A new species of Phoreiobothrium Linton, 1889 (Cestoda: Onchoproteocephalidea) from the spinner shark, Carcharhinus brevipinna (Valenciennes) off the coast of KwaZulu-Natal, South Africa

The extreme sparsity of collection efforts in many parts of the word, including southern Africa, leaves a vast hidden diversity of marine cestode species, such as species of Phoreiobothrium. The examination of a common South African coastal-pelagic shark species, Carcharhinus brevipinna (Valenciennes), resulted in the discovery of a new species of Phoreiobothrium Linton, 1889. Phoreiobothrium martinisp. n. is undoubtedly different from all other congeners in several morphological characteristics, however, it is most easily distinguished from other species by its total length, the size of its hooks, and the number of post vaginal testes. The addition of P. martinisp. n. increases the total number of valid species of Phoreiobothrium to 20 worldwide. Furthermore, P. martinisp. n. marks the description of representatives of only the second onchoproteocephalidean genus from southern Africa, therefore expanding the generic biogeographical representation and introducing new host associations. Apart from the description of this new species of cestode, the discovery of additional species of Phoreiobothrium will be beneficial regarding future ecological investigations. With the high degree of host-specificity found amongst species of Phoreiobothrium, species such as P. martinisp. n. could be used as sentinel species for the accurate identification, separation and diagnosis of commonly misidentified shark species.


Introduction
Research on cestodes of the family Onchobothriidae Braun, 1900 parasitising the spiral intestine of elasmobranch hosts has rapidly increased over the past two decades, particularly in the fields of taxonomy and phylogenetics (Kurashima et al., 2014;. The discovery and description of new taxa, along with the analyses of novel molecular data helped resolve the inaccurate phylogenetic placement represented by taxa previously grouped in the order Tetraphyllidea (sensu . From this, two new orders, Onchoproteocephalidea Caira, Jensen, Waeschenbach, Olsen et Littlewood, 2014and Phyllobothriidea Caira, Jensen, Waeschenbach, Olsen et Littlewood, 2014emerged (Caira et al., 2014. With these advancements, the current knowledge of the unique host-parasite relationships between cestodes and elasmobranchs has greatly improved in most parts of the world . The order Onchoproteocephalidea II (sensu Caira et al., 2017) currently includes 12 genera, all of which include one pair of hooks on each of their four bothridia (with the exception of Prosobothrium Cohn, 1902) (Caira et al., 2017). Amongst these is the genus Phoreiobothrium Linton, 1889 (Caira et al., 2017). Although the World Register of Marine Species (WoRMS, 2022) lists 24 species of Phoreiobothrium worldwide, this genus only comprises 19 valid species (Caira et al., 2021), since revisions by  and Caira and Jensen (2015) concluded that a number of previously described species lack complete and vital descriptive data and are therefore considered species inquirendae. Like many onchobothriideans, the majority of species of Phoreiobothrium, with the exception of P. exceptum Linton, 1924and P. pectinatum Linton, 1924(Caira & Jensen, 2015 are extremely host-specific, exhibiting oioxenous specificity infecting a single host species (Kurashima et al., 2014;Caira et al., 2017;Darvishi & Haseli, 2019). Species of Phoreiobothrium are known to exclusively parasitise sharks, with the majority (13/19) infecting sharks of the family Carcharhinidae, whilst the remainder (6/19) infect sharks from the family Sphyrnidae Caira et al., 2017;Caira et al., 2021;Javadi & Haseli, 2022).
Given that the definitive hosts of the 19 currently accepted species of Phoreiobothrium only represent 25 % (13/52) of recognised carcharhinid and 66 % (6/ 9) of sphyrnid shark species (Froese & Pauly, 2021;Caira et al., 2021), several cestode species await scientific discovery. There are also sparse collection efforts of elasmobranch hosts for parasitic research in many parts of the world, including South Africa (Van Der Spuy et al., 2020;2022;Schaeffner & Smit, 2019). This situation is further impeded by the incorrect identification of elasmobranch hosts, which may cause complications regarding the accuracy of reported hostparasite interactions. Phylogenetic analyses from the NADH2 gene have revealed the existence of speciescomplexes within elasmobranchs (Naylor et al., 2012), therefore, species misidentifications are common. This is especially true in the carcharhinids, where, for instance, the spinner shark, Carcharhinus brevipinna (Valenciennes), is often misidentified as the blacktip shark, C. limbatus (Valenciennes) (Tillett et al., 2012).
With this in mind, the present study introduces a new species of Phoreiobothrium from the spiral intestines of three spinner sharks, located in an under sampled region, using morphological methods. Apart from the description of a new species of cestode, new host-parasite interrelationships are highlighted and a possible solution to the problems associated with either the identification or separation of shark species such as C. brevipinna and C. limbatus are presented, which could prove particularly valuable for future research in this field.

Materials and Methods
Ethical approval for this project was received from the Faculty of Natural and Agricultural Sciences' Ethics Committee (FNASREC) of the North-West University with ethics number NWU-01655-20-A9. Permits for the collection and possession of sharks for the purpose of research were issued by the South African Department of Agriculture, Forestry and Fisheries (permit no. RES2020/20 issued to the KwaZulu-Natal Sharks Board).
Three adult specimens of C. brevipinna, two males and one female ranging between 190 and 230 cm in total length, were collected by the KwaZulu-Natal Sharks Board Maritime Centre of Excellence in March 2020 at Glenmore Beach, Zinkwazi Beach and Banana Beach, KwaZulu-Natal, South Africa. The identity of this shark was determined and confirmed by the KwaZulu-Natal Sharks Board. All three specimens were recovered deceased in gill-nets set within the framework of the South African bather protection programme off the coast of Glenmore, Zinkwazi and Banana Beaches, and were subsequently frozen for approximately 6 months at -20C in the laboratory at the KwaZulu-Natal Sharks Board Maritime Centre of Excellence.
Prior to dissection, sharks were defrosted. Spiral intestines were removed by a mid-ventral incision and approximately two-thirds of each intestinal tract, along with the contents, was fixed in 4 % neutrallybuffered formalin. The intestines and contents were then transferred to 70 % ethanol after a period of two weeks. The remaining third of the intestinal tract was fixed in molecular grade ethanol. However, after going through the ethanol-fixed material numerous times, all Phoreiobothrium specimens detected were used for molecular analyses. Due to the disintegrated nature possibly caused by the freezing process only partial DNA sequences were obtained that could not be used for an accurate phylogenetic analysis. Preserved spiral intestines were scanned for cestodes using a stereomicroscope and specimens were manually removed. These specimens were hydrated in a graded ethanol series, stained with Delafield's haematoxylin, and dehydrated in a graded ethanol series again to 70 % ethanol. Following staining, specimens were placed in 1 % hydrogen chloride to clear the excess stain and then fully dehydrated in a graded ethanol series. The tissue of these specimens was cleared in clove oil, and each specimen was permanently mounted on microscope slides using Canada balsam.
Once the microscope slides were completely dry, morphological observations were conducted using a Nikon Y-TV55 video camera mounted on a Nikon ECLIPSE Ni light microscope (Nikon, Tokyo, Japan). Images of characteristic body structures were taken, from which measurements were obtained, using the image analysis software Image Pro Express (Nikon, Japan). The morphological characterisation of the specimens follows Van Der Spuy et al. (2020). Measurements of internal organs, hooks and body structures follows . Measurements are provided in the description as the range, followed by the mean and standard deviation, followed by the number of specimens examined (N), as well as the number of observations made (n). All measurements are presented in micrometres (lm), except for the total length presented in millimetres (mm). After acquiring all images and measurements, line drawings were made using a drawing attachment tube. Four specimens were subjected to scanning electron microscopy (SEM). Specimens were cleaned in 70 % ethanol from any remaining host tissue or mucus as thoroughly as possible by using a soft, liner brush. Specimens were dehydrated in a graded ethanol series to 100 % ethanol and placed in hexamethyldisilisane (HMDS). Following the drying protocol, specimens were mounted on carbon tape on aluminium stubs and sputter-coated with carbon (Emscope TB500, Quorum Technologies, Puslinch, Ontario, USA), followed by 20 to 30 nm gold/palladium (Eiko IB2 ion coater, Eiko, Japan). Specimens were observed and micrographs of the various individual body regions were taken using a FEI Nova NanoSEM 450 scanning electron microscope (FEI, Hillsboro, Oregon, USA). Micrographs were used to conduct microthrix comparisons of scolex regions and the strobila following Chervy (2009).
Microscope slides of type specimens have been deposited in the following helminthological collections: The National Museum, Bloemfontein, South Africa (NMB); the Institute of Parasitology, Biology Centre of the Czech Academy of Sciences, Č eské Budějovice, Czech Republic (IPCAS); and the Natural History Museum, Geneva, Switzerland (MHNG-PLAT). Stubs containing specimens used for SEM were retained in the parasite collection of the Water Research Group, North-West University, South Africa.
Hooks tri-pronged, with blunt talon embedded in muscular pad of scolex; talon of medial hook longer than talon of lateral hook (Fig. 1B). Hook prongs and talon hollow. Internal channels of all hook prongs continuous, smooth (Fig. 1B). Axial prongs longest.
One particularly interesting feature became highly noticeable in the scanning electron micrographs of the cephalic peduncle of P. martini sp. n. compared to all the other species of Phoreiobothrium. All congeners display densely packed gladiate spinitriches on their cephalic peduncle, while P. martini sp. n. mostly displays papilliform filitriches interspersed with only a few small gladiate spinitriches (Fig. 2). However, the lack of the appearance of spinitriches found on the cephalic peduncle of P. martini sp. n. should not be used as a distinguishing characteristic, since microtriches could have been shed while the host was frozen.
Phoreiobothrium martini sp. n. marks the first species of the genus to be described from South Africa, and increases the number of species of Phoreiobothrium to 20 worldwide (Caira et al. 2021, Javadi & Haseli, 2022.

Discussion
In accordance with the revision of the genus Phoreiobothrium by , there is a general microthrix pattern within this genus. The cephalic peduncle includes a combination of filitriches and gladiate spinitriches are presented by all congeners . The most surprising and peculiar feature of P. martini sp. n., however, is the scarcity of gladiate spinitriches found on the cephalic peduncle. This has not been recorded in any other congener within this genus and could resemble a new morphological characteristic for this genus. However, the former assumption that this could be a new morphological characteristic is highly unlikely and this feature might only be artificial, which resulted from the freezing process of the host after it was obtained, or the effects on parasites through the disintegration of the host tissue prior to its collection. For this reason, this feature must be regarded with caution and not used as a distinguishing character. The collection of new specimens from fresh hosts are therefore required to assess and/ or confirm the microthrix patterns reported herein.
Phoreiobothrium martini sp. n. is the first named species described from C. brevipinna. In a previous study on the diversity of rhinebothriidean cestode larval types by Jensen and Bullard (2010), an undescribed species of Phoreiobothrium (Phoreiobothrium sp. 1A) was detected in a spinner shark, C. brevipinna, collected off the coast of Mississippi in 2006, and used for molecular analyses (GenBank nos.: 82,95). However, this species has never been formally described and named, and as such P. martini sp. n. is the first named species of Phoreiobothrium from C. brevipinna (Pollerspöck & Straube, 2022). Each of the valid species of Phoreiobothrium, excluding P. exceptum and P. pectinatum (Caira & Jensen, 2015), is extremely host specific (oioxenous), known to parasitise a single host species.
Given that known species of Phoreiobothrium exhibit such tight associations towards their definitive hosts (Darvishi & Haseli, 2019), the species found by Jensen and Bullard (2010) might prove to be the same species described herein. However, without a comprehensive molecular analysis, this assumption or the possibility that P. martini sp. n. might have undergone cospeciation within C. brevipinna, as reported for congeners, cannot be proven at this time and requires additional research. Furthermore, given the geographical distance between P. martini sp. n. and the species of Jensen and Bullard (2010), they might be congeners rather than conspecifics. The spinner shark, C. brevipinna, exhibits a wide distribution, inhabiting warm temperate and tropical waters of the Atlantic, Mediterranean Sea and Indo-West Pacific Ocean (Compagno, 1984;Froese & Pauly, 2021). This host has also been recorded from the southwestern Indian Ocean ranging between southern Mozambique and the western coast of Madagascar (Allan & Cliff, 2000). In South Africa, C. brevipinna is a common coastalpelagic species found off the coast of KwaZulu-Natal, ranging as far south as Mossel Bay (Allan & Cliff, 2000). Although having a wide distribution, this shark species prefers shallower inshore waters over the continental shelf with seasonal migratory patterns moving into shallower bays (Froese & Pauly, 2021). Given the rather restricted movement pattern of these sharks, it is unlikely that the specimens observed by Jensen and Bullard (2010) off the coast of Mississippi represent the same species. This means that Phoreiobothrium sp. 1A and P. martini sp. n. might form part of the ''exception category'' including P. exceptum and P. pectinatum known to exhibit synhospitalic congeners. Once again, this only endorses the need for future comprehensive molecular analyses.
The biogeographical realm including the ocean basins of South Africa still holds the potential of an immense hidden cestode diversity (Van Der Spuy et al., 2020;2022), with a large proportion of the regional cestode fauna awaiting scientific discovery. This is not only supported by the present study but also by the fact that studies by Van Der Spuy et al. (2020;2022) and Oosthuizen et al. (2021) have reported and described new species from this region within the last two years, adding to the diversity of cestodes from this region and further highlighting southern Africa as a neglected biogeographic area for marine parasites with the potential of a large hidden diversity. Albeit a limited sample size, this study found that all three sampled sharks hosted specimens of P. martini sp. n., suggesting that P. martini sp. n. is very prevalent in C. brevipinna in South African waters. Apart from increasing the total number of species of Phoreiobothrium to 20 worldwide, the description of P. martini sp. n. marks only the second onchoproteocephalidean genus reported from southern Africa, therefore expanding the generic biogeographical representation and introducing new host associations, which might contribute to environmental and ecological research in future (Caira & Jensen, 2015;Darvishi & Haseli, 2019).
As part of the larger project on cestodes of elasmobranchs in South Africa, all other shark species collected within this area have also been screened yielding no other specimens belonging to the genus Phoreiobothrium. Since P. martini sp. n. represents a new species, and no other congener has been found to infect C. brevipinna, the present study provides insights to support the degree of high host-specificity exhibited by the genus. These results may prove useful as this species could be used as a potential biological tag for the accurate identification of C. brevipinna, since it has often been misidentified as C. limbatus, due to their overall body similarity. This is supported by recent studies, where highly host-specific onchoproteocephalideans, especially species of Phoreiobothrium, were used to aid in the identification of host species belonging to the Rhizoprionodon acutus (Rüppell) species complex (Darvishi & Haseli, 2019;Ganjgah & Haseli, 2020). The discovery of additional species of Phoreiobothrium is likely, with additional sampling efforts of host species that have been either misidentified or which have never been part of a parasitological investigation. This will also be beneficial as the assessment of the species diversity has the potential to provide the resources for future ecological investigations 2015;Darvishi & Haseli, 2019). With the high degree of hostspecificity found amongst species of Phoreiobothrium, these species can be applied as sentinel species for an accurate identification and diagnosis of elasmobranch hosts (Darvishi & Haseli, 2019). The lack of molecular and phylogenetic analyses within this field, renders a great vacuity regarding both phylogenetic relationships amongst congeners, as well as any information on the specific life-cycles of these marine cestodes. Future large-scale surveys, incorporating interdisciplinary approaches in ecology, taxonomy and molecular biology will undoubtedly lead to numerous scientific advances in the field of marine parasitology in South Africa.