Stillabothrium cadenati ( Euzet, 1954 ) Healy et Reyda, 2016

Reyda, Florian B., Healy, Claire J., Haslach, Andrew R., Ruhnke, Timothy R., Aprill, Tara L., Bergman, Michael P., Daigler, Andrew L., Dedrick, Elsie A., Delgado, Illari, Forti, Kathryn S., Herzog, Kaylee S., Russell, Rebecca S. & Willsey, Danielle D., 2016, A new genus of rhinebothriidean cestodes from batoid elasmobranchs, with the description of five new species and two new combinations, Folia Parasitologica (038) 63 (38), pp. 1-28 : 19-22

Stillabothrium cadenati ( Euzet, 1954) Healy et Reyda, 2016 comb. n. Figs. 1 , 12– 14 , 16E

S y n o n y m: Rhinebothrium cadenati Euzet, 1954

I n f o r m a l s y n o n y m s: Rhinebothriinae New genus 3 cadenati of Healy et al. (2009), Caira et al. (2014), Ruhnke et al. (2015), Marques and Caira (2016).

ZooBank number for species:

urn:lsid:zoobank.org:act:9274C612-40F1-4F66-839C-DE145BC0C01C

Redescription (based on specimens collected from Z. schoenleinii consisting of whole mounts of 6 complete mature worms, 5 strobilae and 2 scoleces; cross sections of 1 strobila and longitudinal sections of 2 scoleces [including 1 in situ scolex], and 1 specimen prepared for SEM): Worms euapolytic, slightly craspedote ( Fig. 12A ), 1.04–1.77 mm (1.35 ± 0.3; n = 6) long, greatest width 439–694 (568 ± 96; n = 7) at level of scolex; 5–7 (5.7 ± 0.8; n = 6) proglottids per worm. Cephalic peduncle lacking; small darkly staining germinative zone present. Scolex ( Figs. 12A , 14A ) consisting of scolex proper bearing four stalked bothridia. Stalks 39–128 (86 ± 33; n = 8) long by 40–77 (60 ± 13; n = 8) wide, attached slightly posterior to middle of bothridia. Bothridia ( Figs. 12A,B , 14A,B ) varying in shape with degree of contraction, deeply ( Fig. 12A ), finely deltoid or broadly deltoid ( Figs. 12B , 14A,B ), facially loculated, 285–378 (328 ± 38; n = 6) long by 201–355 (274 ± 64; n = 7) wide. Anterior region of bothridia ( Figs. 12A,B ) with 3 horizontally oriented loculi (i.e. loculi wider than long) with 3–4 (3.8 ± 0.4; n = 6) complete transverse septa; fourth complete but reduced transverse septum ( Fig. 12B ) observed in 5 of 6 specimens. Anteriormost loculus 58–105 (77 ± 14; n = 8) long and 74–118 (108 ± 14; n = 8) wide. Posterior region of bothridia ( Fig. 12A,B ) with 4 (n = 8) nonmedial longitudinal septa dividing bothridia into 5 loculi longer than wide; longitudinal septa in posterior region of bothridia overlapping posteriormost (fourth) transverse septum in anterior region of bothridia ( Fig. 12B ). Muscle fibres other than septa but parallel to bothridial surface also observed on bothridia ( Fig. 12A ). All septa appear as ridges in section with proximal and distal portions different ( Fig. 13 ); proximal portion of septa formed by underlying bothridial wall, consisting of radial muscles oriented with proximal ends of fibres adjacent to each other; distal portion of septa formed by separate muscle bundle; proximal and distal portions of septa separated by a triangular gap.

Loculi ( Fig. 14D ) and septa ( Fig. 14E ) of distal bothridial surfaces bearing coniform spinitriches and capilliform filitriches; spinitriches lacking on distal bothridial margin and rim ( Fig. 14C ). Proximal bothridial rim ( Fig. 14C ) bearing capilliform filitriches greater in length than those on distal bothridial surfaces ( Fig. 14D,E ). Proximal bothridial surfaces away from rim and stalks bearing coniform spinitriches and capilliform filitriches ( Fig. 14F,G ). Strobila bearing capilliform filitriches only.

Strobila with 3–4 (3.2 ± 0.4; n = 6) proglottids wider than long followed by 2–4 (2.5 ± 0.8; n = 6) proglottids longer than wide. Strobila widest at terminal proglottid; terminal proglottid 467–1 005 (n = 11) long by 103–139 (n = 11) wide; genital pore located 64–73% (n = 11) of proglottid length from proglottid posterior margin. Genital atrium expansive, 38–68 (51 ± 10; n = 11) long by 31–49 (40 ± 7; n = 11) wide, with convoluted, muscular walls. Immature proglottids 4–5 (4.5 ± 0.5; n = 6) in number. Mature proglottids 1–2 (1.4 ± 0.5; n = 11) in number, including 0–1 (0.7 ± 0.5; n = 11) vas deferens-mature proglottids.

Testes in mature proglottids 7–13 (10.8 ± 2; n = 12) in total number, 1 layer deep in section, arranged in 1–3 (2 ± 0.4; n = 11) though usually 2, columns ( Fig. 12C ); columns extending from near anterior margin of proglottid to anterior margin of vagina, 22–36 (30 ± 4; n = 11) long by 29–50 (38 ± 6; n = 11) wide. Vas deferens coiled, entering anterior margin of cirrus sac, extending anterior to ovarian isthmus to near anterior vagina. Cirrus sac thick-walled and relatively large, oval and bent posteriorly, extending medially past midline of proglottid; cirrus sac in terminal mature proglottid 85–152 (119 ± 47; n = 2) long by 58–62 (60 ± 3; n = 2) wide; cirrus sac in vas deferens-mature proglottids 103–171 (136 ± 23; n = 8) long by 50–75 (67 ± 8; n = 8) wide. Cirrus spinitriches present, 3.5–6.1 (4.3 ± 0.9; n = 10) long by 2.0–3.7 (2.8 ± 0.5; n = 10) wide at base.

Vagina ( Fig. 12A,C ) thick-walled, sinuous, not overlapping cirrus sac, recurved ( Figs. 12C , 16F ); poral portion expanded; medial and posterior portion abruptly narrowed; extending from ootype past midline to aporal side of proglottid to anterior margin of cirrus sac, then laterally to open into genital atrium anterior to cirrus sac; vaginal sphincter absent. Seminal receptacle present. Ovary near posterior end of proglottid, lobulated, H-shaped in frontal view, tetralobed in cross section, not overlapping cirrus sac; ovarian lobes asymmetrical; poral and aporal ovarian lobes in terminal mature proglottids 125–215 (170 ± 64; n = 2) and 125–225 (175 ± 71; n = 2) long, respectively. Poral and aporal ovarian lobes in vas deferens-mature proglottids 120–350 (222 ± 71; n = 9) and 125–350 (218 ± 69; n = 9) long, respectively. Maximum width of ovary 48–87 (62 ± 13; n = 10). Ovarian isthmus at or anterior to midpoint of ovary; poral lobe of ovary stopping 90–190 (145 ± 35; n = 10) short of genital pore. Mehlis’ gland posterior to ovarian isthmus, 25–40 (33 ± 5; n = 10) long by 16–25 (20 ± 3; n = 10) wide. Vitellarium follicular; vitelline follicles arranged in 1 dorsal and 1 ventral column on each side of proglottid; columns extending from anterior to posterior margin of proglottid, interrupted by terminal genitalia, not interrupted by ovary ( Fig. 12A,C ). Uterus ventral, sacciform, extending from posterior margin of proglottid to near anterior margin of proglottid.

T y p e h o s t: Zanobatus schoenleini [sic!] (= schoenleinii ) (Müller et Henle), striped panray ( Rhinopristiformes : Zanobatidae ).

A d d i t i o n a l h o s t: Rhinobatos rhinobatos (Linnaeus) , Common guitarfish ( Rhinopristiformes : Rhinobatidae ).

Ty p e l o c a l i t y: Gorée Island, Senegal.

A d d i t i o n a l l o c a l i t i e s: Atlantic Ocean off Ouakam (14°42'54''N; 17°29'28''W) ( SE –28) and Soumbédioune (14°40'42''N; 17°27'42''W) ( SE –201) in Dakar, and Joal (14°10'30''N; 16°51'12''W) ( SE –299) and Kafountine (12°55'41''N; 16°45'10''W) ( SE –289), Senegal .

S i t e o f i n f e c t i o n: Spiral intestine.

Ty p e m a t e r i a l: Unknown.

M a t e r i a l e x a m i n e d (v o u c h e r s): LRP Nos. 9000– 9002; 9134–9148 (including molecular vouchers, sections and SEM specimens); MNHN Nos. HEL580–HEL581; USNM No. 1420484.

Remarks. Type specimens of S. cadenati comb. n. were not available for this study. In the original description of S. cadenati, Euzet (1954) did not provide specimen deposition information. It is possible that the specimens were in the personal collection of the late Louis Euzet, a collection now transferred to the Museum National d’Histoire Naturelle (MNHN) in Paris, France. Examination of that collection by the curator, however, did not reveal the presence of any specimens of S. cadenati . The material on which this redescription was based were newly collected, topotypic specimens of S. cadenati The specimens were collected from the type host at Ouakam and Soumbédioune on the west coast of Dakar, Senegal, approximately 12 and seven km, respectively away from Gorée island, the type locality of this species. Gorée lies 2.5 km off the eastern coast of Dakar. Specimens of S. cadenati were also obtained from the type host in Joal, Senegal.

Two specimens of S. cadenati were included in the phylogenetic analysis ( Fig. 1 , Table 1 ). One was from the type host, Z. schoenleinii , and one was from R. rhinobatos (the common guitarfish). Given that R. rhinobatos and the type host, Z. schoenleinii , belong to different orders of elasmobranchs, this host record requires verification, ideally by the examination of additional specimens.

The bothridial morphology of S. cadenati was not comprehensively described by Euzet (1954). While he noted the presence of 3 transverse septa, Euzet (1954) stated that the bothridia of the specimens he examined were tightly folded and clamped in four scallops. Subsequently, when Euzet and co-authors ( Ball et al. 2003) erected the genus Scalithrium Ball, Neifar et Euzet, 2003 , for species of Rhinebothrium that lack a median longitudinal septum, they concluded that the scolex morphology of S. cadenati was too poorly known to allow them to confidently place it in their new genus at that time.

The work conducted here enabled comprehensive characterisation of the bothridial morphology of S. cadenati for the first time. A fourth, less muscular transverse septum ( Figs. 12B , 13 ) lies posterior to the three transverse septa noted by Euzet (1954), and this reduced transverse septum is crossed by some, but not all, non-medial longitudinal septa ( Fig. 12B ). We believe the scalloped appearance in the posterior part of the bothridium of specimens with folded bothridia noted by Euzet (1954) is due to the presence of these non-medial longitudinal septa.

This redescription also provides additional data on the reproductive morphology and microthrix distribution patterns of this species. Some measurements reported by Euzet (1954) suggest that his specimens of S. cadenati were somewhat larger than those examined here. For example, Euzet’s (1954) specimens were 4–6 mm long in total length and possessed 13–16 proglottids, whereas the specimens examined here were 1.04–1.77 mm long and possessed 5–7 proglottids. In spite of these differences, we believe that the specimens we obtained for the current study are conspecific with those examined by Euzet (1954), based on the illustrations he provided, and considering that that the specimens we used for this study are from the same host, and within 11 km of the type locality.

The bothridia of S. cadenati are unlike any previously described Stillabothrium species in that they possess septa that differ in thickness. In S. ashleyae , S. davidcynthiaorum , S. campbelli , S. hyphantoseptum and S. jeanfortiae , all transverse and longitudinal septa appear uniform in thickness. In S. cadenati the 3 anteriormost transverse septa are thicker than the fourth transverse septa and the longitudinal septa ( Fig. 12B ).

Stillabothrium cadenati can also be distinguished from each of its five congeners in its conspicuously different proglottid morphology. Unlike those of its congeners, the genital atrium in S. cadenati has convoluted walls that are more muscular in appearance (compare Fig. 12C with Figs. 2C , 4C , 6C , 8C , 10C ), and the genital pore is located more anteriorly in S. cadenati (64–73% of proglottid length from proglottid posterior margin) than it is in S. ashleyae , S. davidcynthiaorum , S. campbelli , S. hyphantoseptum and S. jeanfortiae (35–46%, 42–54%, 41–55%, 50–61% and 42– 56%, respectively). In S. cadenati , the vitellarium ( Fig. 12C ) occurs along the length of the ovary, whereas in S. ashleyae , S. davidcynthiaorum , S. campbelli , S. hyphantoseptum and S. jeanfortiae , the vitelline columns are interrupted by the ovary ( Figs. 2C , 4C , 6C , 8C , 10C ). The cirrus sac is relatively larger in S. cadenati when compared to its 5 congeners (compare Fig. 12C to Figs. 2C , 4C , 6C , 8C , 10C ), and reaches a greater length (103–171 µm vs 28–38 µm, 32–45 µm, 45–90 µm, 52–79 µm and 80–87 µm, respectively).

Stillabothrium cadenati is the only species of the genus reported from the Atlantic Ocean. Specimens that appear to represent at least two additional species of Stillabothrium were encountered in stingrays of the genus Fontitrygon Last, Naylor et Manjaji-Matsumoto over the course of survey work in Senegal. These remain to be examined in detail.

Four specimens of S. cadenati were included in the phylogenetic analysis ( Fig. 1 , Table 1 ). One of the four specimens (LRP 3924) was previously included in the analysis of Healy et al. (2009) as ‘Rhinebothriinae New genus 3 cadenati ’.

Figures

Fig. 1. Phylogram based on Bayesian analysis of 1084 bp region of D1–D3 28S rDNA. Numbers above branches indicate Bayesian posterior probabilities, numbers below indicate Bootstrap percentage values, based on 1000 replicates. Scale indicates expected num- ber of substitutions per site. Diagrammatic line drawings of fully opened bothridia of the seven described species mapped onto the clades representing those respective species. Host species information in Table 1.

Fig. 12. Line drawings of Stillabothrium cadenati comb. n. from Zanobatus schoenleinii (Müller et Henle). A – whole worm (LRP 9135); B – bothridium (LRP 9134); C – terminal proglottid (LRP 9136).

Fig. 13. Histological sections through bothridium of Stillabothrium cadenati (Euzet, 1954) comb. n. from Zanobatus schoenleinii (Müller et Henle). Arrows indicate divisions between proximal and distal portions of transverse septa. Abbreviations: ATS – an- teriormost transverse septum; IV – intestinal villus; RM – radial musculature; RTS – reduced transverse septum; SM – septal mus- culature; ST – stalk; TM – transverse muscle bundle.

Fig. 14. Scanning electron micrographs of Stillabothrium cadenati (Euzet, 1954) comb. n. from Zanobatus schoenleinii (Müller et Henle). A – scolex, letters indicate locations of other SEMs; B – bothridium; C – distal bothridial surface at rim; D – distal bothridial surface within loculus; E – distal bothridial surface within septa; F – proximal bothridial surface; G – strobila.

Fig. 16. Light micrographs of terminal genitalia of Stillabothrium species, each with genital pore oriented left. A – Stillabothrium ashleyae sp. n. from Dasyatis biasa (Last, White et Naylor); B – Stillabothrium davidcynthiaorum sp. n. from Himantura gerrardi (Gray); C – Stillabothrium campbelli sp. n. from Himantura cf. pastinacoides; D – Stillabothrium hyphantoseptum sp. n. from Pastinachus solocirostris Last, Manjaji et Yearsley; E – Stillabothrium jeanfortiae sp. n. from Himantura australis Last, White et Naylor; F – Stillabothrium cadenati comb. n. from Zanobatus schoenleinii (Müller et Henle).

Fig. 2. Line drawings of Stillabothrium ashleyae sp. n. from Dasyatis biasa (Last, White et Naylor). A – whole worm (holotype; MZUM [P] 2016.6 [H]); B – scolex (LRP 9011); C – terminal proglottid (LRP 9010) with position of sections labeled (uterus not shown); D – section of mature proglottid at level of testes (LRP 9037); E – section of mature proglottid posterior to ovarian bridge (LRP 9030).

Fig. 4. Line drawings of Stillabothrium davidcynthiaorum sp. n. from Himantura heterura (Bleeker). A – whole worm (LRP 9046); B – scolex (holotype, MZUM [P] 2016.9 [H]); C – terminal proglottid (LRP 9046).

Fig. 6. Line drawings of Stillabothrium campbelli sp. n. from Himantura cf. pastinacoides. A – whole worm (holotype; MZUM [P] 2016.11 [H]); B – scolex (LRP 9066); C – terminal proglottid (LRP 9067).

Fig. 8. Line drawings of Stillabothrium hyphantoseptum sp. n. from Pastinachus solocirostris Last, Manjaji et Yearsley. A – whole worm (holotype; MZUM [P] 2016.13 [H]); B – scolex (LRP 9092); C – terminal proglottid (LRP 9088).

Fig. 10. Line drawings of Stillabothrium jeanfortiae sp. n. from Himantura australis Last, White et Naylor. A – whole worm (holotype; QM G235198); B – scolex (LRP 9123); C – terminal proglottid (holotype; QM 235198).