Introduction
Cestodes of the family Rhinebothriidae Euzet, 1953 are specifically known to parasitise batoids. Among them, the genus Rhinebothrium Linton, 1890 stands out as the most diverse taxon, with 58 valid species (Global Cestode Database 2024; Golzarianpour et al. Reference Golzarianpour, Malek, Golestaninasab, Sarafrazi and Kochmann2020a; Menoret & Ivanov Reference Menoret and Ivanov2023; Trevisan & Caira Reference Trevisan and Caira2020) from approximately 50 host species (Global Cestode Database 2024; Menoret & Ivanov Reference Menoret and Ivanov2023; Trevisan & Caira Reference Trevisan and Caira2020).
Although more than 40 batoid species have been recorded in the Persian Gulf and the Gulf of Oman (Jabado et al. Reference Jabado, Kyne, Pollom, Ebert, Simpfendorfer, Ralph and Dulvy2017), only four Rhinebothrium species have been described so far, namely R. persicum Golestaninasab & Malek, 2016, and R. kruppi Golestaninasab & Malek, 2016 from Glaucostegus granulatus; R. atabaki Golzarianpour, Malek, Golestaninasab, Sarafrazi & Kochmann, 2020 from Maculabatis randalli; R. klimpeli Golzarianpour, Malek, Golestaninasab, Sarafrazi & Kochmann, 2020 from Pateobatis fai and Brevitrygon walga (Global Cestode Database 2024; Golzarianpour et al. Reference Golzarianpour, Malek, Golestaninasab, Sarafrazi, Kochmann and Klimpel2020b).
Although the Rhinebothriidea Healy, Caira, Jensen, Webster and Littlewood, 2009 have been primarily recognised as oioxenous parasites (Caira & Jensen Reference Caira and Jensen2017), it has been recently shown that at least some species are not strictly host-specific, e.g., R. atabaki parasitises seven host species (Golestaninasab Reference Golestaninasab2014; Golzarianpour et al. Reference Golzarianpour, Malek, Golestaninasab, Sarafrazi and Kochmann2020a). Although marine Rhinebothrium species typically parasitise one or occasionally two host species (Healy Reference Healy2006), freshwater lineages often parasitise various host species (Laudet et al. Reference Laudet, Reyda and Marques2011). Challenges arise when metastenoxenous species infect hosts that are neither sympatric nor congeneric (Mantovani Reference Mantovani2018). Healy (Reference Healy2006) suggests that our current understanding of host specificity among rhinebothriid species is based on limited sampling and misidentification of hosts and parasites, emphasising the need for additional evidence to evaluate host specificity accurately.
In our previous study for describing two other species, it was verified that there are also two new sister taxa of Rhinebothrium with a very close genetic distance and remarkable morphological similarities. Therefore, by examining more specimens and employing a meticulous morphomeristic approach, we aim to describe these new sister species of Rhinebothrium. For a higher certainty, the host specificity patterns were also studied by examining numerous host species from different geographical locations (a total of 102 individuals including seven different species from four different stations) for these two sister parasite species. Furthermore, a relevant comment on the host specificity is presented in the conclusion.
Material and methods
Host specimen collection
Host specimens were collected as bycatch with the cooperation of local fishermen along the northern coastlines of the Persian Gulf and the Gulf of Oman. Sampling localities are presented in Figure 1. Those specimens that were alive were returned to the sea. The specific localities were as follows: from the Persian Gulf, off Bushehr (28°52’45.6"N 50°43’09.7"E) in March 2011 and August 2017, off Hormuz Island (27°02’52.9"N 56°31’49.1"E) in July 2017, off Bandar Abbas (27°06’41.9"N 56°13’32.6"E) in July 2017, and from the Gulf of Oman, off Djod, Zarabad (25°26’59.4"N 59°30’27.4"E) in June 2010, April 2011, August 2014, January 2016, March and June 2017. A total of 102 individuals representing seven batoid species were examined, comprising Brevitrygon walga (N = 3), Glaucostegus granulatus (N = 23), Glaucostegus halavi (N = 1), Pastinachus sephen (N = 21), Rhynchobatus laevis (N = 1), Maculabatis arabica (N = 11), and Maculabatis randalli (N = 42) (Table 1). Each specimen was designated a unique MM number for consistent identification purposes.
Figure 1. Sampling localities: 1: Off Bushehr; 2: Off Hormuz Island; 3: Off Bandar Abbas; 4: Off Djod, Zarabad.
Table 1. Summary of the examined hosts and the sampling localities
The spiral intestine of each host specimen was excised and longitudinally opened. A section of the intestine was initially fixed in 4% seawater-buffered formalin, and after two weeks transferred to 70% ethanol for morphological examination, the remaining portion was preserved in 96% ethanol for any relevant study in future. The taxonomic classification of batoids adheres to the guidelines outlined by Last et al. (Reference Last, White and Naylor2016). Moreover, the hosts’ identity was molecularly analysed in our previous study (Golzarianpour et al. Reference Golzarianpour, Malek, Golestaninasab, Sarafrazi, Kochmann and Klimpel2020b); the details are provided in Table 2.
Table 2. Details of the molecularly analysed host and parasite specimens (Golzarianpour et al., Reference Golzarianpour, Malek, Golestaninasab, Sarafrazi and Kochmann2020a; Golzarianpour et al., Reference Golzarianpour, Malek, Golestaninasab, Sarafrazi, Kochmann and Klimpel2020b)
Abbreviations: COI, cytochrome c oxidase subunit I; LSU, large subunit ribosomal RNA gene; ND2, NADH dehydrogenase subunit 2 gene; SSU, small subunit ribosomal RNA gene.
a Rhinebothrium cf. oligotesticulare as mentioned in Golzarianpour et al. Reference Golzarianpour, Malek, Golestaninasab, Sarafrazi and Kochmann2020a.
b Rhinebothrium sp. A as mentioned in Golzarianpour et al. Reference Golzarianpour, Malek, Golestaninasab, Sarafrazi and Kochmann2020a.
Parasite specimen preparation
Specimens underwent preparation as whole mounts and for scanning electron microscopy (SEM) following the protocols outlined by Healy (Reference Healy2006) and Golzarianpour et al. (Reference Golzarianpour, Malek, Golestaninasab, Sarafrazi and Kochmann2020a), respectively. Prepared whole mounts were subjected to measurement and examination using Leica Application Suite V.3 software installed on a Leica DM500 microscope (Buffalo Grove, Illinois, USA) equipped with a Leica ICC50 HD built-in camera. Measurements of all genitalia were conducted on the terminal proglottid, except in cases where specimens exhibited a terminal proglottid with atrophied testes and expanded vas deferens, wherein the testes were measured on the mature subterminal proglottid. The parasites identity was molecularly analysed in our previous study (Golzarianpour et al. Reference Golzarianpour, Malek, Golestaninasab, Sarafrazi and Kochmann2020a) and the respective details are provided in Table 2. All measurements were performed using Digimizer software v. 6.4.0 (MedCalc Software Ltd., Belgium). The scolex and remaining proglottids of each specimen included in the molecular analysis were mounted as a voucher and subjected to measurement. Measurement data are presented as range followed by a parenthesis including the mean and standard error. All measurements are in μm unless stated otherwise; number of specimens measured as N and total number of measurements as n.
Line drawings were generated utilizing a line tube attached to Richert Biovar Microscope and optimised in Adobe Illustrator CC 2022. Scanning electron micrographs were captured using a field emission SEM (4160102HIT, Hitachi, Tokyo, Japan). Microthrix terminology follows the conventions outlined by Chervy (Reference Chervy2009). The corrections provided by Coleman et al. (Reference Coleman, Beveridge and Campbell2018) on the orthography of specific epithets of species of Rhinebothrium were followed throughout this manuscript.
Abbreviations utilised are DW, disk width; INPM, Iranian National Parasitology Museum, Tehran, Iran; MM, Prof. Masoumeh Malek’s parasite collection; ZMB, Museum für Naturkunde, Berlin, Germany; ZUTC, Collection of Zoological Museum, University of Tehran, Iran.
Results
Hosts Descriptive Report
Of 11 individuals of Maculabatis arabica, three were female and eight were male. Total length, disk width and disk length of hosts were 56–119.4 cm (86.2 ± 23.3 cm), 17–47 cm (32.6 ± 12.1 cm), and 15.5–46 cm (29.8 ± 11.9 cm), respectively.
Of 42 individuals of Maculabatis randalli, 23 were female and 19 were male. Total length, disk width and disk length of hosts were 64.4–128.2 cm (99.7 ± 17.9 cm), 19.2–45.3 cm (32.9 ± 6.5 cm), and 15.8–41.4 cm (27.5 ± 5.8 cm), respectively.
Description of Parasites
Rhinebothrium gossi sp. nov. ( Figs 2 - 3 )
Figure 2. Line drawing of Rhinebothrium gossi sp. nov. from it’s host stingray Maculabatis arabica: A and B, the holotype (ZUTC Platy. 1900, slide MM1441F4); C and D, paratype (ZUTC Platy. 1904, slide MM1547F8). A, Whole worm; B, scolex; C, the terminal proglottid; D, a mature proglottid.
Figure 3. SEM of Rhinebothrium gossi sp. nov. from it’s host stingray Maculabatis arabica. A (MM1531P2S2 voucher) and B (MM1547P1S2 voucher), Scolex; C, The middle region of loculi on the adhesive distal surface of bothridium; D, Non-adhesive proximal surface of bothridium; E, Transverse and longitudinal septa on the adhesive surface; F, Bothridium rim on the non-adhesive surface. Scale: A = 200 μm; B = 300 μm; F, D, C = 3 μm, and E = 9 μm. Note: both vouchers were molecularly assigned to the given species.
Zoobank code. http://zoobank.org/urn:lsid:zoobank.org:act: B3418FFD-BE0C-47D9-9C27-85BE3068B686
Diagnosis (Figs. 2–3). The distinctive features of Rhinebothrium gossi sp. nov. are the euapolytic reproductive strategy; bearing a single loculus at the posteriormost end of the bothridia, 50 loculi in each bothridium, loculi absent at the bothridia hinge; six to eight testes limited to the anterior field of the genital pore; position of the genital pore in the anterior region of the mature proglottids; vitelline glands interrupted at the level of genital pore, not interrupted neither at the aporal level of genital pore nor by the ovary; cirrus sac containing coiled armed cirrus.
Description (Figs. 2A–B, 3A–B). Based on whole mounts of 10 mature worms, four scoleces prepared for SEM, four strobila of SEM vouchers, and three molecular vouchers. Rhinebothriidae: worms euapolytic, slightly craspedote proglottids; total length 7.2–12.4 mm (9.1 ± 0.7 mm; N = 10), maximum width 0.2–1.2 mm (0.7 ± 0.1 mm; N = 10) at the level of scolex, with 33–145 (65.3 ± 16.6; N = 10) proglottids. Scolex consists of scolex proper with four stalked bothridia, stalklet and myzorhynchus lacking. Bothridia 237.7–464.8 (320.2 ± 19.8; N = 10; n = 15) long by 129.2–293.5 (143.4 ± 15.3; N = 10; n = 13) wide, no loculi at the hinge site, anterior and posterior halves of bothridia almost equal in size (Fig. 2B), each divided by 11 pairs of transverse septa and a single conspicuous medial longitudinal septum into two columns of transversely orientated loculi, with a single loculus at the tip of each half of bothridium, in total 50 loculi (N = 14; n = 43) per bothridium, widest in the middle of each bothridium, marginal loculi lacking; posteriormost loculus single 18.1–44.4 (32.6 ± 2.5; N = 10) in length and 39.7–65.6 (52.4 ± 3; N = 10) in width. Stalk 89.5–449.2 (194.9 ± 19.6; N = 10; n = 21) long by 38.1–156.7 (105.5 ± 7.1; N = 10; n = 21) wide, attaching to the middle region of bothridium. Cephalic peduncles vary in constriction state 40.1–221 (90.2 ± 33.5; N = 10) long, shorter than bothridium stalks in most.
Strobila (Figs. 2A). Immature proglottids numerous, 21–137 (53.8 ± 17.5; N = 10), wider than long in the anterior half, 11.8–403.2 (96.9 ± 15.9; N = 18; n = 38) in length, 14.9–241.9 (126.2 ± 7.1; N = 18; n = 38) in width. Fewer mature proglottids 7–16 (11 ± 1.4; N = 10) apparently longer than wide, with a length of 183.1–958.8 (390.8 ± 35.6; N = 18; n = 25), and a width of 116.8–290.8 (175.4 ± 9.9; N = 18; n = 25), usually beginning at the posterior one third, mostly with atrophying testes; terminal proglottid spindle-shaped with atrophied testes, 267.8–958.8 (575.9 ± 78.3; N = 13) long and 140.9–288.5 (201.5 ± 20.6; N = 13) wide. No gravid proglottids observed.
Reproductive system (Figs 2C–D). Genital pores lateral, irregularly alternating, anteriorly positioned at 69–73.3% (70.4% ± 1.4; N = 10) of proglottid length from posterior end; genital atrium conspicuous, non-muscular. Testes 6–8 (6.7 ± 0.3; N = 13) in number, 11.3–46.2 (27.5 ± 1; N = 13; n = 52) long by 11.2–67 (40.1 ± 2.2; N = 13; n = 52) wide, in single field anterior to genital pore, arranged in two columns and oval shaped, gradually atrophying in most mature proglottids; postporal testes lacking. Vas deferens duct coiled, entering cirrus sac from anterior margin; cirrus sac small, oval shaped, reaching the ovarian level, 114.8–193.5 (154.3 ± 8.4; N = 10) long by 28.9–103.6 (65 ± 6.3; N = 10) wide in the terminal proglottid. Cirrus sac crossing proglottid midline. Cirrus coiled, bearing conspicuous spinitriches. Vagina connecting common atrium anterior to cirrus, thick-walled in mature and terminal proglottids, extending anteriorly with relatively even width from genital atrium far from middle line of the proglottid, then bending towards posterior region along the aporal margin of cirrus sac, extending to the ootype, slightly overlapping cirrus sac margins in some, the proximal region of the vagina has a vaginal sphincter. Ovary Ɐ-shaped, almost symmetrical, lobular, occupied 39.3–59.8% (47.2 ± 3.4; N = 10) of the terminal proglottid, poral lobe 89.6–396.1 (215.6 ± 27.7; N = 15; n = 18) long by 22.5–77.8 (46.4 ± 4.4; N = 15; n = 18) wide, aporal lobe 87.2–400.4 (203.5 ± 25.3; N = 15; n = 18) long by 18.2–61.3 (39.9 ± 2.9; N = 15; n = 18) wide; ovarian isthmus close to posterior apex of the ovary. Mehlis’ gland, and seminal receptacle anterior to ovarian isthmus. Vitellaria follicular, follicles with irregular shapes, 3.2–37.5 (15.9 ± 1.4; N = 17; n = 38) long by 3.1–26.1 (12.6 ± 0.9; N = 17; n = 38) wide, occupying two lateral bands in two dorsal and ventral columns, extending from anterior extent of testicular field to posterior margin of proglottid far from the level of ovary, interrupted at the level of genital pore, uninterrupted neither at the aporal level of genital pore nor by the ovary; uterus saccate, obvious at the mature terminal proglottids, extending from the posterior region of the proglottid to the anteriormost margin of the testes field.
SEM (Fig 3). Distal surfaces of anterior and posterior regions of bothridia covered with varying densities of small gladiate spinitriches and acicular or capilliform filitriches; distal surfaces of the middle part of bothridia covered with small gladiate spinitriches and capilliform filitriches. Proximal surfaces of bothridia covered with capilliform filitriches. An obvious rim encircles the bothridium.
Taxonomic summary
Classification. Rhinebothriidea (Order), Rhinebothriidae (Family)
Type materials. Holotype: (ZUTC Platy. 1990), slide MM1441F4; paratypes: seven whole mounts (ZUTC Platy. 1901–1907), one whole mount (INPM.ACC.2023.C.29), slide MM1412F4, one whole mount (ZMB E.7759), slide MM1412F1, one SEM (ZUTC Platy. 1908), and DNA hologenophores (ZUTC Platy. 1912).
Other Material Examined. Three SEM (ZUT Platy. 1909–1911) and two DNA vouchers (ZUT Platy. 1913–1914).
Type host. Maculabatis arabica Manjaji-Matsumoto and Last, Reference Manjaji-Matsumoto and Last2016 (Myliobatiformes: Dasyatidae), MM1441: DW=46.7 cm, female.
Additional host. Maculabatis randalli (Last, Manjaji-Matsumoto & Moore, 2012).
Prevalence. 45.5% (five of 11 individuals) in M. arabica, 7.1% (three of 42 individuals) in M. randalli.
Mean Intensity. 3.4 ± 1.4 in M. arabica, 1 in M. randalli.
Type locality. Off Djod (25°26’59.4"N 59°30’27.4"E), Zarabad, Gulf of Oman, Iran.
Additional localities. Off Bandar Abbas (27°06’41.9"N 56°13’32.6"E), Hormuzgan, Persian Gulf, Iran.
Site in host. Spiral intestine.
Etymology. The species is named in honor of Professor Greg Goss, University of Alberta for his invaluable and significant research on fish physiology and toxicology.
Remarks. Rhinebothrium gossi sp. nov. was determined by Golzarianpour et al. (Reference Golzarianpour, Malek, Golestaninasab, Sarafrazi and Kochmann2020a) as Rhinebothrium cf. oligotesticulare (Subramaniam, 1940) and they mentioned that a more detailed consideration on this species is nessessary. By having a single posteriormost loculus (vs. paired loculi at the posterior end of the bothridium) it differentiates from R. brooksi Reyda & Marques, 2011, R. copianullum Reyda, 2008, R. corbatai Menoret & Ivanov, 2011, R. fulbrighti Menoret & Ivanov, 2011, R. ghardaguense Ramadan, 1984, R. jaimei Marques & Reyda, 2015, R. mistyae Menoret & Ivanov, 2011, R. margaritense Mayes & Brooks, 1981, R. paratrygoni Rego & Dias, 1976, R. rhinobati Dailey & Carvajal, 1976, R. setiensis Euzet, 1955, R. taeniuri Ramadan, 1984, R. tetralobatum Brooks, 1977, and R. tumidulum (Rudolphi, 1819). More details are provided in Table 2. Of the remaining 42 species, it compares with taxa bearing more than four and fewer than 10 testes. By having 6–8 testes, R. gossi sp. nov. resembles R. bunburyense Coleman, Beveridge & Campbell, 2019, R. gravidum Friggens & Duszynski, 2005, R. maccallumi Linton, 1924, R. oligotesticulare (Subramaniam, 1940), R. palmeri sp. nov., R. ruhnkei Trevisan & Caira, Reference Trevisan and Caira2020, R. urobatidum Young, 1955, R. vandiemeni Coleman, Beveridge & Campbell, 2019, and R. walga Shipley & Hornell, 1906. However, it differs in the number of loculi; i.e., 50 in R. gossi sp. nov. vs. 34 in R. bunburyense, 29–31 in R. maccallumi, 34–50 as mentioned by Coleman et al. (Reference Coleman, Beveridge and Campbell2018) or 9–13 pairs of loculi on each half bothridium as mentioned by Subhapradha (Reference Subhapradha1955) in R. oligotesticulare, 42 in R. palmeri sp. nov., 68–78 in R. ruhnkei, 38–42 in R. urobatidum, 38 in R. vandiemeni, and 20 in R. walga.
Although R. gravidum have 40–56 loculi, euapolytic vs. apolytic strategy, higher number of proglottids (9–21 vs. 33–145), and acraspedote vs. slightly craspedote segments distinguish it from R. gossi sp. nov. The characteristics of the R. gossi sp. nov. are very similar to the R. oligotesticulare, but this species has 9–13 pairs of loculi on each half bothridium as mentioned by Subhapradha (Reference Subhapradha1955) or 34–50 as mentioned by Coleman et al. (Reference Coleman, Beveridge and Campbell2018), which is a larger range, whereas in this newly introduced species only 12 pairs of loculi on each half of bothridium was observed. Moreover, the R. oligotesticulare was introduced with the total length of the body as 14.5–20 mm (vs. 7.2–12.4 mm in R. gossi sp. nov.) and 66 proglottids (vs. 33–145 in R. gossi sp. nov.). Furthermore, R. oligotesticulare has 4–7 testes and an H or X-shaped ovary while R. gossi sp. nov. bears 6–8 testes and an Ɐ-shaped ovary. Rhinebothrium oligotesticulare was reported from Glaucostegus granulatus while R. gossi sp. nov. was discovered from Maculabatis arabica. Accordingly, it is reasonable to conclude that the worm is distinct from R. oligotesticulare.
Rhinebothrium palmeri sp. nov. (Figs 4–5)
Figure 4. Line drawing of Rhinebothrium palmeri sp. nov. from it’s host stingray Maculabatis randalli: A and B, holotype (ZUTC Platy. 1915, slide MM1486F1); C and D, paratype (ZUTC Platy. 1926, slide MM1485F2). A, whole worm, scale = 200 μm; B, scolex; C, a mature proglottid; D, the terminal proglottid.
Figure 5. SEM of Rhinebothrium palmeri sp. nov. from it’s host stingray Maculabatis randalli (MM1531S3 voucher). A, Scolex; B, the single loculus at the anterior tip of bothridium; C, the middle region of loculi on the distal adhesive surface of bothridium; E, transverse septum on adhesive surface; F, bothridium stalk; G, non-adhesive proximal surface of bothridium; H, terminal proglottid with the protruding cirrus [detached from MM1413S1 voucher]; I, the short cephalic peduncle; J, the margin of bothridium; K, the margin of bothridium on the non-adhesive surface; L, the margin of bothridium on the adhesive surface; A = 300 μm; B = 10 μm; C, E = 2 μm; F, 3 μm; G = 2 μm; H = 200 μm; I = 9 μm; K = 2 μm; L = 5 μm.
Zoobank code. http://zoobank.org/urn:lsid:zoobank.org:act:1330CA70-DF04-4751-A9BC-AE9B55B9012B
Diagnosis (Figs 4–5). The distinctive features of R. palmeri sp. nov. are the euapolytic reproductive strategy; bearing a single loculus at the posteriormost end of the bothridia, 42 loculi in each bothridium, loculi absent at the bothridia hinge; 8–14 testes in the anterior field of the genital pore; the presence of the genital pore in the anterior region of the mature proglottids; vitelline glands interrupted at the level of genital pore, not interrupted neither at the aporal level of genital pore nor by the ovary; cirrus sac containing coiled armed cirrus.
Description (Figs. 4A–B, 5A–B). Based on whole mounts of 35 mature worms, four scoleces prepared for SEM, four strobila of SEM vouchers, and five molecular vouchers. Rhinebothriidae: Worms euapolytic, slightly craspedote proglottids, total length 5.6–19.6 mm (10.6 ± 0.7 mm; N = 28), maximum width 0.7–1.3 mm (1.0 ± 0.04 mm; N = 35) at the level of scolex, with 40–173 (92.3 ± 7.0; N = 28) proglottids. Scolex consists of scolex proper with four stalked bothridia, stalklet and myzorhynchus lacking. Bothridia 173.8–523.7 (329.2 ± 7.3; N = 35; n = 91) long by 113.4–282.1 (177.7 ± 4.1; N = 35; n = 75) wide; no loculi at the hinge site, anterior and posterior halves of bothridia almost equal in size (Fig. 4B), each divided by 10 pairs of transverse septa and a single conspicuous medial longitudinal septum into two columns of transversely orientated loculi, with a single loculus at the tip of each half of bothridium, in total 42 loculi (N = 39; n = 141) per bothridium, widest in the middle of each bothridium, marginal loculi lacking; Posteriormost loculus single 16.8–45.9 μm (32.6 ± 0.8; N = 33; n = 62) in length and 23.9–83.1 (49.9 ± 1.4; N = 33; n = 62) in width. Stalks 96.1–584.8 (241.1 ± 10.5; N = 35; n = 75) long by 63.1–230.9 (115.9 ± 4.3; N = 35; n = 75) wide, attaching to the middle region of bothridium. Cephalic peduncles vary in constriction state 18.6–107.0 (48.5 ± 3.5; N = 34) long, shorter than bothridium stalks in most.
Strobila (Figs. 4A). Immature proglottids numerous, 38–160 (86.1 ± 6.5; N = 35), wider than long in the anterior half, 12.4–435.8 (140.6 ± 7.8; N = 38; n = 137) in length, 49.2–466.5 (168.3 ± 6.4; N = 38; n = 137) in width. Fewer mature proglottids 1–17 (6.2 ± 0.7; N = 35) apparently longer than wide, with a length of 224.1–859.4 (454.9 ± 24.9; N = 45; n = 40), and a width of 83.4–441.1 (222.3 ± 14.0; N = 45; n = 40), usually beginning at the posterior one third, mostly with atrophying testes; terminal proglottid spindle-shaped with atrophied testes, 448.0–1074.1 (732.5 ± 43.9; N = 40) long and 115.9–253.2 (192.6 ± 13.5; N = 40) wide. No gravid proglottids observed.
Reproductive system (Figs 4C–D). Genital pores lateral, irregularly alternating, anteriorly positioned at 53.9%–76.6% (66.3% ± 2.3; N = 26) of proglottid length from posterior end; genital atrium conspicuous, non-muscular. Testes 8–14 (10.3 ± 1.3; N = 35) in number, 11.3–46.2 (28.3 ± 1.1; N = 35; n = 60) long by 11.2–70.2 (39.6 ± 2.1; N = 35; n = 60) wide, in single field anterior to genital pore, in an irregular arrangement and oval shaped, gradually atrophying in most mature proglottids; postporal testes lacking. Vas deferens duct coiled, entering cirrus sac from anterior margin; Cirrus sac small, oval shaped, reaching the ovarian level, 114.8–273.7 (173.3 ± 17.1; N = 28; n = 30) long by 28.9–166.9 (78.6 ± 11.1; N = 28; n = 30) wide in the terminal proglottid. Cirrus sac crossing proglottid midline. Cirrus coiled, bearing conspicuous spinitriches. Vagina connecting common atrium anterior to cirrus, thick-walled in mature and terminal proglottids, extending anteriorly with relatively even width from genital atrium far from middle line of the proglottid, then bending towards posterior region along the aporal margin of cirrus sac, extending to the ootype, slightly overlapping cirrus sac margins in some, the proximal region of the vagina has a vaginal sphincter. Ovary Ɐ-shaped, almost symmetrical, lobular, occupied 31.9%–53.3% (42.2% ± 2.7; N = 35) of the terminal proglottid, 100.0–581.9 (323.4 ± 27.2; N = 30; n = 36) long by 30.5–101.0 (62.5 ± 5.7; N = 30; n = 36) wide, aporal lobe 119.2–572.3 (316.9 ± 22.1; N = 30; n = 36) long by 20.2–95.9 (55.4 ± 5.2; N = 30; n = 36) wide; ovarian isthmus close to posterior apex of the ovary. Mehlis’ gland, and seminal receptacle anterior to ovarian isthmus. Vitellaria follicular, follicles with irregular shapes, 7.7–28.3 (15.5 ± 0.4; N = 40; n = 76) long by 4.2–20.6 (9.1 ± 0.5; N = 40; n = 77) wide, occupying two lateral bands in two dorsal and ventral columns, extending from anterior extent of testicular field to posterior margin of proglottid far from the level of ovary, interrupted at the level of genital pore, uninterrupted at neither the aporal level of genital pore nor by the ovary; uterus saccate, obvious at the mature terminal proglottids, extending from the posterior region of the proglottid to the anteriormost margin of the testes field.
SEM (Fig 5). Distal surfaces of anterior and posterior regions of bothridia covered with varying densities of small gladiate spinitriches and acicular or capilliform filitriches; distal surfaces of the middle part of bothridia covered with small gladiate spinitriches and capilliform filitriches. Proximal surfaces of bothridia covered with capilliform filitriches. An obvious rim encircles the bothridium.
Taxonomic summary
Classification. Rhinebothriidea (Order), Rhinebothriidae (Family)
Type materials. Holotype: (ZUTC Platy. 1915), slide MM1486F1; Paratypes: 32 whole mount (ZUTC Platy. 1916–1947), one whole mount (INPM.ACC.2023.C.30), slide MM1567F4, one whole mounts (ZMB E.7760), slide MM1486F5, one SEM (ZUTC Platy. 1948), and DNA hologenophores (ZUT Platy.1952).
Other Material Examined. Three SEM (ZUT Platy.1949–1951) and four DNA vouchers (ZUT Platy.1953–1956).
Type host. Maculabatis randalli (Myliobatiformes: Dasyatidae), MM1486: DW=37.4 cm, Male.
Additional host. Maculabatis arabica (Myliobatiformes: Dasyatidae).
Prevalence. 40.5% (17 of 42 individuals) in M. randalli. 9.1% (1 of 11 individuals) in M. arabica.
Mean Intensity. 2.4 ± 0.3 in M. randalli and 2 in M. arabica.
Type locality. Off Hormuz Island (27°02’52.9"N 56°31’49.1"E), Persian Gulf, Iran.
Additional localities. Off Djod (25°26’59.4"N 59°30’27.4"E), Zarabad, Gulf of Oman, Iran.
Site in host. Spiral intestine.
Etymology. The species is named in honor of Professor Rich Palmer, University of Alberta, for his many years of contribution towards ecology and evolution of marine invertebrates.
Remarks. This new species was mentioned in Golzarianpour et al. (Reference Golzarianpour, Malek, Golestaninasab, Sarafrazi and Kochmann2020a) as Rhinebothrium sp. A. It is also found in Maculabatis arabica. It differs from all but seven (i.e., R. euzeti Williams, 1958, R. gravidum, R. gossi sp. nov. (Fig. 6), R. hawaiiense Cornford, 1974, R. ruhnkei, R. urobatidium (Young, 1955), and R. verticillatum (Subhaprada, 1955) of 58 species of the genus Rhinebothrium because of bearing fewer than 15 testes and more than seven testes. Those marine and freshwater species that possess paired loculi at the posterior end of the bothridium (vs. single in R. palmeri sp. nov.) were excluded from the comparison (see Table 2). It has fewer number of loculi than R. euzeti, R. gossi sp. nov., R. ruhnkei, and R. verticillatum (42 vs. 78, 50, 68–78, and 48–50, respectively). It has more loculi than R. hawaiiense (42 vs. 23–25). It differs from R. gravidum and R. urobatidium in greater size (5.6–19.6 vs. 1.8–5.3 and 3.1–3.4, respectively), higher number of proglottids (40–173 with average 97 vs. 9–21 and 30–41, respectively), and location of genital pore (53.9–76.6% vs. 50–60% and a genital pore in the posterior half of the mature segments, respectively). Moreover, R. palmeri sp. nov. distinguishes from R. gravidum by its reproductive strategy (euapolytic vs. apolytic).
Figure 6. Light micrograph of the Holotypes: Left, Rhinebothrium gossi sp. nov. from it’s host stingray Maculabatis arabica (ZUTC Platy. 1990, slide MM1441F4); Right, Rhinebothrium palmeri sp. nov. from it’s host stingray Maculabatis randalli (ZUTC Platy. 1915, slide MM1486F1); Scale = 0.5 mm.
Discussion
Both new species described here (Fig. 6) are distinguishable from four other congeneric species previously described from the given region, the type species R. flexile and other valid species of Rhinebothrium by a combination of a single posteriormost loculus and number of testes along with some other morphological characteristics discussed earlier. A brief report of the main morphomeristic characteristics of these two species and the other related congeners with less than 15 testes has been reported in Table 3. In this table, the species have been sorted based on the state of the posteriormost loculus (being paired instead of single) and the testicular number. Therefore, it provides a proper insight into distinguishing the close species from each other with more ease. Some other species, including R. setiense Euzet, 1955 and R. ghardaguense also bear paired loculi at the posteriormost part of the bothridium, but they were removed from the list because of having higher than 15 testes in their mature proglottids. These two characteristics are reliable to delimit species, whereas other traits show a mosaic inconsistent pattern among the present taxa. Of 38 listed species, 26 taxa have a single loculus at the posteriormost end. Among them, seven species namely R. urobatidum, R. ruhnkei, R. gravidum, R. gossi sp. nov., R. tumidulum, R. euzeti, R. hawaiiense, and R. verticillatum have such a number of testes, which match with the testicular range of Rhinebothrium palmeri sp. nov. The number of loculi of the bothridium, as the third distinctive trait, is robust enough to distinguish this new species from those taxa. The same order of characteristics usage could be applied to Rhinebothrium gossi sp. nov. for differentiating it from close species.
Table 3. The main morpho-meristic characteristics, hosts, and ecological regions of Rhinebothrium species with less than 15 testes, including Rhinebothrium gossi sp. nov. and Rhinebothrium palmeri sp. nov
-, characteristic not mentioned in the original paper; *, just one specimen reported.
In this study, we reported two shared parasite species from two different but genetically close hosts, Maculabatis arabica and M. randalli. Taxonomic relationships of the hosts were analysed using the NADH2 marker, with verification through using the reference samples (Figures 7 and 1 in Golzarianpour et al. Reference Golzarianpour, Malek, Golestaninasab, Sarafrazi, Kochmann and Klimpel2020b). The accession numbers of these host individuals are MN602003 and MN602004 for M. arabica; MN602006 and MN602007 for M. randalli (the details are mentioned in Table 2). In the current study, the introduced parasites were obtained from the same host individuals which were analysed molecularly in our previous study. Notably, the interspecific mean p-distance between them was as low as 3% in the NADH2 marker (Table 3 in Golzarianpour et al. Reference Golzarianpour, Malek, Golestaninasab, Sarafrazi, Kochmann and Klimpel2020b).
Figure 7. Phylogenetic relationships tree of Rhinebothriidae adapted from Golzarianpour et al. (Reference Golzarianpour, Malek, Golestaninasab, Sarafrazi and Kochmann2020a); Note: the sister relationship is shown in the red rectangle.
On the basis of K2P distance from NADH2 marker, the average intraspecific and interspecific distance values for elasmobranchs are 0.27%, and 10.81%, respectively (Naylor et al. Reference Naylor, Caira, Jensen, Rosana, White and Last2012). Golzarianpour et al. (Reference Golzarianpour, Malek, Golestaninasab, Sarafrazi, Kochmann and Klimpel2020b) have mentioned that the distribution range of M. arabica is broader than what was discussed by Manjaji-Matsumoto & Last (Reference Manjaji-Matsumoto and Last2016) and Fernando et al. (Reference Fernando, Bown, Tanna, Gobiraj, Ralicki, Jockusch, Ebert, Jensen and Caira2019), and documented the presence of this species in the Persian Gulf. These findings confirm that M. arabica and M. randalli coexist in the northern regions of the Persian Gulf and the Gulf of Oman, where they have overlapped dispersion. Although there is no geographical barrier between these stingrays in their present distribution, this close relationship could be caused by a recent divergence in the northwest region of the Indian Ocean. As mentioned by Martin et al. (Reference Martin, Naylor and Palumbi1992) and because of this slight interspecific mean p-distance, the speciation phenomenon may have been occurred in the Persian Gulf during the early Pleistocene, when the level of water in the oceans was changed repeatedly. This could be considered as a geographical barrier in this region which was resulted into a reproductive barrier. They may extend their distribution subsequently and diversify in the Holocene, when the Persian Gulf was connected once again (Jabado et al. Reference Jabado, Al Ghais, Hamza, Shivji and Henderson2014).
Although new parasite species presented here (i.e., Rhinebothrium gossi sp. nov. and Rhinebothrium palmeri sp. nov.) are morphologically differentiable they were found to have only diminutive genetic distance (Table 2 in Golzarianpour et al. Reference Golzarianpour, Malek, Golestaninasab, Sarafrazi and Kochmann2020a). As it was analysed in our previous study, the minimum genetic distance between Rhinebothrium gossi sp. nov. and Rhinebothrium palmeri sp. nov. (which were named as Rhinebothrium cf. oligotesticullaris and Rhinebothrium sp. A, respectively) was 0.9% (12–13 bp of 1259), 0.3% (6 bp of 1930), and 11.7% (61–64 bp of 570) for 28s rDNA, 18s rDNA, and COI genes, respectively (Golzarianpour et al. Reference Golzarianpour, Malek, Golestaninasab, Sarafrazi and Kochmann2020a). Additionally, the molecular hologenophore and paragenophores from the aforementioned study were used as a basis for the morphological descriptions (Golzarianpour et al. Reference Golzarianpour, Malek, Golestaninasab, Sarafrazi and Kochmann2020a). The accession numbers of these parasites’ individuals are MT153860 for COI, MT033095-MT033095 for SSU and MT032161-MT032162 for LSU in Rhinebothrium gossi sp. nov.; and MT153864 for COI, MT033105 for SSU and MT032171-MT032172 for LSU in Rhinebothrium palmeri sp. nov. The details are mentioned in Table 2.
This observation highlights a very close relationship between the species, like their host species as illustrated in Figure 1 of Golzarianpour et al. (Reference Golzarianpour, Malek, Golestaninasab, Sarafrazi, Kochmann and Klimpel2020b). In the phylogenetic tree for Rhinebothriidae presented by Golzarianpour et al. (Reference Golzarianpour, Malek, Golestaninasab, Sarafrazi and Kochmann2020a) (Fig. 7), the two sister taxa, Rhinebothrium cf. oligotesticullaris and Rhinebothrium sp. A, are markedly distinct. These were described here as new species: Rhinebothrium gossi sp. nov. and Rhinebothrium palmeri sp. nov., respectively. Notably, these worms were occasionally found in the same host individual. As previously mentioned, only minor genetic differentiation was observed between their hosts.
Each tapeworm parasitises both hosts in nature; however, the infection rate is unbalanced. Rhinebothrium gossi sp. nov. was isolated from eight hosts, 62.5% of which was Maculabatis arabica, whereas Rhinebothrium palmeri sp. nov. was obtained from 18 hosts, 94.4% of which was M. randalli. The overall intensity was 2.6 and 3.4 for Rhinebothrium gossi sp. nov. and Rhinebothrium palmeri sp. nov., respectively. Both parasite species were found simultaneously in one host M. randali (MM1531: 43.5 cm DW, male) from the Gulf of Oman. It can be concluded that R. gossi sp. nov. was more frequent in M. arabica, but M. randalli was mostly infected by R. palmeri sp. nov.
This fact that different host species are infected by the same parasite species is contrary to the common belief (Caira & Jensen Reference Caira and Jensen2014; Fyler Reference Fyler2009; Mantovani Reference Mantovani2018; Pickering Reference Pickering2012). First, it provides more evidence that the genus Rhinebothrium may not be an oioxenous worm. Golzarianpour et al. (Reference Golzarianpour, Malek, Golestaninasab, Sarafrazi and Kochmann2020a) well explained about other parasite species in the region. These findings confirm that different patterns of host specificity, including oioxenous, mesostenoxenous, and euryxenous, shape host-parasite association in the genus Rhinebothrium at least in the Persian Gulf and the Gulf of Oman. Second, it could be an example of co-speciation in a host-parasite system in which when the host species, namely M. arabica and M. randalli have been diverged, their parasites also speciated concurrently. This possible scenario can explain the ultimate answer to the way that such hosts are infected by these parasites. To reveal other possibilities or proximate answers, why Rhinebothrium gossi sp. nov. and Rhinebothrium palmeri sp. nov. have respectively chosen M. arabica and M. radalli as their main host, we need to have a more comprehensive view of their life cycle and many diverse factors such as the immune system capabilities, physiologic condition of the spiral intestine of different hosts, and foraging behaviors, which cause a parasite to select a special host (Johnson et al. Reference Johnson, Calhoun, Riepe and Koprivnikar2019). On the other hand, it is possible that a given host bears different parasite species in different localities because of the environmental conditions, feeding behaviors, and available intermediate hosts (Golzarianpour et al. Reference Golzarianpour, Malek, Golestaninasab, Sarafrazi and Kochmann2020a; Healy Reference Healy2006). For example, although R. leopardensis, R. nandoi, and R. ruhnkei were introduced from Himantura leoparda in Australian waters (Trevisan & Caira Reference Trevisan and Caira2020), no Rhinebothriid cestodes have been recorded from this species from the region so far. However, we are aware that more host samples should be investigated.
Considering the great diversity of batoid species in the Indo-Pacific region and the limited parasitological studies in the mentioned areas, describing new species was not far from the mind. According to the present knowledge, of 58 Rhinebothrium species found globally, 22 of them have been introduced from water bodies connected to the Indian Ocean (Menoret & Ivanov Reference Menoret and Ivanov2023; Trevisan & Caira Reference Trevisan and Caira2020). The present study increases this number to 24 and the global number to 60 species. Additional taxonomic works are essential to shed more light on the phylogeny of the genus Rhinebothrium in the extended global view.
Acknowledgements
We express our appreciation to people of Djod village for their hospitality. We thank Hussein Salari and local fishermen that provided invaluable assistance during sampling off Djod. We appreciate Skipper Fazel and his anonymous fishers for providing us with his fishing boat to sample the waters of Bushehr.
Funding
This study was financially supported by the National Science Foundation of Iran [grant number: 4030722].
Competing interest declaration
The authors declare none.
