Metacestodes of Elasmobranch Tapeworms in Octopus vulgaris (Mollusca, Cephalopoda) from Central Mediterranean—SEM and Molecular Data

Simple Summary Information on the role of cephalopods in the life cycle of elasmobranch tapeworms and other parasites transmitted through the food web is limited. Such knowledge is useful to gain further understanding of the biology and ecology of this group of mollusks and would inform a correct management of wild cephalopod stocks for conservation and fishery purposes. In the present work, we aimed to characterize tapeworms infecting the common octopus Octopus vulgaris, one of the most widely distributed and commercially important cephalopod species, with morphological and molecular methods. Our results suggest a possible important role of O. vulgaris in the transmission of elasmobranch tapeworms and add valuable information on the host-range and distribution of the tapeworms identified. Abstract Cephalopods are intermediate/paratenic hosts in the life cycle of elasmobranch tapeworms, nevertheless most records of infection in this group of mollusks are outdated and fragmentary. The present work aimed to investigate the cestode fauna of the common octopus Octopus vulgaris from the Tyrrhenian Sea (Central Mediterranean). The parasitic stages were characterized by light and Scanning Electron Microscopy (SEM) and sequencing of 28S rDNA. Three cestode taxa were identified to the genus level: the onchoproteocephalidean Acanthobothrium sp. (prevalence 28%), the “tetraphyllidean” Anthobothrium sp. (prevalence 13%) and the trypanorhynch Nybelinia sp. (prevalence 3%). The remarkable prevalence observed for gastrointestinal cestodes highlight a possible important role of O. vulgaris in the transmission of elasmobranch tapeworms, particularly Onchoproteocephalideans. Furthermore, the present work provides, for the first time, detailed morphological (SEM) and molecular support to confirm the occurrence of Anthobothrium sp. in cephalopod hosts. In order to gain higher taxonomic resolution for the identified taxa, we stress the need to collect further morphological and molecular data of adult cestodes infecting their elasmobranch definitive hosts.


Introduction
Cephalopods can host a diversity of larval cestodes, which mainly infect their digestive tract [1][2][3][4] but can also occur free in the mantle cavity or encysted in the mantle musculature [5,6]. Knowledge of

Sample Collection and Preservation
Fresh dead adult specimens of O. vulgaris (n = 32) were obtained from different areas in the Gulf of Naples (Tyrrhenian Sea, Mediterranean Sea) from local fishermen in the period March 2015-February 2016. Octopuses were dissected and examined for the presence of helminths in the body cavity and in organs of the digestive tract. Collected worms were washed in saline (8% ) and stored in 70% ethanol for further analyses. For permanent mounts, fixed specimens were stained with Semichon's acetocarmine, destained in 1% acid ethanol, dehydrated in a graded ethanol series and mounted in Canada balsam. Line drawings were made with the help of a camera lucida and all measurements were taken with the imaging software NIS-Elements.
The terminology for larval stages follows Chervy [21] and Palm [10]. The description of tegument surface and microtriches appearance by SEM analysis follows Chervy [22].

Scanning Electron Microscopy (SEM)
For SEM, specimens were fixed in 2.5% glutaraldehyde (0.1 M sodium cacodylate buffer, pH 7.4), washed in the same buffer and post-fixed in buffered 1% osmium tetroxide, dehydrated through a graded ethanol series, critical point dried and sputter coated with platinum. Observations were made using a JEOL JSM 6700F scanning electron microscope operating at 5.0 kV(JEOL, Basiglio, Italy).

Results
Cestodes larvae were recovered in 44% of examined octopuses (14 specimens), with intensity values ranging from 1 to 3. Particularly, two larval forms were found in the digestive tract and were assigned to two distinct orders on the basis of their general morphology: 19 Onchoproteocephalidea larvae were recovered from the caecum and intestine of 9 octopuses (prevalence 28%, mean intensity 2.11) and 5 "Tetraphyllidea" larvae were found in the caecum and intestine of 4 octopuses (prevalence 13%, mean intensity 1.25). Furthermore, a single specimen (prevalence 3%), assigned to the Trypanorhyncha, was recovered from the mantle cavity of a single octopus, adhering to the external wall of the crop.
Coinfection of different larval forms was not observed. All the collected larvae were alive, moving vigorously by rapid contractions and extensions of their body. Descriptions and morphometric features in micrometers (µm; mean followed by the range in parentheses) are provided below.

Molecular Results
The 28S rDNA of 8 specimens, 7 Acanthobothrium sp. and 1 Anthobothrium sp. were successfully sequenced and were 1763-1799 bp in length, with a mean divergence 0-0.12% among them. The BLAST analysis of the 7 specimens identified morphologically as Acanthobothrium sp.  Figure 6) generated aligning our sequences with species of Acanthobothrium spp. and Anthobothrium spp. 28S rDNA retrieved from GenBank showed our Acanthobothrium specimens forming a monophyletic group separated but closely related and well supported with the species A. santarosaliense, A. wedli and Acanthobothrium sp. Anthobothrium from the present study is included in the cluster of the Anthobothrium sp. (mean distance 0-0.07%). Animals 2020, 10, x 7 of 13

Molecular Results
The 28S rDNA of 8 specimens, 7 Acanthobothrium sp. and 1 Anthobothrium sp. were successfully sequenced and were 1763-1799 bp in length, with a mean divergence 0-0.12% among them. The BLAST analysis of the 7 specimens identified morphologically as Acanthobothrium sp.  of the 7 Acanthobothrium showed they were all identical to each other except for one specimen showing some transition and transversion. The Maximum-Likelihood tree ( Figure 6) generated aligning our sequences with species of Acanthobothrium spp. and Anthobothrium spp. 28S rDNA retrieved from GenBank showed our Acanthobothrium specimens forming a monophyletic group separated but closely related and well supported with the species A. santarosaliense, A. wedli and Acanthobothrium sp. Anthobothrium from the present study is included in the cluster of the Anthobothrium sp. (mean distance 0-0.07%).

Discussion
Understanding the diversity and life cycle of elasmobranch tapeworms is often problematic due to difficulties in the morphological identification of larval forms [9] and in performing experimental life-cycle studies, insufficient information available in the literature and the frequent reporting of currently unaccepted larval names (e.g., Scolex pleuronectis) in the intermediate hosts.
For several orders of elasmobranch tapeworms (e.g., "tetraphyllideans"), the identification of larvae is particularly troublesome, since their scoleces remain less differentiated until they develop in the definitive host, therefore the use of molecular data is often required in order to clarify their taxonomy [8].
In a study aimed at generating a molecular sequence library for a wide range of larval cestodes, in order to elucidate morphological features useful for their identification [8], Acanthobothrium and Anthobothrium larvae infecting teleosts from the Gulf of Mexico have been characterized with morphological and morphometric analyses and through amplification and sequencing of partial 28S rDNA gene. Their size range and general morphology are in accordance with our description of larval Acanthobothrium and Anthobothrium from Mediterranean O. vulgaris.
Plerocercoids tentatively assigned to the genus Acanthobothrium based on the morphology of the bothridia were recovered from the intestine of O. vulgaris collected in Roscoff (Northeastern Atlantic, France) [26]. Similar larvae, reported as Scolex polymorphus, were found infecting the digestive tract of Sepia officinalis Linnaeus, 1758 and O. vulgaris collected in the Bay of Arcachon (Northeastern Atlantic, France) [27]. Larvae described by Dollfus [26,27] are similar to the larvae of Acanthobothrium described in the present study with respect to their general morphology and the presence of bright red regions in the anterior part of the body, posteriorly to the scolex. However, their bothridia differ in the number of septa dividing the loculus posterior to the accessory sucker: bothridia of larvae described by Dollfus are provided with an anterior accessory sucker and a posterior loculus divided in two parts by one transversal septum, while in our Acanthobothrium larvae the posterior loculus is divided in three parts by two transversal septa. In larvae of Acanthobothrium identified with molecular methods the number of visible septa in the posterior loculus of bothridia can vary from one to two [8]; based on this evidence we speculate that our Acanthobothrium larvae and those described by Dollfus [26,27] in O. vulgaris from northeastern Atlantic may belong to the same species of Acanthobothrium. Possible routes of infection with Acanthobothrium in O. vulgaris may be represented by bivalve and gastropod mollusks and decapod crustaceans, that are part of the diet of this octopus species and in which different Acanthobothrium spp. have been repeatedly reported [28][29][30].
With 201 Acanthobothrium species described so far [31], this genus is the most speciose in the order Onchoproteocephalidea and among tapeworms parasitizing elasmobranchs [32] and includes a majority of species that parasitize batoids and some that parasitize sharks [31,33]. Unfortunately, sequence data are currently available for only 16 Acanthobothrium species, therefore the identification to the species level of larvae belonging to this genus remains problematic. Our samples apparently belong either to not yet molecularly characterized species of Acanthobothrium or to a new species.
The genus Anthobothrium is currently classified in the non-monophyletic order "Tetraphyllidea" and has not been assigned to a family; the most recent classification scheme assigns this genus to the Clade 2 of the "Tetraphyllidea" [33]. The taxonomy of the "Tetraphyllidea" have more recently been revised in the light of new molecular data, with the creation of new eucestode orders-namely Rhinebothriidea [37], Onchoproteocephalidea and Phyllobothriidea [38]-while other species remain in the non-monophyletic "Tetraphyllidea" until additional molecular data become available [33]. With respect to previous work carried out in cephalopods, the description of our Anthobothrium sp. larva is consistent with the description of Scolex sp. infecting Loligo vulgaris Lamarck, 1798 from Brest, France [26] and from the Bay of Arcachon [27]. Nevertheless, morphological data alone do not allow to assign these larval stages to a genus, since larvae with unilocular bothridia could be tentatively assigned to genera belonging to different cestode orders [8,39]. To the authors' knowledge, no confirmed report of Anthobothrium in cephalopod hosts was so far available in the literature.
Similarly to Acanthobothrium, the identification of our Anthobothrium larvae to the species level is hampered by the scarcity of molecular information: this genus currently includes 16 valid species [33], however sequence data of only two species (A. laciniatum, A. caseyi) are available.
In the Tyrrhenian Sea, two species of Anthobothrium are reported in different elasmobranchs: A. auriculatum (Rudolphi, 1819) in T. torpedo [40], in T. marmorata and in S. squatina [41][42][43] and A. cornucopia Van Beneden, 1850 in S. squatina [43]. The diet of these host species consists of a variety of vertebrate and invertebrate preys, including cephalopods [44,45]. A few additional Anthobothrium species are reported from different parts of the Mediterranean Sea (Supplementary Table S1).
Among Trypanorhyncha, the species Nybelinia lingualis and Tentacularia coryphaenae Bosc, 1802 are the most commonly encountered in cephalopods from Mediterranean and extra-Mediterranean areas. Particularly, N. lingualis has been reported in O. vulgaris from the Mediterranean Sea and English Channel [1, [18][19][20][46][47][48]. Scolex features, body size and general morphology of the Nybelinia specimen characterized in the present study are consistent with the description of N. lingualis reported in O. vulgaris and Todaropsis eblanae (Ball, 1841) from NE Atlantic [1, 27,49]; particularly, detailed observations by SEM allowed to observe the form and arrangement of hooks over the tentacles and the appearance and distribution of microtriches, adding further detailed information on the microthrix pattern over bothrial surface. Nevertheless, due to the scarcity of material available (only one specimen) and to the lack of molecular data it is not possible to confirm the identity of the collected larva to the species level.

Conclusions
Our results suggest a possible important role of O. vulgaris in the transmission of elasmobranch tapeworms, particularly Onchoproteocephalideans. With respect to tripanorhynchs, this role is arguably less important as compared to ommastrephid squids, where prevalence can be higher than 90% and more than 200 larvae can be found in a single squid [14].
Furthermore, the present work provides, for the first time, detailed morphological (SEM) and molecular support to confirm the occurrence of Anthobothrium sp. in cephalopod hosts and the presence of Acanthobothrium sp. infection in O. vulgaris from the Mediterranean Sea.
Supplementary Materials: The following are available online at http://www.mdpi.com/2076-2615/10/11/2038/s1, Table S1: List of the sequences used for build the ML tree, with details of the developmental stage, host, locality and specific references.