The Mediterranean Mussel (Mytilus galloprovincialis) as Intermediate Host for the Anisakid Sulcascaris sulcata (Nematoda), a Pathogen Parasite of the Mediterranean Loggerhead Turtle (Caretta caretta)

Sulcascaris sulcata (Anisakidae), a pathogenic nematode of sea turtles, may cause ulcerous gastritis with different degrees of severity. Previous studies demonstrated a high prevalence of infection in the Mediterranean loggerhead turtle (Caretta caretta), although no data on the potential intermediate hosts of this nematode has been published thus far from the Mediterranean basin. Here, using molecular analyses, we demonstrated that the cross sections of nematode larvae observed histologically in Mediterranean mussels (Mytilus galloprovincialis) collected from a farm along the Tyrrhenian coast of southern Italy belong to S. sulcata. The BLAST analysis of sequences at the ITS2 region of rDNA and mtDNA cox2 gene loci here obtained from samples of two Mediterranean mussels containing nematode larvae showed 100% homology with those at the same gene loci from the adults of S. sulcata collected from the Mediterranean Sea and deposited in GenBank. To our knowledge, this study is the first to present data on a potential intermediate host of S. sulcata in the Mediterranean basin and to report a nematode parasite from the Mediterranean mussel.


Introduction
The anisakid nematode Sulcascaris sulcata is a pathogenic parasite of the esophagus and stomach of sea turtles being able to cause ulcerous gastritis with different degrees of severity predominantly depending on the intensity of infection. S. sulcata infects the loggerhead turtle (Caretta caretta), green turtle (Chelonia mydas), and Kemp's ridley turtle (Lepidochelys kempii) in the Mediterranean and Caribbean Seas, and the South Atlantic, Western Atlantic, and Western Pacific Oceans [1].
Berry and Cannon [2] demonstrated experimentally that hatchling loggerhead turtles become infected by ingesting scallops infected with fourth stage larvae S. sulcata. Larvae attach at the base of the esophagus where four molts occur about three weeks after infection and mature to adults in at least 5 months. Adult parasites live in the stomach of sea turtles and eggs are shed in the marine Table 1. Marine molluscan hosts for larval forms of Sulcascaris sulcata updated by Lichtenfels et al. [3].

Polinices sordidus
Experimental Australia [2] While studying the occurrence of protozoan parasites in the Mediterranean mussel (Mytilus galloprovincialis) along the coast of Campania region of southern Italy, larval forms of nematodes were observed histologically in the tissues of Mediterranean mussels. Herein, using molecular analysis, we report for the first time the occurrence of larvae of S. sulcata in Mediterranean mussels from the Tyrrhenian Sea.

Results
A total of five (1.4%) individual Mediterranean mussels collected on February (one mussel), May (one mussel), July (two mussels), and August (one mussel) were histologically positive to one (n = 3), two (n = 1), and three (n = 1) cross sections of nematode larvae, respectively. Cross sections of larvae (n = 4) measured in mean 290.5 µm (range: 178 to 430.9) x 202.5 µm (range: 149.4 to 292.8). Larvae were encysted within the foot of Mediterranean mussels extending to the digestive gland and revealed host inflammatory reaction in all cases. In most cases, larvae appeared to be viable and were surrounded by well-defined hemocytic capsules.
The BLAST analysis of the ITS2 of rDNA and mtDNA cox2 sequences produced here from samples of two Mediterranean mussels containing nematode larvae showed 100% homology with those of adult stages of S. sulcata from the Mediterranean Sea, previously deposited in GenBank (Figures 1 and 2). Sequences obtained in the present study were deposited in GenBank under accession numbers MN736715.1 and MN736716.1 for ITS2 and MN991208 and MN991209 for cox2.

MN699444_Adult1
A

MN699444_Adult1
C

Discussion
The Mediterranean mussel has been intensively studied for pathogens in the whole Mediterranean as well as the Campanian coastal areas [19][20][21], but to date, our study describes the first finding of a parasite nematode in this bivalve species. According to McElwain et al. [22], parasitic nematodes are uncommon in marine bivalves. In mussels, the only data is by Lauckner [23], reporting in North Atlantic an infection by an anisakid larva thought to be Phocanema (Pseudoterranova) decipiens in a North Atlantic Mytilus edulis.
In the Mediterranean Sea, the occurrence of S. sulcata in loggerhead turtles seems to be limited to its eastern basin and the Tyrrhenian Sea [1,24]. Recently, we observed that all Sulcascaris positive loggerhead turtles from the Tyrrhenian came from the coastal sites located between Castel Volturno and the Naples Gulf (it includes also Monte di Procida), where all Mediterranean mussel farms and wild bivalve beds, registered along the Campania coast are concentrated [1]. According to Berry and Cannon [2], it is plausible to think that the farmed Mediterranean mussels may have been infected by filtering seawater contaminated with S. sulcata eggs and/or larvae laid with feces by an infected loggerhead turtle while feeding on farm ropes of Mediterranean mussels.   A