Echinocephalus caniculus n. sp. (Nematoda: Gnathostomatidae Railliet, 1895) from the lesser spotted dogfish Scyliorhinus canicula (L.) (Elasmobranchii: Scyliorhinidae Gill, 1862) off Tunisia, with a key to species of the genus Echinocephalus

Echinocephalus caniculus n. sp. (Nematoda, Gnathostomatidae Railliet, 1895) was isolated from the spiral valve of the lesser spotted dogfish Scyliorhinus canicula (L.) from the waters off Kalaat El Andalous, North East Tunisia. This new species is mainly characterized by a cephalic bulb armed with 31–39 transverse rows of uncinated hooks, a comparatively long oesophagus, short spicules and the presence of a gubernaculum. The new species differs from its congeners by having four cervical sacs of almost equal length, a higher oesophagus/body length ratio, the arrangement of the caudal papillae, the absence of a medioventral preanal organ and numerous scattered `pores´ limited to the lateral side of the posterior part of the body. This is the first report of a member of the genus Echinocephalus Molin, 1858 from the Tunisian coast, and a new host and locality record for the Gnathostomatidae. A key to the species of Echinocephalus is provided.

The objective of this study is to describe a new species of Echinocephalus of the lesser spotted dogfish Scyliorhinus canicula off Kalaat El Andalous, NE Tunisia.

Materials & methods
Between 6 th -23 rd April 2019, 17 specimens of Scyliorhinus canicula were collected by fishermen from the Sea of Kalaat El Andalous, Gulf of Tunis (37°07'23.8''N 10°23'40.7''E), NE Tunisia. Fish were transported on ice directly to the Laboratory of Diversity, Management and Conservation of Biological Systems, Faculty of Sciences of Tunis, University of Tunis El Manar, Tunisia and studied for endohelminths. Fish nomenclature and classification followed Froese and Pauly (2020).
Six nematodes were found alive inside the spiral valve of the infected host, washed in physiological solution and stored in vials containing 70% ethanol. Morphometric studies were performed on 4 specimens (2 males and 2 females) while another 2 were used for Scanning Electron Microscopy (SEM). For light microscopic examination, 2 males and 2 females were cleared in lactophenol. Drawings were made with a drawing tube attached to an Olympus BX53 DIC microscope. For SEM, samples were dehydrated in an ascending series of ethanol and transferred to 100% acetone, critical point dried, mounted on aluminium SEM-carrier with adhesive conductive carbon tape (PLANO) and coated with gold (10 nm-20 nm layer) under vacuum (EM SCD 004, BALTE). Nematodes were analysed by a field emission scanning electron microscope (FE-SEM, MERLIN Ò VP Compact, Zeiss), and SEM-images were taken from the selected regions (conditions like applied detector, accelerating voltage, magnification, working distance are given in the figures). All measurements are in micrometers unless otherwise stated. Etymology: This species is named after its type-host species name, canicula where it was discovered and described for the first time.

Remarks
The present nematode species belongs to the genus Echinocephalus based on the unarmed body, two trilobed pseudolabia, a cephalic bulb provided with transverse rows of spines, a vulva close to the posterior end of the body and a didelphic, prodelphic uterus. So far, the taxonomy of this genus is incompletely resolved due to several incomplete descriptions.  (Moravec & Justine, 2021). However, because of their poor descriptions, they were designated as species inquirendae by Moravec and Justine (2021). Moreover, E. carpiae Abdel-Ghaffar, Bashtar, Mehlhorn, Abdel-Gaber, Al Quraishy and Saleh, 2013 and E. crassostreai were described based on their larval stages only (Cheng, 1975;Abdel-Ghaffar et al., 2013). Larval Echinocephalus have undeveloped morphological characteristics compared to adults, e.g., the number of cephalic hooks differs and the reproductive systems are not developed. Moravec and Justine (2021) did not consider E. carpiae and E. crassostreai as valid species but pointed out their stati as species inquirendae or species dubiae.
In terms of number of valid species, Bezerra et al. As it possesses more than 30 transverse rows of hooks on the cephalic bulb, the present species differs from E. multidentatus Baylis &Lane, 1920, E. pseudouncinatus Millemann, 1951 andE. southwelli Baylis & Lane, 1920 which have 10-21 rows; and E. diazi, E. pteroplateae Wang et al., 1978 andE. sinensis Ko, 1975 which have 25-30 rows. Echinocephalus pteroplateae and E. sinensis have been reported from the same host order (Myliobatiformes) and zoogeographical region (China) and show similar numbers of transverse rows and length of body (Ko, 1975;Wang et al., 1978). There are minor variations in the distance between the nerve ring, oesophagi and deirids from the cephalic end (considered to be interspecific variation) while other data is not available for further comparison based on their poor descriptions. Therefore, we suggest that E. sinensis is a junior synonym of E. pteroplateae and, consequently, we have not included E. sinensis in our key to the species. However, Bezerra et al. (2020) and Moravec and Justine (2021) still considered E. pteroplateae and E. sinensis as separate species.
In possessing more than 30 transverse rows of hooks, the present species is similar to E. daileyi, E. janzeni Hoberg, Brooks & Urena, 1998, E. inserratus Moravec & Justine 2021, E. overstreeti Deardorff and Ko 1987, E. spinosissimus von Linstow, 1905 uncinatus but differs in the combination of different morphological features, host and geographical location (see the key to species). The new species is different from E. daileyi (from freshwater stingrays, South America, Deardorff et al., 1981) and E. janzeni (from Pacific chupare, Styracura pacifica (Beebe & Tee-Van) Hoberg et al., 1998) as it has lower number of caudal papillae (7 pairs vs. 9 pairs), different hosts (Carcharhiniformes vs. Myliobatiformes) and localities (North Africa (Tunisia) vs. Central and South America).
By having interlabia with cuticular striations, the new species can be easily distinguished from E. inserratus. Moreover, the new species has been discovered in a different geographical location (North Africa vs. New Caledonia). The new species differs from E. spinosissimus by having a lower number of caudal papillae (7 pairs vs. 8 pairs) and a longer distance between the vulva and the tip of the tail (1.8-2.4 mm vs. 1.1-1.4 mm). Similarly, the new species is reported from a different location to E. spinosissimus (North Africa vs. Indian Ocean) (Baylis & Dubney, 1920;Beveridge, 1985). By having equal spicules and lower numbers of cloacal papillae, the new species can be differentiated from E. uncinatus which has unequal spicules and a higher number of cloacal papillae.
Again, the new species has been found in different region (North Africa vs. North Atlantic region) (Beveridge, 1985).
Echinocephalus overstreeti most closely resembles the new species but differs in that it has cervical sacs of almost equal length of (vs. unequal), a higher oesophagus-body length ratio (16-22% vs. 11-16%), a ventral surface of the male tail (annulations vs. smooth) and reported different hosts and localities (see the key to species) (Deardorff and Ko, 1983;Beveridge, 1987;Moravec & Justine 2006). Besides, E caniculus n. sp. differs in possessing numerous porelike structures scattered laterally on the posterior part of the body.
The new species is also compared with those species that have been reported from similar geographical regions. E. spinosissimus has been recorded only from the Adriatic Sea (Baylis & Lane, 1920), which is in a similar geographical region to our new species. As mentioned above, E. caniculus n. sp. is distinct from E. spinosissimus.
Echinocephalus carpiae [species inquirenda], E. janzeni, E. inserraus, E. overstreeti and E. sinensis [junior syn. of E. pteroplateae] have been studied by scanning electron microscopy (Hoberg et al., 1998;Moravec & Justine, 2006Abdel-Ghaffar et al., 2013), illustrating these specimens more precisely. We conclude that there are 12 valid species in the genus Echinocephalus and the key to each species is provided below. Consent for publication Not applicable.
Research involving human participants and/or animals No alive marine fish was treated or killed for the purpose of the present research, but were collected after their death for human consumption. All applicable international, national and/or institutional guidelines for the care and use of animals were followed. Fish species is not listed in CITES or CMS and listed under Least Concern in IUCN Red List Status.
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