A New Species of Alexandercestus Ruhnke and Workman, 2013

J Parasit Dis (Jan-Mar 2020) 44(1):110–115 https://doi.org/10.1007/s12639-019-01168-6

ORIGINAL ARTICLE

A new species of Alexandercestus Ruhnke and Workman, 2013 (Cestoda: Phyllobothriidea) from the sickleﬁn lemon shark, Negaprion acutidens (Elasmobranchii: Carcharhinidae), in the Gulf of Oman

1 2 3 Loghman Maleki • Tooraj Valinasab • Harry W. Palm

Received: 18 May 2019 / Accepted: 4 October 2019 / Published online: 16 October 2019 Ó Indian Society for Parasitology 2019

Abstract A new species, Alexandercestus masoumehae n. from N. acutidens to five and the described tapeworms in sp., is described from the sicklefin lemon shark, Negaprion the Persian Gulf and Gulf of Oman region to 65. acutidens (Ru¨ppell), from the Iranian coast of the Gulf of Oman. The new species is the third in this genus, all par- Keywords Alexandercestus Á Negaprion acutidens Á asitizing species of Negaprion Whitley. Based on mor- Gulf of Oman Á New species Á Morphological characters phological characters and scanning electron microscopy the new species is different from its congeners of in a combination of characters including total length, terminal Introduction proglottid length, cirrus sac length, and genital pore position. Compared to Alexandercestus gibsoni Ruhnke & Recent studies on the tapeworm fauna of elasmobranchs in Workman, 2013 and A. manteri Ruhnke & Workman, 2013 the Iranian coast of the Persian Gulf and Gulf of Oman the specimens of the new species are smaller have revealed several new species, especially from rays (7.08–8.5 mm), with a smaller terminal proglottid length to (see Haseli et al. 2010; Caira et al. 2011; Haseli and Palm width ratio (1:0.7–1.3 vs. 1.4–3.2:1 and 1.2–3.1:1, 2015; Aminjan and Malek 2016). Recently, Maleki et al. respectively), and have a more anterior genital pore (2013, 2015) described six new Acanthobothrium Blan- (70–73% of proglottid length vs. 42–66% and 41–65%, chard, 1848 species from two batoid hosts, Pastinachus cf. respectively). In addition, A. masoumehae possesses less sephen (Forsska˚l) and Rhynchobatus cf. djiddensis (For- foliose bothridia compared with A. gibsoni, and more sska˚l). However, less studies have focused on the tape- densely packed vitelline follicles in comparison with A. worms of sharks from Iranian waters. Haseli et al. (2010) manteri. The SEM examination of the scolex in the new reported trypanorhynch cestodes from Carcharhinus cf. species reveals a specific pattern of microtriches adding dussumieri (Mu¨ller and Henle), Rhizoprionodon acutus new details in our understanding of the surface morphology (Ru¨ppell) and Carcharhinus cf. sorrah (Mu¨ller and Henle). of members of Alexandercestus. Finally, the new species Malek et al. (2010) described two species of Paraoryg- increases the total number of described tapeworm species matobothrium Ruhnke, 1994 and Caira et al. (2011), a species of Doliobothrium Caira, Malek & Ruhnke, 2011 both from C. cf. dussumieri. Examination of the spiral intestine from a single speci- & Loghman Maleki men of Negaprion acutidens (Ru¨ppell) in the Gulf of Oman [email protected] resulted in collecting a new cestode species. This new 1 Department of Biological Sciences, Faculty of Science, species described herein is the third member of the genus University of Kurdistan, Sanandaj, Iran Alexandercestus Ruhnke & Workman, 2013, all of which 2 Iranian Fisheries Research Organization, Tehran, Iran parasitize sharks of the genus Negaprion Whitley. In addition to describing the new species, detailed 3 Aquaculture and Sea-Ranching, Faculty of Agricultural and Environmental Sciences, University Rostock, Justus-von- information is provided regarding the surface Liebig-Weg 6, 18059 Rostock, Germany 123 J Parasit Dis (Jan-Mar 2020) 44(1):110–115 111 ultrastructure, by adding new information about the surface Type-material: Holotype (ZCUOK 114), one paratype morphology of Alexandercestus member. (ZMB E.7628), one SEM voucher (ZCUOK 116), one paratype (ZUTC 6172). Etymology: This species is named after Dr. Masoumeh Materials and methods Malek in the University of Tehran, for her valuable con- tribution to the knowledge of Iranian elasmobranch Four cestode specimens were collected from a female parasites. specimen of N. acutidens collected by gill net with the help of fishermen in May 2015 from Jod village, off the Iranian Description (Figs. 1a–c, 2a–n) coast of the Gulf of Oman. The spiral intestine was removed, opened with a longitudinal incision, and placed (Based on three whole-mounts of mature specimens, one in 10% formalin buffered with seawater. After a week, the scolex examined for SEM and whole-mount of its strobila). cestodes were removed from the spiral intestine and Worms euapolytic, 7.08–8.5 mm (7.9 ± 0.7; 3; 3) long, transferred to 70% ethanol for storage. The specimens greatest width at level of immature proglottids in middle of prepared for light microscopy were hydrated in a graded strobila, 997–1219 (1104 ± 95.3; 4; 4) wide (Fig. 1b). ethanol series, stained with Delafield’s hematoxylin, Scolex tetrabothridiate, 566–1097 (868 ± 272; 3; 3) long dehydrated in a graded ethanol series, cleared in methyl by 488–1075 (797 ± 294; 3; 3) wide, with free posterior salicylate, and mounted on glass slides in Canada balsam. and lateral margins, scolex proper with cruciform apex. A scolex of one specimen was prepared for scanning Bothridia uniloculate, petaloid form with single apical electron microscopy (SEM), with its strobila processed as a sucker, 543–834 (701 ± 127; 3; 4) long by 277–361 whole-mount to be served as voucher. (313 ± 37; 3; 4) wide (Figs. 1a, 2a). Apical sucker 79–178 The specimen examined with SEM was hydrated in a (113 ± 44; 3; 4) long by 64–105 (83 ± 21; 3; 4) wide. graded ethanol series, post-fixed in 1% osmium tetroxide Margins of bothridia on distal surface covered with overnight, dehydrated in a graded ethanol series, dried capilliform filitriches (Fig. 2b, c), acicular filitriches using hexamethyldisilazane (HMDS), and mounted on interspersed with cilia of regularly arranged sensory double-sided adhesive carbon tape on an aluminum stub. receptors (Fig. 2d). Towards the centre of bothridia, distal The specimen was coated with 15 nm of gold and exam- bothridial surface covered with small aristate serrate ined with a SEM model TESCAN MIRA3. gladiate spinitriches (Fig. 2f) and acicular filitriches inter- Illustrations were made with the help of a drawing tube. spersed with capilliform filitriches and cilia (Fig. 2e). Images were taken with a digital camera and Cellsens Distal bothridial surfaces in centre of bothridia covered Dimension software attached to an OLYMPUS BX51 with acicular filitriches (Fig. 2g). Proximal bothridial sur- compound microscope. Measurements are given in faces (Fig. 2h) near bothridial rim covered with acicular micrometers except the total worm length (in millimeters). filitriches and acicular filitriches interspersed with aristate The range is given, followed by the mean, standard devi- gladiate spinitriches (Fig. 2i, j). Surface of apex covered ation, number of measured worms (N) and number of with capilliform filitriches (Fig. 2k). Surface of entire measurements (n) taken in parentheses. Microthrix termi- strobila scullate (Fig. 2l, m), scutes covered with tightly nology follows Chervy (2009). Museum abbreviations are arranged capilliform filitriches (Fig. 2n). as follows: ZUTC, Collection of the Zoological Museum, Proglottids slightly craspedote, proglottids 125–135 University of Tehran, Tehran, Iran; ZCUOK, Zoological (130 ± 5; N = 3; n = 3) in number, immature proglottids collection, University of Kurdistan, Sanandaj, Iran and much wider than long. Terminal proglottid mature, ZMB, the Natural History Museum Berlin, Germany. 460–763 (625 ± 147; N = 4; n = 4) long by 473–777 Order Phyllobothriidea Caira, Jensen, Waeschen- (624 ± 135; N = 4; n = 4) wide; length to width ratio bach, Olson & Littlewood, 2014 0.7–1.33 (1 ± 0.2; N = 4; n = 4):1. Testes irregularly oval Genus Alexandercestus Ruhnke & Workman, 2013 in ventral view, 127–245 (170 ± 55; N = 3; n = 3) in Alexandercestus masoumehae n. sp. number in terminal proglottid, 20–40 (33 ± 6.2; N = 4; Type-host: Negaprion acutidens (Ru¨ppell), sicklefin n = 10) long by 39–60 (45 ± 6.9; N = 4; n = 10) wide. lemon shark (Carcharhiniformes: Charcharhinidae). Cirrus sac reniform, 44–111 (72 ± 28; N = 4; n = 4) long Type-locality: Off Jod, Gulf of Oman (25°2604900 N, by 237–366 (324 ± 60; N = 4; n = 4) wide, contains 59°3002000 E). coiled cirrus; cirrus armed with small microtriches. Vas Site of infection: Spiral intestine. deferens coiled, medial to cirrus sac; cirrus expanded at Date of collection: May 2015 base. Genital pores lateral, alternating irregularly, 70–73% (71 ± 1.2; N = 4; n = 4) of proglottid length from posterior end of proglottid. Vagina median, slightly sinuous, 123 112 J Parasit Dis (Jan-Mar 2020) 44(1):110–115

Fig. 1 Alexandercestus masoumehae n. sp. a Scolex; b entire worm; c terminal proglottid. Scale-bar: a 300 lm; b 600 lm; c 100 lm

extending from ootype along median line of proglottid to vagina, extends from anterior margin of ovary to anterior level of anterior margin of cirrus sac, then turns laterally level of testes. Mehlis’ gland between anterior half of along anterior margin of vas deferens and cirrus sac to ovarian lobes. Uterine duct dorsal to uterus, median, enter the genital atrium. Genital atrium small. Vaginal extends anteriorly, entering anterior third of uterus. Vitel- sphincter absent. Seminal receptacle not seen. Ovary larium follicular; in 2 lateral bands, each band consisting of located at or near posterior end of proglottid, lobulated, 4–7 ventral columns of follicles, extended entire length of H-shaped in dorsoventral view. Maximum width at anterior mature proglottid, interrupted by cirrus sac and vagina, margin, 88–172 (131 ± 47; N = 4; n = 4) long by follicles oval in ventral view, 9–18 (13 ± 2.9; N = 4; 292–622 (490 ± 137; N = 4; n = 4) wide. Ovicapt at n = 8) long by 15–37 (23 ± 8; N = 4; n = 8) wide, folli- posterior margin of ovarian bridge. Uterus ventral to cles. Eggs not seen. Excretory ducts lateral.

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Fig. 2 Scanning electron micrographs of Alexandercestus masoumehae n. sp. a Scolex; note: small numbers on figures correspond to the figures showing higher magnification images of these surfaces; b distal bothridial surface (arrows shows knob- shaped protrusions of bothridial surface); c rim of bothridium; d microtriches on edge of distal bothridial surface; e projections on surface of distal bothridial surface; f serrate gladiate spinitriches on distal bothridial surface; g distal bothridial surface; h enlarged view of proximal bothridial surface; i proximal bothridial surface; j proximal surface at bothridial base; k apex of scolex; l surface of anterior region of strobila; m surface of scutes; n microtriches on scutes. Scale- bars: a 200 lm; b, h, l 20 lm; d–f, i–m 2 lm; c, g, n 1 lm

Remarks species of Alexandercestus, having foliose and ﬂeshy bothridia with a single sucker, a unique pattern of micro- The new species, A. masoumehae n. sp., is similar to the triches, and an anteriorly curved cirrus sac, more than 100 monotypic phyllobothriid Hemipristicola Cutmore, Theiss, testes per proglottid and 5–7 rows of vitelline follicles on Bennett & Cribb, 2011, based on scolex morphology and each side of the terminal proglottid. Furthermore, it can be proglottid characteristics, and to the Paraorygmatoboth- distinguished from its congeners (A. gibsoni and A. man- rium, based on scolex morphology. It can be distinguished teri) by the following suite of characters. A. masoumehae n. from Hemipristicola by the lack of a deep central cavity on sp. is shorter (7.08–8.5) than A. gibsoni (17–45) and A. its bothridia. It differs from Paraorygmatobothrium in the manteri (11–36), possesses less foliose bothridia, fewer lack of serrate gladiate spinitriches on the proximal both- proglottids (125–135) than A. gibsoni (150–250), and a ridial surfaces and having an anteriorly curved cirrus sac. shorter terminal proglottid (460–777) compared to A. gib- The new species is morphologically most similar to the soni (1080–2050). In addition, the position of the genital

123 114 J Parasit Dis (Jan-Mar 2020) 44(1):110–115 pore also differs compared with A. gibsoni and A. manteri slightly triangular in shape and less serrated, compared to (70–73% vs. 42–66 and 41–65%, respectively). Alexan- the microtriches of Paraorygmatobothrium species, Nan- dercestus manteri possesses vitelline follicles which are docestus guariticus Reyda, 2008, Hemipristicola gunterae considerably less densely packed compared with A. gib- Cutmore, Theiss, Bennett & Cribb, 2011, Ruhnkecestus soni. Further, the vitelline follicles of A. masoumehae n. sp. latipi Caira & Durkin, 2006. The serrate spinitriches of the are more similar to those in A. manteri. Although the new species are more similar to those of A. gibsoni detected species distinguishing the characters within although the marginal serrations are restricted to the Alexandercestus appears subtle, both congeners also have proximal half of the blade length, while the serrate gladiate been collected from different zoogeographical regions spinitriches of A. gibsoni seem to be uniformly serrated (Atlantic and Pacific Ocean). along the entire length of the blades. The neck and strobila surfaces in the new species were completely covered with scutes. This pattern is different from the species of Do- Discussion liobothrium, Nandocestus, Paraorygmatobothrium and Ruhnkecestus where the scutes seem to be restricted only to Alexandercestus masoumehae n. sp. is the third species of the neck region. Alexandercestus to be described. The description of this The elasmobranch genus Negaprion includes two spe- new species from Iran reveals a global distribution pattern cies, the lemon shark [N. brevirostris (Poey, 1868)] in the for the genus, occurring in the three main zoogeographical western Atlantic and the sicklefin lemon shark (N. acuti- regions, the Indian (A. masoumehae n. sp.), Pacific (A. dens) in the Indo-West Pacific (Schultz et al. 2008). Ne- gibsoni) and Atlantic (A. manteri) Ocean. Cutmore et al. gaprion brevirostris has already been reported as the host (2017) reported A. gibsoni from Carcharhinus leucas for A. manteri, Callltetrarhynchus gracilis Pintner, 1931, (Mu¨ller and Henle) from Australia, extending the known C. speciosus (Linton, 1897), Dasyrhynchus giganteus host range of Alexandercestus beyond Negaprion. (Diesing, 1850), D. variouncinatus (Pintner, 1913), Flori- The new species is different from its congeners in its ceps saccatus Cuvier, 1817 (as C. caballeroi Cruz-Reyes, scolex morphology with the possession of less foliose 1977), Paraorygmatobothrium roberti Ruhnke & Thomp- although it has more robust and fleshy bothridia, and the son, 2006, Phoreiobothrium anticaporum Caira, Richmond strobila characteristics. The possession of serrate gladiate & Swanson, 2005, Platybothrium harpago Healy, 2003, P. spinitriches, along with capilliform and acicular filitriches hypoprioni Potter, 1937 and Pseudogrillotia perelica on the distal bothridial surface and aristate gladiate spini- (Shuler, 1938). Negaprion acutidens has been infected with triches along with acicular filitriches on the proximal A. gibsoni, Otobothrium sp., Paraorygmatobothrium bothridial surfaces in A. masoumehae n. sp. are similar to arnoldi Ruhnke & Thompson, 2006, Phoreiobothrium its congener A. gibsoni. The microtriches are arranged in perilocrocodilus Caira, Richmond & Swanson, 2005 and concentric circles on the distal bothridial surface, despite Triloculatum andersonorum Caira & Jensen, 2009 (Caira the fact that the scolex surface is not smooth and possesses et al. 2005; Ruhnke and Thompson 2006; Palm 2004). The transverse grooves and small projections. Based on the description of A. masoumehae n. sp. increases the number regular arrangement, these unique structures may not have of the described species from N. acutidens to five and the formed due to the fixation method. It is worth noting that number of cestode species in the genus Negaprion to 17. the cilia are numerous, and arranged regularly, especially at Description of the new species here increases the total the distal bothridial margins. The free lateral and posterior number of reported tapeworm species of elasmobranchs bothridial margins allow undulating movements and from the Persian Gulf and Gulf of Oman to 65, among probably the migration of the worm inside the spiral which 11 (17%) are from six sharks species (Malek et al. intestine. Obviously, these free margins have significant 2010; Caira et al. 2011). At least 26 species of sharks are sensory function in A. masoumehae n. sp. The possibility to known from the region, with 16 of them belonging to the find more worms for molecular studied was not possible. Carcharhinidae (Moore et al. 2012). Thus, it is much likely One of the features of this genus is that it bears smaller that many more so far undescribed cestode species occur in gladiate spinitriches than typically seen in other phyl- the region. lobothriideans. Phyllobothriid serrate spinitriches are rep- Acknowledgements We are grateful to local fishermen in Jod village resented by several variations in blade size as well as for their help collecting the shark specimen. We thank the Central number and position of the serrations on each side of the Laboratory at University of Kurdistan for preparing Scanning Elec- blade (see Ruhnke et al. 2006; Ivanov 2008; Cutmore et al. tron Microscopy images. Financial support was provided in part by 2011; Ruhnke and Workman 2013). Alexandercestus the Grant No. 4/1279-1395/12/15 through the Vice Chancellor for Research, University of Kurdistan. masoumehae n. sp. possesses smaller blades, which is

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Compliance with ethical standards Haseli M, Malek M, Palm HW (2010) Trypanorhynch cestodes of elasmobranchs from the Persian Gulf. Zootaxa 2492:28–48 Conflict of interest On behalf of all authors, the corresponding Ivanov VA (2008) Orygmatobothrium spp. (Cestoda: Tetraphyllidea) author states that there is no conflict of interest. from triakid sharks in Argentina: redescription of Orygmato- bothrium schmittii and description of a new species. J Parasitol Ethical approval All applicable institutional, national and interna- 94:1087–1097 tional guidelines for the care and use of animals were followed. Malek M, Caira JN, Haseli M (2010) Two new species of Paraorygmatobothrium Ruhnke, 1994 (Cestoda: Tetraphyllidea) from the carcharhinid shark Carcharhinus cf. dussumieri (Mu¨ller & Henle) in the Persian Gulf. Syst Parasitol 76:59–68 References Maleki L, Malek M, Palm HW (2013) Two new species of Acanthobothrium (Tetraphyllidea: Onchobothriidae) from Pasti- Aminjan AR, Malek M (2016) Two new cestode species of nachus cf. sephen (Myliobatiformes: Dasyatidae) from the Tetragonocephalum Shipley & Hornell, 1905 (Lecanicephalidea, Persian Gulf and Gulf of Oman. Folia Parasitol 60:448–456 Tetragonocephalidae) from Himantura randalli Last, Manjaji- Maleki L, Malek M, Palm HW (2015) Four new species of Matsumoto & Moore (Myliobatiformes, Dasyatidae) from the Acanthobothrium van Benden, 1850 (Cestoda: Onchoproteo- Gulf of Oman. ZooKeys 623:1–13 cephalidea) from the guitarfish, Rhynchobatus cf. djiddensis Caira JN, Richmond C, Swanson J (2005) A revision of Phoreioboth- (Elasmobranchii: Rhynchobatidae), from the Persian Gulf and rium (Tetraphyllidea: Onchobothriidae) with descriptions of five Gulf of Oman. Folia Parasitol 62:12 new species. J Parasitol 91:1153–1174 Moore AB, Ward RD, Peirce R (2012) Sharks of the Persian Caira JN, Malek M, Ruhnke TR (2011) A new genus of Phylloboth- (Arabian) Gulf: a first annotated checklist (Chondrichthyes: riidae (Cestoda: Tetraphyllidea) in carcharhiniform sharks from Elasmobranchii). Zootaxa 3167:1–16 Iran and Australia. J Helminthol 85:40–50 Palm HW (2004) The Trypanorhyncha Diesing, 1863. IPB-PKSPL Caira JN, Jensen K, Waeschenbach A, Olson PD, Littlewood DTJ Press, Bogor (2014) Orders out of chaos–molecular phylogenetics reveals the Ruhnke TR, Thompson VA (2006) Two new species of Paraoryg- complexity of shark and stingray tapeworm relationships. Int J matobothrium (Tetraphyllidea: Phyllobothriidae) from the lemon Parasitol 44:55–73 sharks Negaprion brevirostris and Negaprion acutidens (Car- Chervy L (2009) Unified terminology for cestode microtriches: a charhiniformes: Carcharhinidae). Comp Parasitol 73:35–41 proposal from the International Workshops on Cestode System- Ruhnke TR, Workman R (2013) Two new species and a new atics in 2002–2008. Folia Parasitol 56:199–230 phyllobothriid cestode genus from sharks of the genus Ne- Cutmore SC, Theiss SM, Bennett MB, Cribb TH (2011) Hemipris- gaprion Whitley (Carcharhiniformes). Syst Parasitol 85:37–48 ticola gunterae gen. n., sp. n. (Cestoda: Tetraphyllidea: Ruhnke TR, Healy CJ, Shapero S (2006) Two new species of Phyllobothriidae) from the snaggletooth shark, Hemipristis Paraorygmatobothrium (Cestoda: Tetraphyllidea) from weasel elongata (Carcharhiniformes: Hemigaleidae), from Moreton sharks (Carcharhiniformes: Hemigaleidae) of Australia and Bay, Australia. Folia Parasitol 58:187–196 Borneo. J Parasitol 92:145–150 Cutmore SC, Bennett MB, Miller TL, Cribb TH (2017) Patterns of Schultz JK, Feldheim KA, Gruber SH, Ashley MV, McGovern TM, specificity and diversity in species of Paraorygmatobothrium Bowen BW (2008) Global phylogeography and seascape genet- Ruhnke, 1994 (Cestoda: Phyllobothriidae) in Moreton Bay, ics of the lemon sharks (genus Negaprion). Mol Ecol Queensland, Australia, with the description of four new species. 17:5336–5348 Syst Parasitol 94:941–970 Haseli M, Palm HW (2015) Dollfusiella qeshmiensis n. sp. (Cestoda: Trypanorhyncha) from the cowtail stingray Pastinachus sephen Publisher’s Note Springer Nature remains neutral with regard to (Forsskal) in the Persian Gulf, with a key to the species of jurisdictional claims in published maps and institutional affiliations. Campbell & Beveridge, 1994. Syst Parasitol 92:161–169

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