Eniochobothrium acostae, Oosthuizen & Naidoo & Smit & Schaeffner, 2022

Oosthuizen, Geraldine, Naidoo, Kristina, Smit, Nico J. & Schaeffner, Bjoern C., 2022, Adding one more to the list: A new species of Eniochobothrium (Cestoda: Lecanicephalidea) from the Oman cownose ray in South Africa, International Journal for Parasitology: Parasites and Wildlife 19, pp. 138-147 : 140-146

3.1. Eniochobothrium acostae n. sp. Oosthuizen, Smit & Schaeffner, 2022 ( Figs. 1 and 2 )

Our observation of E. acostae n. sp. indicated the presence of two morphotypes infecting the same host individual, namely large and small. These are highly variable worms ranging greatly in size and morphological characteristics (suppl. Table 1 ). The diagnosis is based on both morphotypes (40 whole mounts of 25 immature worms, six mature and nine gravid worms [all lacking scoleces] and two immature specimens [one with scolex] prepared for SEM).

Adult worms apolytic (all lacking scoleces), 1318–6007 (3534 ± 1428; n = 15) long; maximum width either at level of trough or posteriormost proglottid 155–921 (540 ± 243; n = 15); total number of proglottids 22–47 (36 ± 7; n = 14) ( Figs. 1A and 2A ). Strobila divided into anterior trough region consisting of non-reproductive proglottids, expanding laterally to form U-shaped trough and posterior reproductive region consisting of reproductive proglottids with internal reproductive organs in mature development stages ( Fig. 1A ). Scolex from scanning electron microscopy 91 (n = 1) long by 79 (n = 1) wide, bearing four acetabula ( Fig. 2B ). Acetabula in form of sessile suckers, 47–61 (54 ± 10; n = 1; 2) long by 34–36 (35 ± 1; n = 1; 2) wide ( Figs. 1E and 2B ). Apical modification of scolex proper in form of narrowed extension with small terminal apical aperture ( Fig. 2B ), with small, mostly glandular, inextensible and irreversible apical organ. Apical organ 14 (n = 1) long by 20 (n = 1) wide ( Figs. 1E and 2B ). Cirrus covered with small, triangular microtriches visible at opening of the genital pore ( Fig. 2C ). Cephalic peduncle not observed. Non-reproductive and reproductive proglottids craspedote, non-laciniate ( Figs. 1A and 2A ). Trough 402–980 (730 ± 172; n = 15) long by 125–736 (451 ± 215; n = 14) wide, consisting of 12–22 (18 ± 3; n = 14) non-reproductive proglottids ( Fig. 1A, D , 2A, D ). Reproductive region of strobila 933–5471 (2811 ± 1320; n = 15) long by 151–921 (525 ± 242; n = 15) wide, consisting of 10–26 (17 ± 5; n = 15) reproductive proglottids ( Figs. 1A and 2A ). Immature proglottids 9–25 (16 ± 5; n = 15) in number, initially wider than long, becoming longer than wide; posteriormost immature proglottid 164–1150 (619 ± 323; n = 15) long by 370–844 (565 ± 154; n = 15) wide ( Figs. 1A and 2A ). Mature proglottids 0 or 1 in number, longer than wide, 591–2172 (1062 ± 638; n = 6) long by 151–667 (317 ± 202; n = 6) wide ( Fig. 1A and B ). Gravid proglottids 0 or 1 in number, 1208–2511 (1805 ± 475; n = 9) long by 418–921 (654 ± 148; n = 9) wide ( Fig. 1A, C ). Total number of testes 16–34 (21 ± 7; n = 6), arranged in two distinct groups of aporal and poral testes extending from anterior part to middle of proglottid in both mature and gravid proglottids ( Fig. 1B and C ). Aporal testes extend from anterior part of proglottid to corresponding group of vitelline follicles, 11–27 (15 ± 6; n = 6) in number; poral testes 3–7 (5 ± 2; n = 6) in number ( Fig. 1B and C ). Testes 15–30 (24 ± 5; n = 6) long by 6–27 (18 ± 7; n = 6) wide, anterior to ovary, in several irregular columns in dorso-ventral view ( Fig. 1B and C ). Vas deferens with glandular wall observed at level of cirrus sac, entering cirrus sac at distal end visible along lateral margin of proglottid, just posterior to margin of U-shape of cirrus sac, 206–765 (376 ± 227; n = 6) long by 18–115 (52 ± 36; n = 6) wide ( Fig. 1B–C, F ). External seminal vesicle absent. Internal seminal vesicle present, small, 31–72 (53 ± 16; n = 6) long by 15–64 (37 ± 18; n = 6) wide ( Fig. 1B–C, F ). Cirrus sac Ushaped, thick-walled, 251–935 (468 ± 267; n = 6) long by 41–111 (71 ± 24; n = 6) wide, containing a long, inverted cirrus ( Fig. 1A–C, F ). Cirrus armed, 222–889 (403 ± 275; n = 6) long by 21–46 (30 ± 10; n = 6) wide ( Fig. 1B–C, F ). Ovary H-shaped in dorso-ventral view, 197–650 (374 ± 177; n = 6) long by 49–159 (85 ± 48; n = 6) wide ( Fig. 1B and C ). Ootype between bases of ovarian lobes, large, ovoid, 61–161 (91 ± 41; n = 6) long by 32–103 (55 ± 34; n = 6) wide ( Fig. 1B and C ). Vagina absent. Genital pores lateral, irregularly alternating, 69–86% (75 ± 6; n = 6) of proglottid length from posterior margin ( Fig. 1A–C ). Uterus medial, saccate, extending from posterior margin of ovary to near posterior margin of cirrus sac, 213–799 (465 ± 284; n = 5) long by 37–54 (43 ± 7; n = 5) wide; uterine duct not observed; uterine pore absent ( Fig. 1B and C ). Vitellaria arranged in two lateral bands with multiple columns, extending from middle of cirrus sac to level of ovarian isthmus; vitelline follicles 7–37 (18 ± 13; n = 6) long by 4–24 (11 ± 10; n = 6) wide ( Fig. 1B and C ). Two lateral pairs of excretory vessels present ( Fig. 1B–C, F ). Eggs in cocoons; total number of cocoons 41–79 (62 ± 14; n = 5) ( Fig. 1C, G ). Each cocoon contains 30–42 (35 ± 6; n = 5) eggs; free cocoons 56–71 (62 ± 5; n = 6) long by 44–52 (48 ± 3; n = 6) wide ( Fig. 1G ). Eggs subspherical, thin-walled, 14–15 (14 ± 1; n = 6) long by 11–12 (12 ± 1; n = 6) wide ( Fig. 1G ).

3.2. Taxonomic summary

Type host: Oman cownose ray, Rhinoptera jayakari Boulenger ( Myliobatiformes , Rhinopteridae ).

Type locality: South-western Indian Ocean off Scottburgh (28 ◦ 78 ′ 0 ′′ S, 30 ◦ 76 ′ 0 ′′ E), KwaZulu-Natal Province, South Africa.

Additional locality: South-western Indian Ocean off Richards Bay (28 ◦ 78 ′ 07 ′′ S, 32 ◦ 03 ′ 83 ′′ E), KwaZulu-Natal Province, South Africa.

Site of infection: Spiral intestine.

Prevalence and intensity of infection: Prevalence 67% (2 out of 3 R. jayakari ); intensity> 70 worms per host.

Specimens deposited: Holotype in NMB ( NMB P-883); paratypes in IPCAS (IPCAS C-916), MNHG (MHNG-PLAT-0138936–0138937) and NMB (NMB P-884–898). The specimen used for SEM is retained in the parasite collection of the Water Research Group, North-West University.

Representative DNA sequences: Partial sequences of 28S rRNA 1229–1389 bp in length (GenBank accession numbers: ON972441; ON972440; ON972442); partial sequences of mtCOI 536–555 bp in length (GenBank accession numbers: ON964522, ON964530, ON964533). Paragenophore in NMB (NMB P-882).

ZooBank registration: The Life Science Identifier (LSID) of the article is urn:lsid:zoobank.org:pub:F0C0720A-66CE-40F4-A621-4BCD1EC233B7 . The LSID for the new name Eniochobothrium acostae n. sp. is urn:lsid:zoobank.org:act: B6CB02C9-B528-49AA-894B-4F41D24333B2.

Etymology: The species name is dedicated to Dr. Aline Angelina Acosta for her contributions to the systematics of parasitic platyhelminths.

3.3. Remarks

Eniochobothrium acostae n. sp. closely resembles congeners within Eniochobothrium , namely E. gracile , E. qatarense and E. euaxos , in morphological characteristics. However, the new species presents the largest specimen recorded in total body length (without scolex) exceeding that of E. qatarense (including scolex) by more than 300 μm ( Table 2 ). Eniochobothrium acostae n. sp. can be further distinguished from E. euaxos in possessing only postporal testes on the poral side of the proglottid while the distribution of testes in E. euaxos is both posterior and anterior of the genital pore. In addition, E. acostae n. sp. has slightly fewer testes than E. euaxos (16–34 vs. 35–48) and smaller cocoons ( Table 2 ). A morphological differentiation based on metrical features is impeded between E. acostae n. sp. and E. gracile due to a scarcity of morphological information provided in the original description ( Shipley and Hornell, 1906). However, E. acostae n. sp. can be differentiated from E. gracile in lacking the region described and illustrated as a “short neck of three segments” ( Shipley and Hornell, 1906). In the description of E. gracile, Shipley and Hornell (1906) referred to the apex of the scolex as the rostrum, whereas E. acostae n. sp. possesses a rather noticeable apical organ. Eniochobothrium acostae n. sp. differs from E. gracile in the number of mature proglottids (0 or 1 vs. ± 6–8, respectively). In addition, E. acostae n. sp. differs from E. qatarense in possessing slightly fewer testes (16–34 vs. 35–43) and fewer mature proglottids (0 or 1 vs. 4–6), respectively. Eniochobothrium acostae n. sp. has slightly smaller eggs than E. qatarense (14–15 vs. 17–24, respectively). In contrast, cocoons of E. acostae n. sp. contain 30– 42 eggs, whereas E. qatarense is described as possessing “egg balls” (sensu Al Kawari et al., 1994) containing approximately ten eggs. Eniochobothrium acostae n. sp. can also be distinguished from E. qatarense in having a larger ootype (61–161 μm vs. 40–60 μm, respectively) and a much smaller internal seminal vesicle (31–72 μm vs. 250–310 μm, respectively) ( Table 2 ). In the description of E. qatarense , the apex of the scolex is referred to as “a weak proximal pyramidal rostellum” (sensu Al Kawari et al., 1994), while E. acostae n. sp. has an apical organ. Additional differences in metrical features between E. acostae n. sp. and congeners are listed in Table 2 .

Even though we observed the presence of two morphotypes, the molecular data of smaller and larger morphotypes verified that these belong to the same species (isolate 1 and 2 – large; isolate 3 and 4 – small) ( Figs. 3 and 4 ). Regrettably, the only obtained scolex of E. acostae n. sp., which has been examined with scanning electron microscopy, was lost after the picture was taken, emphasising just how fragile the connection between the scolex and the anterior trough region of the strobila really is.

3.4. Phylogenetic relationships

Both ML and BI topologies for 28S and COI recovered the same relationships among the taxa included in the phylogenetic analyses. Figs. 3 and 4 show the ML phylogram for each analyses (28S and COI, respectively). The phylogenetic analyses of the 28S showed the clade encompassing Eniochobothrium spp. (including the new sequences of E. acostae n. sp.) as a monophyletic group (supported by BI analyses [0.99]), supporting the Eniochobothriidae that was proposed by Jensen et al. (2016) to accommodate this genus. The relationship among Eniochobothrium spp. is well supported (see Fig. 3 ). ML analyses recovered a support value of 73 between isolates 2 and 3 of E. acostae n. sp. Nevertheless, the partial sequences of the 28S of the three isolates of E. acostae n. sp. are identical (p -distance 0%, 0 difference in bp). The new South African species appeared more closely related to Eniochobothrium n. sp. 1. The Eniochobothriidae appeared more closely related to the Lecanicephalidae and the Cephalobothriidae ( Fig. 3 ). The newly generated partial mtCOI sequences of E. acostae n. sp. were compared to E. euaxos and the taxa Eniochobothrium n. sp. 1, 2 and 3. The phylogenetic analysis of the mtCOI sequences of Eniochobothrium spp. showed the new species as sister to Eniochobothrium n. sp. 1, with strong support, which mirrors the results of the 28S rRNA analysis ( Fig. 4 ).

The estimates for evolutionary divergences for 28S rRNA were compared using the newly generated and the available sequences of Eniochobothrium spp. from GenBank. The p -distances were 2.1–2.2% (27–29 bp) between E. acostae n. sp. and E. euaxos ; 0.2% (3 bp) between E. acostae n. sp. and Eniochobothrium n. sp. 1; and 5.9–7.3% (77–91 bp) between E. acostae n. sp. and Eniochobothrium n. sp. 2 and 3. The estimates for evolutionary divergences for mtCOI were compared using partial sequences of E. acostae n. sp. with the four available sequences of Eniochobothrium and one sequence of H. folifer used as an outgroup retrieved from GenBank, with p -distances varying from 0 to 22.4%. The p -distance values between E. acostae n. sp. and E. euaxos were 18.1–18.6% (96–102 bp); E. acostae n. sp. and Eniochobothrium n. sp. 1 were 8.7–8.9% (44–46 bp); and between E. acostae n. sp. and Eniochobothrium n. sp. 2 and 3 were 19.2–19.9% (92–106 bp).

4. Discussion

According to Jensen (2005), Eniochobothrium is presently one of two genera restricted to parasitising a single batoid genus. All species of Eniochobothrium currently considered valid have been described as adults from the batoid genus Rhinoptera . Eniochobothrium euaxos was described from the Australian cownose ray, Rhinoptera neglecta Ogilby , from Dundee Beach, Fog Bay, Australia; E. gracile has been reported from the flapnose ray Rhinoptera javanica Müller and Henle , from Dutch Bay, Sri Lanka; and E. qatarense infects R. javanica (as Rhinoptera adspersa Müller and Henle ) from the Arabian Gulf, Qatar. Additional host-parasite associations have been reported for three undescribed species of Eniochobothrium : Eniochobothrium n. sp 1 ( Caira et al., 2014) from the Pacific cownose ray, Rhinoptera cf. steindachneri Evermann and Jenkins , from Ship Island, Mississippi, USA; Eniochobothrium n. sp. 3 ( Jensen et al., 2016) from R. neglecta , from Dundee Beach, Fog Bay, Australia; and Eniochobothrium n. sp. 2 ( Jensen et al., 2016) from Rhinoptera sp. , from St. Louis, Senegal. In his Masters Thesis (2007), Garrett Call reports two species, Eniochobothrium overstreeti and Eniochobothrium sedlockae , from Rhinoptera bonasus Mitchill , yet these species and corresponding species names have never been officially described (Unpublished data; https://kuscholarworks.ku.edu/handle/1808/5536).

Ebert et al. (2021) verified that there is only a single species of Rhinoptera in South African waters, R. jayakari . Previous records (see Compagno et al., 1989; Compagno, 1986, 1999; Ebert et al., 2015; Heemstra and Heemstra, 2004; Smith, 1952, 1961; Wallace, 1967) mentioned the occurrence of R. javanica . Furthermore, all the voucher material from South African specimens is deposited under R. javanica . Jensen et al. (2017) estimated that 24 lecanicephalidean species parasitise rhinopterid hosts globally. Thus far, four of the eight rhinopterid hosts have been examined for Eniochobothrium , with each species hosting one to two unique species of Eniochobothrium . We therefore estimate that the actual species diversity of Eniochobothrium in this host group most likely ranges between eight and 16 species worldwide. Even considering E. acostae n. sp. from R. jayakari , only 63% of the potential rhinopterid hosts (five out of eight species) have been examined for the presence of Eniochobothrium species. Rhinoptera adspersa (Indo-West Pacific: off India, Malaysia, and East Indies), Rhinoptera brasiliensis Müller (southern tip of Brazil to western Florida) and Rhinoptera marginata Geoffroy Saint-Hilaire (western coast of Africa and Mediterranean Sea) still await parasitological examination.

The phylogenetic analyses of the 28S rRNA presented herein support the allocation of the new species within Eniochobothrium , which formed a strongly supported clade with its congeners ( Fig. 3 ). In the present study, the analyses of 28S rRNA sequences of selected lecanicephalideans corroborate the results of Jensen et al. (2016). These authors presented a concatenated analysis of lecanicephalidean sequences of four genes (complete 28S rRNA, partial 18S rRNA, partial mtCOI, and partial 16S rRNA). Their combined phylogram of the concatenated analyses recovered eight lecanicephalidean clades, similar to the analyses presented herein. The eight clades of Jensen et al. (2016) correspond to the prior existing families Lecanicephalidae , Polypocephalidae , Tetragonocephalidae , and Cephalobothriidae , and their proposed families Aberrapecidae , Eniochobothriidae , Paraberrapecidae and Zanobatocestidae . The proposal of the family Eniochobothriidae for species of Eniochobothrium by Jensen et al. (2016) was supported by their phylogenetic analyses, since this clade appeared as one of the most molecularly divergent groups. Such results were also verified in the present study ( Fig. 3 ), in which the addition of a new Eniochobothrium species did not alter the topology of lecanicephalidean families of the former authors. In addition, the present study highlights the importance of including both morphological and molecular analyses on newly collected specimens to aid the support of their phylogenetic position.

The tegument of lecanicephalideans is extremely intriguing and has value for taxonomic and presumably phylogenetic studies. According to Jensen (2005), lecanicephalideans possess a unique character trait involving a specific microthrix form described as “long, pointed filiform” (sensu Caira et al., 1999) found on different external surfaces. Microthrix pattern examinations of Jensen (2005) revealed that this unique character state of lecanicephalideans was not observed in any of the> 80 specimens forming part of the tetraphyllideans and other outgroup taxa examined by Caira et al. (1999, 2001). The description of the microthrix morphology of E. acostae n. sp., as well as comparison to that of E. euaxos , was impeded by the freezing and thawing of the host material, which seemed to negatively affect microtriches on individual body regions. Collection of fresh material from the type host and, preferably, the type locality are needed to describe the microthrix pattern of E. acostae n. sp. in the future. The microthrix patterns have been examined in only one species of Eniochobothrium (see Jensen, 2005). Therefore, additional studies focusing on the surface ultrastructure of members of Eniochobothrium can add more detailed characteristics for the diagnosis and species circumscription of representatives of the Eniochobothriidae .

Lecanicephalideans have a global distribution, known from eight of the 12 marine biogeographic regions identified by Spalding et al. (2007). The Central Indo-Pacific has the highest species diversity (69%) followed by the Western Indo-Pacific (14%). Other biogeographical realms present a much lower number of reported species [Temperate Northern Pacific, Tropical Atlantic, and Temperate Northern Atlantic (5% each), Eastern Indo-Pacific, Tropical Eastern Pacific, Temperate South America, Temperate Australasia (1% each)]. Up until now, lecanicephalideans have not been reported from the marine regions of the Arctic, Southern Ocean, and Temperate Southern Africa ( Jensen et al., 2017). Eniochobothrium acostae n. sp. is the first species of the order Lecanicephalidea reported from southern Africa. Partial sequences of 28S rRNA and mtCOI genes are provided for the new species, adding relevant data for the genus and thus aiding future studies. Phylogenetic analyses support the validity of Eniochobothriidae for species of Eniochobothrium by Jensen et al. (2016). When taking into consideration that only 63% of the potential rhinopterid hosts have been examined for the presence of Eniochobothrium species, it is clear that a considerable number of representatives might still remain unknown and await future discovery and description.

Note

Nucleotide sequence data reported in this paper are available in the GenBank™, EMBL and DDBJ databases under the accession numbers: ON972441, ON972440, ON972442, ON964522, ON964530, ON964533.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Figures

Fig. 1. Line drawings of Eniochobothrium acostae n. sp. from the South-western Indian Ocean off Scottburgh and Richards Bay, KwaZulu-Natal Province, South Africa. A, outline of entire cestode; B, mature proglottid; C, early gravid proglottid; D, trough formed by non-reproductive proglottids of the anterior strobila; E, scolex; F, terminal genitalia; G, cocoon with eggs. Abbreviations: c (cirrus); cs (cirrus sac); ex (excretory canal); gp (genital pore); isv (internal seminal vesicle); ot (ootype); ov (ovary); t (testes); u (uterus); vd (vas deferens); vf (vitelline follicle).

Fig. 2. Scanning electron micrographs of an immature specimen of Eniochobothrium acostae n. sp. from the South-western Indian Ocean off Scottburgh and Richards Bay, KwaZulu-Natal Province, South Africa. A, entire strobila; B, scolex; C, genital pore; D, trough formed by non-reproductive proglottids of the anterior strobila.

Fig. 3. Maximum likelihood phylogram based on partial sequences of the large subunit 28S rRNA gene. Nodal support is shown as posterior probability and bootstrap. GenBank accession number precedes species name. Branch length scale bar indicates the number of substitutions per site. (//) Branch length reduced to one time the scale bar; (///) branch length reduced to two times the scale bar. Squares represent Posterior Probability values while circles represent Bootstrap values.

Fig. 4. Maximum likelihood phylogram based on partial sequences of the mitochondrial cytochrome oxidase subunit I (mtCOI) gene. Nodal support is shown as posterior probability and bootstrap. GenBank accession number precedes species name. Branch length scale bar indicates the number of substitutions per site. (//) Branch length reduced to one time the scale bar. Squares represent Posterior Probability values while circles represent Bootstrap values.