The survey of ectoparasites on two species of Triakids (Mustelus mustelus and M. punctulatus) sharks from Tunisian coasts.

Between 2018 and 2020, 696 fish belonging to two species of sharks from the Family Triakidae (Mustelus mustelus and M. punctulatus) were collected from the coasts of Tunisia and inspected for parasites. Six copepod taxa (Perissopus dentatus Steenstrup & Lütken, 1861, Eudactylinella alba Wilson, 1932, Kroyeria lineata Van Beneden, 1853, Nesippus orientalis Heller, 1865 and Lernaeopoda galei Krøyer, 1837, Kroyeria sp.), four isopod species (Anilocra physodes (Linnaeus, 1758), Emetha audouini (H. Milne Edwards, 1840), Ceratothoa parallela (Otto, 1828) and Ceratothoa oestroides (Risso, 1816)) and two monogenean species (Erpocotyle sp1. And Erpocotyle sp2) were collected. A large number of global host records was reported, including the occurrence of E. audouini on M. mustelus and of Erpocotyle sp.2 on M. punctulatus. The study of the diversity of parasites per host species revealed that M. mustelus had a higher parasitic richness compared to M. punctulatus . In this study, it was provided for the first records on ectoparasites on Triakidae sharks from Tunisian coasts and their infection indices.

Introduction

The Tunisian coast of the Mediterranean Sea is known to harbour a great richness of chondrichthyans fish, with at least 62 described species (Bradaï et al., 2012). The Family Triakidae comprises four species, namely Galeorhinus galeus (Linnaeus, 1758), Mustelus mustelus (Linnaeus, 1758) M. asterias Cloquet, 1819, and M. punctulatus Risso, 1827 (Saidi et al., 2009). However, the taxonomic identification of species appurtenant to the genus Mustelus sp. remains cumbersome, given the unclear and sometimes contradictory diagnosis traits. The species of this genus are normally sympatric, potentially explaining their strong morphologic similarities (Compagno, 1984, Ebert and Stehmann, 2013, Marino et al., 2018). According to Bradaï et al. (2012) the species Mustelus punctulatus and M. mustelus are common along the Tunisian coasts and have similar diet (e.g., crustaceans, cephalopods and bony fishes) (Compagno, 1984, Saidi et al., 2009). Both species are of great commercial and gastronomic value in Tunisia; as such, as a consequence of their extensive consumption, they became vulnerable and even threatened in some areas, according to the International Union for Conservation of Nature and Natural Resources (IUCN).

The body of a shark confers many attachment sites for other organisms (Dippenaar and Molele, 2015). However, the studies of their parasites are scarce and with a main focus on single taxonomic group, such as copepods (Essafi, 1975, Benmansour and Youssef, 2019, Youssef et al., 2019) and monogenea (Neifar, 2001), disregarding other phyla. The aim of the current work was to provide original data on the parasitic taxa found on Mustelus mustelus and M. punctulatus bodies within Tunisian waters in order to analyse their taxonomic richness on both host species and to better understand the associations with their hosts.

Material and methods

Prospected area and specimen examination

Between 2018 and 2020, a total of 480 individuals of Mustelus punctulatus and 216 of M. mustelus, respectively, were examined for ectoparasites (Table 1). The fish were collected weekly from local Tunisian fishermen in Bizerte Bay, Gulfs of Tunis, Hammamet, and from Gabes (Sfax and Zarzis) (Fig. 1). The fish were transported for further laboratory analysis and identified according to Compagno (1984), Fischer et al., 1987, Serena, 2005. The host nomenclature followed data in Froese and Pauly (2019).

Table 1

Sampling details of Mustelus mustelus and M. punctulatus

Host	Sampling location	Season	Number of examined fish
Mustelus mustelus	Bay of Bizerte37°16′25″N-9°53′14″E20 to 100 m	Autumn	22
		Winter	32
		Spring	30
		Summer	36
	Gulf of Tunis37°00′0.00″N-10°29′59.99′’E20 to 100 m	Autumn	30
		Winter	30
		Spring	30
		Summer	30
	Gulf of Hammamet36°04′60.00″N-10°44′59.99″E20 to 100 m	Autumn	30
		Winter	15
		Spring	47
		Summer	28
	Gulf of Gabes 34°00′0.00″N-10°30′0.00″E20 to 100 m	Autumn	30
		Winter	25
		Spring	25
		Summer	40
Mustelus punctulatus	Bay of Bizerte37°16′25″N-9°53′14″E20 to 100 m	Autumn	7
		Winter	6
		Spring	9
		Summer	11
	Gulf of Tunis37°00′0.00″N-10°29′59.99′’E20 to 100 m	Autumn	8
		Winter	7
		Spring	10
		Summer	12
	Gulf of Hammamet36°04′60.00″N-10°44′59.99″E20 to 100 m	Autumn	7
		Winter	7
		Spring	11
		Summer	9
	Gulf of Gabes34°00′0.00″N-10°30′0.00″E20 to 100 m	Autumn	24
		Winter	28
		Spring	30
		Summer	30

Fig. 1

Sampling sites, indicated by red circles.

Parasitological investigations

For every fish individual, all body traits (i.e. skin, mouth, gills, fins, cloaca, and nasal cavity) were thoroughly examined. Gills were placed afterwards in seawater in Petri dishes and arches of each gill carefully examined. Information about sampling date, prospected areas, taxonomic nomenclature and shape and corpulence of the host fishes and their parasite’s microhabitats were noted.

Copepods and isopods were extracted from hosts and fixed in ethanol (70%), afterwards cleared for 2 h in lactic acid before being examined observed undera 50× stereomicroscope (Model WildHeerbrugg M5A) and a Nikon DS-Fi2 camera coupled with a Nikon microscope (Image Software NIS Elements Analysis Version 4.0 Nikon 4.00.07–build 787–64 bit). The specimens were dissected and temporary mounted on slides in lactophenol (Yamaguti, 1963). The taxonomic identification of parasites was done based on taxonomic keys of Deets, 1994, Kabata, 2003, Ho and Kim, 2004, Boxshall and Halsey, 2004.

The examination of the collected isopods comprises the observation of their morphologic features and of different appendices in order to identify them to species level. Gender, life stage and fixing site of collected specimens were also identified. The taxonomic identification of isopods was performed according to the keys of Trilles, 1979, Bruce, 1986, Keable and Bruce, 1997, Horton, 2000, Charfi-Cheikhrouha et al., 2000.

Iron acetocarmine was necessary to stain monogeneans to be examined in permanent mounts, using Canada balsam (Bailenger and Neuzil, 1953). Monogeneans species identification was carried using the keys of Sproston, 1946, Neifar et al., 1998, Tazerouti et al., 2011, Chero et al., 2018.

Data analyses

The infestation rates were assessed through prevalence (P = N/H × 100) and mean intensity (MI = n/N) [N: number of infected hosts; H: total number of hosts examined; n: total number of collected individuals of one parasitic species] (see Margolis et al., 1982, Bush et al., 1997). The software Plymouth Routines In Multivariate Ecological Research ‘PRIMER’v.5.0 was used to quantify the parasites’ taxonomic diversity and evenness on their distribution on hosts, following the calculation of the indices Margalef’s species richness (SR), Shannon index (H′) and Pielou’s evenness (E).

Results

Taxonomic diversity

A total of twelve parasites, comprising copepods, isopods and monogeneans were identified (Table 2). Copepods comprised the most diverse group, namely:Eudactylinella alba Wilson, 1932, Kroyeria sp., Kroyeria lineata Van Beneden, 1853, Perissopus dentatus Steenstrup and Lütken, 1861, Nesippus orientalis Heller, 1865 and Lernaeopoda galei Krøyer, 1837 (Table 2, Fig. 2). Kroyeria sp. could comprise a new species that awaits further investigations. It was also observed that M. mustelus had a higher number of parasites, namely six species (Fig. 3) compared to M. punctulatus, which was the hosted of a single copepod species, Lernaeopoda galei.

Table 2

List of ectoparasites species and their hosts. NEF: number of examined fish; P (%): prevalence; MI: mean intensity.

Host	NEF	Parasite taxonomic group	Parasite family	Parasite species	Microhabitat	P (%)	MI
Mustelus mustelus	480	Copepods	Eudactylinidae	Eudactylinella alba	Gills	1.4	1.1
			Kroyeriidae	Kroyeria lineata	Gills	3.95	1
				Kroyeria sp.	Gills	1.04	1
			Lernaeopodidae	Lernaeopoda galei	Cloacae cavity	2.50	1.2
			Pandaridae	Nesippus orientalis	Mouth	0.41	1
				Perissopus dentatus	Dorsal fin	0.83	1
		Isopods	Cymothoidae	Anilocra physodes	Body side	0.41	1
				Emetha audouini	Body side	0.41	1
				Ceratothoa parallela	Cloacae cavity	1.8	1.16
		Monogenea	Hexabothriidae	Erpocotyle sp. 1	Gills	6.04	2
Mustelus punctulatus	216	Copepods	Lernaeopodidae	Lernaeopoda galei	Cloacae cavity	3.24	1
		Isopods	Cymothoidae	Ceratothoa oestroides	Gills	2.32	1
				Ceratothoa parallela	Cloacae cavity	1.5	1.5
		Monogenea	Hexabothriidae	Erpocotyle s p. 2	Gills	5.55	1.5

Fig. 2

Mustelus mustelus and its parasites; a: Eudactylinella alba; b: Kroyeria lineata; c: Kroyeria sp.; d: Lernaeopoda galei; e Nesippus orientalis; f: Mustelus mustelus; g: Perissopus dentatus; h: Anilocra physodes; i: Emetha audouini; j: Ceratothoa parallela; k: Erpocotyle sp.

Fig. 3

Mustelus punctulatus and its parasites; a: Eudactylinella alba; b: Mustelus punctulatus; c: Erpocotyle sp.; d: Ceratothoa oestroides; e: Ceratothoa parallela.

Both hosts were infected by four isopod species (Table 2). M. mustelus was the host of three species (i.e. Anilocra physodes (Linnaeus, 1758), Emetha audouini (H. Milne Edwards, 1840) and Ceratothoa parallela (Otto, 1828)) whereas M. punctulatus of two (i.e. Ceratothoa parallela and Ceratothoa oestroides (Risso, 1816)).

Two monogenean species were also collected from both hosts: Erpocotyle sp. 1 and Erpocotyle sp. 2.

Parasitological indices of the studied parasite taxa

Data on the prevalence, mean intensity and infestation area are given in Table 2. Data analysis suggested that prevalence was low for most copepod and isopods but slightly higher for the monogeneans (Table 2). Among copepods, the species K. lineata found on M. mustelus presented the highest prevalence (P = 3.95%). Moreover, the species Lerneaopoda galei presented the second highest prevalence degree on M. punctulatus (P = 3.24%) and M. mustelus (P = 2.50%). This parasite had the highest mean intensity (MI = 1.2) among copepods (Table 2).

Nevertheless, the species N. orientalis, found on M. mustelus, presented the lowest prevalence (P = 0.41%) (Table 2).

The isopods had also low parasitological indices. C. oestroides on M. punctulatus had the highest prevalence (P = 2.32%), whereas C. parallela, found on both hosts, had a relatively higher prevalence on M. mustelus (P = 1.80%) and on M. punctulatus (P = 1.50%). Moreover, both parasites displayed a relatively high mean intensity, varying between 1 and 1.5 (Table 2).

The species A. physodes and E. audouini were found on M. mustelus and had the lowest parasitological indices (P = 0.41% and MI = 1) (Table 2).

The Monogeneans presented the highest parasitological indices recorded during this survey. The prevalence of Erpocotyle sp 1. on M. mustelus was P = 6.04% and of Erpocotyle sp.2 on M. punctulatus P = 5.55%. The mean intensity of both monogeneans was also high and varied between 1.5 and 2 (Table 2).

Parasitic richness per host taxa

The parasitic richness on both hosts revealed that M. mustelus was the richest (SR = 10), by hosting six copepods species, three species of isopods and one monogenean species (Table 3), whereas M. punctulatus had a lower parasitic richness (SR = 4), with one copepod, two isopods and one monogenean species (Table 3).

Table 3

Parasites’ diversity indice for Mustelus mustelus and Mustelus punctulatus. SR: Species richness, H′: Shanon-Weaver index, E: Pielou index of evenness.

Host	Index
	SR	H′	E
Mustelus mustelus	10	1,69	0,73
Mustelus punctulatus	4	0,89	0,64

The maximum value of Shannon index (H′ = 1.69) of the parasite assemblage was found on M. mustelus. A value of 0.89 for Shannon-Weaver index was found for M. punctulatus (Table 3), confirming that the former host possessed the highest parasitic richness.

The study of the Pielou index of evenness revealed that M. mustelus was the host of a highly diverse community of parasites (E = 0.73), followed by M. punctulatus (E = 0.64).

Parasites microhabitats

The parasites presence was intimately related to distinct microhabitats, as described in Table 2. Most parasites were collected from hosts’ gills. In turn, few other species were collected from cloaca (i.e.L. galei) or externally, from skin (i.e.,A. physodes and E. audouini). The species N. orientalis was the only parasite species collected from the mouth of M. mustelus.

Discussion

The current work revealed a high taxonomic diversity of ectoparasites on both species of sharks along the Tunisian coast, comprising a total of 12 species. The results obtained are in accordance with those of Raibaut et al. (1998), which confirmed the fact that the sharks appartenant to Families Carcharhinidae (i.e.Prionace glauca (Linnaeus, 1758)) and Triakidae (i.e. M. mustelus) were frequently associated with the maximum richness for copepods among elasmobranch taxa in the Mediterranean Sea.

The copepods collected from both host species were the dominant taxonomic group. Indeed, the copepods comprised the highest diversity, with six species, followed by isopods with four species and monogeneans with two species. According to Carrier et al. (2012), among the ectoparasites of elasmobranchs, the subclass of copepods presented the highest diversity.

Among the parasite copepods of M. mustelus, K. lineata had the highest prevalence (P = 3.95%), whereas N. orientalis had the lowest values (P = 0.41%).Is known that species from Kroyeria sp. genus are associated with a higher parasite load on their hosts compared to other copepods (Deets, 1994).

Among the six identified copepods, only the species L. galei was collected from both sharks (M. mustelus and M. punctulatus). This species is euryxenous (sensu Euzet and Combes, 1980) and is a common parasite among elasmobranchs. Moreover, this species was previously reported from various host taxa (Raibaut et al., 1998, Henderson et al., 2003, Dippenaar, 2004, Karaytug et al., 2004, Gaevskaya, 2012). Moreover, according to previous investigations, this species appears to have a preference for Triakids (see Raibaut et al., 1998, Dippenaar, 2004, Karaytug et al., 2004 for M. mustelus and Raibaut et al. (1998) for M. punctulatus). This parasite had a higher prevalence for M. punctulatus (P = 3.24%) compared to M. mustelus (P = 2.50%).

E. alba was found on the gills of M. mustelus. This copepod species is euryxenous, infesting numerous species of fish (Izawa, 2011). As previously described by Raibaut et al. (1998) from Mediterranean Sea and by Essafi (1975), it was also found on Dasyatis pastinaca from the Tunisian coasts. This is the first record of this parasite on M. mustelus in Tunisia as elsewhere.

K. lineata was another parasite found on the gills of M. mustelus. This species is cosmopolite and was previously reported from various locations worldwide, such as: Pacific Ocean (Izawa, 2008) and the Atlantic Ocean (Canadian coasts, see Deets, 1994, Benz, 1994). In Tunisia, K. lineata was found on M. asterias (Cloquet, 1918), and on M. mustelus (Essafi, 1975). This copepod species is stenoxenous, infesting mostly Triakidae species.

Kroyeria sp. was found on the gills of M. mustelus with low prevalence (P = 1.04%). The morphological study revealed that this species presented many differences compared to the other species known from the same genus from the Mediterranea Sea and the Atlantic Ocean. However, further studies are needed to support this hypothesis.

N. orientalis, found in the mouth of M. mustelus, is a well-known parasite of chondrichthyans (Cressey, 1967, Cressey, 1970). This euryxenous species has a large geographical distribution and was reported in the South African coasts by Dippenaar and Jordaan (2012) and from the Mediterranean Sea by Raibaut et al. (1998) on Alopias vulpinus (Bonnaterre, 1788), M. mustelus and M. punctulatus. In Tunisia, this species was found on M. mustelus and M. punctulatus by Essafi (1975).

P. dentatus is euryxenous and was found in South African coast (Dippenaar and Jordaan, 2007). This copepod presents a large Mediterranean geographical distribution. P. dentatus was inventoried by Raibaut et al. (1998) and by Essafi (1975) in Tunisia water from M. mustelus.

Among isopods, it wasobserved that M. mustelus had a slightly higher isopod richness (three species) than M. punctulatus (two species). The latter host was exclusive for Ceratothoa sp. species, whereas the former was the host of a more diverse parasitic fauna (A. physodes, E. audouini and C. parallela).

The genus Ceratothoa sp. had the highest parasitological indices are known from a large number of chondrichthyans sharks (Trilles, 1994).

C. parallela has euryxenous specificity and was reported from various Mediterranean areas (Trilles, 1994, Papapanagiotou and Trilles, 2001, Ramdane et al., 2007, Öktener and Trilles, 2004, Ferri et al., 2008). In Tunisia, this parasite was observed by Charfi-Cheikhrouha et al. (2000) on teleosteen fishes. According to Trilles (1994), this species was found on chondrichthyan fishes (i.e.Raja clavata Linnaeus, 1758 and R. asterias Delaroche, 1809) on both females and males. According to Pollerspöck and Straube (2018), this parasite has never been encountered before on M. mustelus and M. punctulatus in the world.

C. oestroides is a ubiquitous and euryxenic species. It was reported in the Mediterranea area (Trilles, 1994, Horton, 2000, Öktener and Trilles, 2004, Bariche and Trilles, 2005, Ramdane et al., 2007). In Tunisia, this isopod was found on various species of teleosteans (Bradaï, 2000); however, this is its first record on cartilaginious fish in the Mediterraneaarea, as elsewhere (Pollerspöck and Straube, 2018).

A. physodes and E. audouini are euryxenic species, with a wide host range and marked preference for teleosteans (Trilles, 1979). The former species has also a marked preference for Sparids and Maenids (Trilles, 1979) and was reported from various locations in the Mediterranean Sea, such as in Algeria (Trilles, 1994, Ramdane et al., 2007), Turkey (Öktener and Trilles, 2004, Koc et al., 2018) and Lebanon (Bariche and Trilles, 2005). In Tunisia, Charfi-Cheikhrouha et al. (2000) reported the occurrence of this parasite on several species of Teleosteans. The species E. audouini was found in several species of fish worldwide (Ramdane, 2007; Öktener and Trilles, 2004). The current record is the first for Tunisian coasts and on the shark M. mustelus.

The marked preference of both isopods for teleost fishes may explain their low prevalence (P = 0.41%) found in the current survey.

Chondrichthyans are known to host an extremely large number of monogeneans (Neifar, 2001, Boudaya and Neifar, 2016). However, it was only possible to collect two parasite species from the sharks M. mustelus and M. punctulatus. For M. punctulatus, a new species of Monogene, Triloculotrema euzeti n.sp. has been reported by Boudaya and Neifar (2016) in Tunisia. However, this parasite was found only in small host individuals in which the total length (TL) was less than 60 cm. In our study, all the fish examined had a length greater than 60 cm. For this reason, we did not find this monogene.

The two identified species belong to Erpocotyle genus. However, they appear to be different to E. catenulata (Guberlet, 1933) and E. laevis Van Beneden and Hesse, 1863, which are well-known parasites of M. mustelus (Lambert and Millard 1979; Llewellyn et al. 1984). Moreover, the current record of Erpocotyle species is the first for the shark M. punctulatus.

Host, microhabitat or site selections are displayed to various degrees throughout parasite taxa and groups (Kabata, 2003). Globally, all parasites exhibit high specificities in terms of attachment site and host (Kabata, 2003). Erpocotyle species are typically found on the gills of their host (Sproston, 1946).

The Family Cymothoidae is known for its binding site rather than host specificity. This family comprises species with unique morphologic features, adapted to specific attachment sites on hosts and some genera with precise location (Fogelman and Grutter, 2008). Only E. audouini is known to be found in the mouth of its host (Ramdane, 2010). The current survey presents a new binding site, whereas the rest of other isopod species were found in their typical binding sites.

The physiologic and morphologic mechanisms that determine the selection of a given site by a specific copepod are still unidentified for most species (Kabata, 2003). However, most of copepods were encountered on gills on both hosts. It was noted that L. galei was found in the same microhabitat (cloaca) on both hosts, suggesting that it may be the preferential binding site to this anatomic part on Triakidae sharks.

Conclusions

In the current study it was observed that the parasite richness of M. mustelus and M. punctulatus differs, even if both host species have similar ecologic and ethologic characteristics. This is probably the cause of intrinsic characters of each host species.

The results could be related to the lower population abundance of M. punctulatus compared to M. mustelus off the Tunisian coasts. In fact, the density of both sharks is considered as one of the universal key components in driving the interspecific parasite diversity.

This current survey completed the known data on parasitic infections for sharks M. mustelus and M. punctulatus in Tunisian coasts, supporting the further use of parasites for a better understanding of the biology and ecology of their hosts.

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.