65 (43) and 38–97 (59) wide at anterior, mid-length and posterior region, respectively. Glandular oesophagus 589–1051 (736) long, 15–29 (18) % of body length; 27–78 (47), 36–104 (60) and 34–97 (56) wide at anterior, mid-length and posterior region, respectively. Length of entire oesophagus and buccal capsule 24–45 (29) % of body length. Nerve ring 144–223 (178) from anterior end. Excretory pore 155–357 (250) from

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anterior end. Deirids 124–251 (185) from anterior end. Caudal end, ventrally curved with narrow alae and papillae: eight to nine pairs precloacal pedunculate papillae ( Fig. 3D and D (i)), one pair adcloacal sessile papillae (slightly anterior to cloacal opening) ( Fig. 3D ), four pairs postcloacal papillae (three pairs postcloacal and ventral ( Fig. 3D ), one pair further dorsolateral). Two simple unequal spicules ( Fig. 3E–F ), both with long and thin cylindrical shaft, with blunt, rounded distal ends and thin, poorly sclerotised, wing-like velum. Right spicule with widened base. Right (large) spicule 41–148 (93) long, left (small) spicule 19–57 (41) long ( Fig. 3G and G (i)). Gubernaculum indistinct, recorded in only five specimens, 36–141 (77) long. Tail 38–85 (56) long.

Remarks. The morphometry of specimens described here strongly agree with the species description of P. (P.) pseudolaeviconchus by Moravec and van As (2015a). It also agrees with other records of this taxon ( Moravec, 2019; Svitin et al., 2019), as well as those reported as P. (P.) laeviconchus from the same host ( Moravec, 1975; Boomker, 1982; Barson and Avenant-Oldewage, 2006). Morphology of isolated buccal capsules, right (large) and left (small) spiculae are presented for the first time. The region above the basal ring lacks a uniform term. Moravec and van As (2015a) described a thickening of the capsule wall situated anteriorly to the basal ring, whereas Svitin et al. (2019) refers to it as two step-like folds, and Moravec (2019) as a narrow ring. Following Moravec (2019), the term narrow ring was used. Vulva, with lips, shown for the first time using SEM. The number of digit-like processes varied, with adult females bearing three and fourth-stage larvae four processes. The number of cloacal papillae in males varied in previous studies, those observed in the present study overlapping with most.

Genus Paracamallanus Yorke and Maplestone, 1926 Paracamallanus cyathopharynx ( Baylis, 1923) Syns. Camallanus cyathopharynx Baylis (1923) ; Paracamallanus senegalensis Vassiliad`es, 1970

Host: African sharptooth catfish, Clarias gariepinus (Burchell, 1822) ; Siluriformes ; Clariidae .

Other hosts: According to Moravec (2019): clariids Channallabes apus (Günther, 1873) , Clariallabes teugelsi Ferraris, 2007 , Clarias alluaudi Boulenger, 1906 , C. anguillaris , Clarias buthupogon Sauvage, 1879 , Clarias ebriensis Pellegrin, 1920 , Clarias liocephalus Boulenger, 1898 , Clarias stappersi Boulenger, 1915 , Clarias theodorae Weber, 1897 , Clarias werneri Boulenger, 1906 , Clarias sp. and Heterobranchus longifilis Valenciennes, 1840 . Additionally, it has been recorded from silurids Clarotes laticeps (Rüppell, 1829) , Schilbe intermedius Rüppell, 1832 , Synodontis schall (Bloch and Schneider, 1801) , Synodontis zambezensis Peters, 1852 ; and a characid Hydrocynus vittatus Castelnau, 1861 .

Locality: Lake Heritage (25 ◦ 57 ′ 31.7 ′′ S, 27 ◦ 51 ′ 20.2 ′′ E), Crocodile

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River, Muldersdrift, South Africa.

Site of infection: Distal intestine and rectum.

Prevalence: 94.1% (CI = 87.7–100%); 48 out of 51 infected.

Intensity: 1–187 (28.3 ± 4.7).

Abundance: 0–187 (26.6).

Specimens deposited: A total of 15 voucher specimens were deposited: 5 (2 females and 3 males) in each of the 3 museums; Institute of Parasitology , Czech Academy of Sciences , Ceske ˇ´Budˇejovice, Czech Republic ( IPCAS N-12 ), the Natural History Museum, London ( NHMUK 2022.10 .13.6–13.10), and Iziko Museum of South Africa (SAMC- A094709–13) .

Genetic data: All genetic material produced deposited to GenBank, five 18S rDNA sequences (OP602982 to OP602986) and twelve CO1 mtDNA sequences (OP602970 to OP602981).

General description. Medium-sized nematodes, sexually dimorphic; males smaller than females. Elongated and thin body, widest at mid-body. Cuticle continuous with prominent horizontal striations along body length. Anterior end with terminal, slit-like oral opening surrounded by eight cephalic submedian papillae (two circular arrangements of four papillae each) ( Fig. 4A and B ). Pair of amphids, located dorsal and ventral to mouth ( Fig. 4A ). Sclerotised buccal capsule, yellow-orange, consisting of anterior and posterior parts separated by isthmus ( Fig. 4C (i)). Anterior buccal capsule rounded, characterized by; dorsal and ventral valves, each valve with two sclerotized plates superficially surrounding mouth ( Fig. 4C ), superficial pair of large posteriorly extending lateral tridents distal to mouth with middle prong longest ( Fig. 4C ). Interior surface of each valve with nine distinct longitudinal ridges ( Fig. 4D ). Posterior buccal capsule (pharynx) comprised of two parts ( Fig. 4C (i)); anterior long, widened towards top and lined with thick walls; posterior substantially smaller with thin walls leading to small, sclerotized and round oesophageal cup (wider than it is tall) ( Fig. 4B ). Muscular oesophagus claviform with basal region wider. Nerve ring situated slightly posterior to oesophageal cup, encircling muscular oesophagus. Pair of inconspicuous, simple, blunt lateral deirids at level of nerve ring ( Fig. 4E ). Excretory pore posterior to nerve ring. Glandular oesophagus cylindrical and longer than muscular oesophagus. Long, straight intestine, narrow rectum. Tail tapered, rounded tip.

Female ( Fig. 4D–E ; supplementary file C). Morphometrics based on 16 specimens for LM; ten whole specimens studied using SEM. Body length 5–10 (8) mm; maximum width 103–187 (147). Entire buccal capsule (excluding oesophageal cup) 107–146 (122) long. Anterior buccal capsule 66–85 (74) long, 52–100 (78) wide. Posterior buccal capsule (pharynx) 34–56 (42) long, 61–98 (77) wide. Posterior part of posterior buccal capsule 12–31 (19) long, 28–53 (40) wide. Tridents 58–93 (73) long. Muscular oesophagus 456–680 (573) long, 6–12 (8) % of body length, 32–80 (50), 42–96 (66) and 12–125 (72) wide at anterior, mid-length and posterior region, respectively. Glandular oesophagus 436–989 (736) long, 5–17 (10) % of body length, 35–86 (59), 30–106 (67) and 25–104 (64) wide at anterior, mid-length and posterior region, respectively. Entire oesophagus and buccal capsule 1.2–1.8 (1.43) mm long; 14–32 (19) % of body length. Nerve ring 153–220 (181) from anterior end. Excretory pore 172–347 (245) from anterior end. Deirids 145–251 (192) from anterior end. Vagina directed posteriorly, causing slight elevation anterior to vulva. Vulva situated postequatorially on body, directed posteriorly, indistinct lips, 2–6 (4) mm from anterior end, 30–63 (55) % of body length. Tail 235–401 (320) long, three or four digit-like processes ( Fig. 4F and G ).

Male ( Fig. 5A–F ; supplementary file D). Morphometrics based on 14 specimens for LM; ten whole specimens and 25 specimens for spicule isolation, studied using SEM. Body length 3.29–6.20 (4.87) mm; maximum width 74–146 (112). Entire buccal capsule (excluding oesophageal cup) 77–101 (91) long. Anterior buccal capsule 50–67 (57) long, 46–71 (58) wide. Posterior buccal capsule (pharynx) 22–62 (32) long, 28–64 (50) wide. Posterior part of posterior buccal capsule 10–17 (12) long, 21–33 (27) wide. Tridents 49–68 (59) long. Muscular oesophagus 353–497 (423) long, 7–12 (9) % of body length, 28–64 (44), 41–74 (53) and 45–97 (67) wide at anterior, mid-length and posterior region, respectively. Glandular oesophagus 416–863 (572) long, 10–14 (12) % of body length, 40–83 (52), 36–108 (59) and 32–104 (58) wide at anterior, mid-length and posterior region, respectively. Entire oesophagus and buccal capsule 845–1397 (1086) long; 18–28 (23) % of body length. Nerve ring 113–164 (143) from anterior end. Excretory pore 140–218 (178) from anterior end. Deirids 124–186 (154) from anterior end. Ventrally curved caudal end, narrow alae and papillae: five pairs precloacal pedunculated papillae ( Fig. 5A ), two pairs adcloacal papillae ( Fig. 5B ), six pairs postcloacal papillae (three pairs grouped together short distance posterior to cloaca, two pairs at mid region of tail and one pair close to tail tip) ( Fig. 5B ). Two spicules, unequal in length. Right (large) spicule 157–323 (264) long, sclerotized with sharply curved, lanceolate distal part 28–56 (38) long ( Fig. 5C–E ), miniscule barb at ventral base of distal part 2–9 (6) long ( Fig. 5E ). Left (small) spicule 52–197 (99) long, acerose, less sclerotised, sharply pointed distal end

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( Fig. 5F ). No openings or channels visible in either of the spicules. Tail 54–103 (70) long, two digit-like processes ( Fig. 5A (i)).

Remarks. The morphometry of specimens described here conform to the species description of P. cyathopharynx by Baylis (1923), the redescription by Moravec (1974b) and the studies by Moravec and van As (2015b), Moravec (2019), Svitin et al. (2019) and Rindoria et al. (2020). Slight differences were recorded compared to Baylis (1923) regarding the length of the tridents and number of longitudinal ridges in the anterior buccal capsule. Isolated buccal capsules in the current study are similar to those in Rindoria et al. (2020), however, the anterior small trident was not observed in any of the current specimens. Morphology of the isolated right (large) and left (small) spiculae studied using SEM are presented for the first time. Variation was observed in the caudal end of females regarding the number of digit-like processes of either three or four. In terms of male digit-like processes, some discrepancy was noted between the studies of Moravec and van As (2015b) and Rindoria et al. (2020) in which the current study corresponds with that described by Moravec and van As (2015b). Lastly, variation was also observed in the number of cloacal papillae described by Rindoria et al. (2020) to the current and other studies.

Figures

Fig. 2. Scanning electron micrographs of Procamallanus (Procamallanus) pseudolaeviconchus Moravec and van As, 2015 from Clarias gariepinus (Burchell, 1822). A-anterior end, lateral view; B- anterior end, apical view, solid arrow shows smooth peribuccal flange, dashed arrow shows marginal elevation; Cisolated buccal capsule, lateral view, arrow shows oesophagus; D-isolated buccal capsule, apical view, solid arrows show marginal elevations; E– buccal capsule interior, apical view, dashed arrow shows narrow ring, solid arrow shows basal ring; F- microdissected buccal capsule, lateral view, dashed arrow shows narrow ring, solid arrow shows basal ring; Ganterior region, lateral view, arrow shows excretory pore; G(i)- excretory pore, lateral view; H- lateral deirid, lateral view; I- lateral deirid, apical view. a = amphid; s = submedian papilla.

Fig. 3. Scanning electron micrographs of Procamallanus (Procamallanus) pseudolaeviconchus Moravec and van As, 2015 from Clarias gariepinus (Burchell, 1822). A-postequatorial region of female, vulva, ventral view; B- vulva, lateral view, arrows show lips; C- first-stage larva exiting vulva; D-posterior end of male, ventral view, solid arrows show pre-cloacal papillae, dashed arrows show post-cloacal papillae, circles show adcloacal papillae; D(i)- pedunculate papilla; E– isolated right spicule, lateral view, solid arrow shows shaft, dashed arrow shows spicule tip; E (i)- right spicule tip, arrow shows velum; F- posterior end of male, ventrolateral view, arrow shows right spicule; G-isolated left spicule, lateral view; G(i)- left spicule tip, arrow shows velum.

Fig. 4. Scanning electron micrographs of Paracamallanus cyathopharynx (Baylis, 1923) from Clarias gariepinus (Burchell, 1822). A-anterior end, apical view; B- anterior end, apical view, arrow shows oesophagus; C- isolated buccal capsule, lateral view, arrow shows oesophagus; C(i)- isolated buccal capsule, lateral view, double arrow shows isthmus; D-microdissected anterior buccal capsule, ventral view of longitudinal ridges. E- lateral view of deirid; F, G, G(i)- posterior end of female, apical view, digit-like processes. a = amphid; s = submedian papilla; sc = sclerotised plate; t = trident; 1 = anterior buccal capsule; 2 = anterior part of posterior buccal capsule; 3 = posterior part of buccal capsule.

Fig. 5. Scanning electron micrographs of male Paracamallanus cyathopharynx (Baylis, 1923) from Clarias gariepinus (Burchell, 1822). A-posterior end, ventrolateral view, arrows show precloacal papillae; Bposterior end, ventral view, solid arrow shows right spicule, dashed arrow shows pair of adcloacal papillae, double arrows show postcloacal papillae; Cisolated right spicule, ventrolateral view, arrow shows shaft; D-right spicule tip, dorsal view; E– right spicule tip, ventral view, arrow shows ventral barb; Fisolated left spicule.