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

Mario Santoro 1,*, Erica Marchiori 2, Marialetizia Palomba 3, Barbara Degli Uberti 4, Federica Marcer 2 and Simonetta Mattiucci 3

1 Department of Integrative Marine , Stazione Zoologica Anthon Dohrn, Villa Comunale, 80121 Naples, Italy 2 Department of Medicine, Production and Health, University of Padua, Viale dell’Università 16, Legnaro, 35020 Padova, Italy; [email protected] (E.M.); [email protected] (F.M.) 3 Department of Public Health and Infectious Diseases, Sapienza University of Rome, Piazzale Aldo Moro 5, 00185 Rome, Italy; [email protected] (M.P.); [email protected] (S.M.) 4 Department of Animal Health, Istituto Zooprofilattico Sperimentale del Mezzogiorno, via Salute 2, Portici, 80055 Naples, Italy; [email protected] * Correspondence: [email protected]

 Received: 7 January 2020; Accepted: 11 February 2020; Published: 13 February 2020 

Abstract: Sulcascaris sulcata (), a pathogenic nematode of sea turtles, may cause ulcerous gastritis with different degrees of severity. Previous studies demonstrated a high prevalence of 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.

Keywords: anisakid nematode; mussel parasite; Mytilus galloprovincialis; Sulcascaris sulcata; Tyrrhenian Sea

1. Introduction The anisakid nematode Sulcascaris sulcata is a pathogenic parasite of the esophagus and 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 are shed in the marine

Pathogens 2020, 9, 118; doi:10.3390/pathogens9020118 www.mdpi.com/journal/pathogens Pathogens 2020, 9, 118 2 of 8 environment with host . Two molts occur in the until the development of the third stage , which spontaneously hatches and develops in bivalve and gastropod mollusks to fourth stage larva after 3–4 months. The duration of the natural cycle could be of two years [2]. Recently, we described the pathological changes caused by S. sulcata in two loggerhead populations inhabiting the Tyrrhenian and the Adriatic Seas in the Mediterranean basin [1]; however, no data exists on its intermediate hosts in this geographical area. Published data of intermediate hosts of S. sulcata exists only for Australian and American waters where at least 17 taxa of mollusks have been found infected or have been experimentally infected including six gastropods and 11 bivalves (listed in Table1).

Table 1. Marine molluscan hosts for larval forms of Sulcascaris sulcata updated by Lichtenfels et al. [3].

Natural/Experimental Molluscan Host Geographical Sites References Infection Bivalves Queensland, Shark Bay Amussium balloti Natural [2,4,5] (Western Australia) Florida, Southeast coast of United Argopecten gibbus Natural [6–10] States Argopecten irradians Natural North and South Carolina [7,8,11,12] Chlamys sp. Natural Queensland [4] Melina ephippium Experimental Australia [2] Pecten sp. Natural North Carolina [13] Pecten ziczac Natural Southeastern Brazil [14] Pinna menkei Natural Moreton Bay, Australia [15] Pinctada sp. Experimental Australia [2] Spisula solidissima Natural Massachusetts to North Carolina [7,8,16–18] Spondylus ducalis Natural Bundaberg, Australia [15] Gastropods Busycon canaliculatum Natural Virginia [8,17] Cypraea tigris Natural Great Barrier Reef, Australia [15] Fasciolaria lilium hunteria Natural Florida [3] Lunatia heros Natural Virginia [8,17] Pleuroploca gigantea Natural Florida [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.

2. 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 (Figures1 and2) . 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. Pathogens 2020, 9, 118 3 of 8 Pathogens 2020, 9, 118 3 of 8 Pathogens 2020, 9, 118 3 of 8 10 20 30 40 50 60 70 80 90 100 110 120 . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | MN699444_Adult1 C G C A G C C A G C T G C G A T A A A T A G T G C G A A T T G C A G A C A C A T T G A G C A C T A A A A A T T C G A A C G C A C A T T G C G C C A T C G G G T T C A T T C C C G T T G G C A C G T C T G G C T G A G G G T C G A A A T A T T G A MN699445_Adult2 ...... 10 20 30 40 50 60 70 80 90 100 110 120 MN699446_Adult3 ...... | ...... | ...... | ...... | ...... |...... | ...... | ...... |...... | ...... | ...... |...... | ...... | ...... | ...... | ...... |...... | ...... | ...... |...... | ...... | ...... |...... | ...... | ...... MN699447_Adult4MN699444_Adult1 C.G.C.A .G .C C. A. G. C. T. G. C. G. A. T.A.A .A .T A. G. T. G. C. G. A. A. T.T.G.C .A G. A. C. A. C. A. T. T.G.A.G.C.A .C .T A. A. A. A. A. T. T. C.G.A.A .C G. C. A. C. A. T. T. G. C.G.C.C.A .T .C G. G. G. T. T. C. A. T. T.C.C.C .G .T .T G. G. C. A. C. G. T.C.T.G .G .C .T G. A. G. G. G. T. C.G.A.A.A .T .A .T T. G. A...... MN736715_Larva1MN699445_Adult2 ...... MN736716_Larva2MN699446_Adult3 ...... MN699447_Adult4 ...... MN736715_Larva1 ...... 130 140 150 160 170 180 190 200 210 220 230 240 MN736716_Larva2 ...... |...... | ...... |...... | ...... | ...... |...... | ...... |...... | ...... | ...... |...... | ...... |...... | ...... | ...... |...... | ...... | ...... | ...... | ...... |...... | .. . . . | . . . . | MN699444_Adult1 A T A C T A T C C G C A A T G C G T T G T T T C A A G A T T T T G T C T T G T T T A G C G C G C G G A A C T T T C G G C G A G C A G T G A C G C T G C G A G C A C T A A G G T G C G A G C G C G C G C T C C T T G C T T A G G T T G A T T G C A MN699445_Adult2 ...... 130...... 140...... 150...... 160...... 170...... 180...... 190...... 200...... 210...... 220...... 230...... 240...... MN699446_Adult3 . . . . | . . . . .| . . . . | . . . . .| . . . . |. . . . . | . . . . .| . . . . |. . . . . | . . . . .| . . . . |. . . . . | . . . . .| . . . . | . . . . .| . . . . |. . . . . | . . . . .| . . . . |. . . . . | . . . . .| . . . . |. . . . . | . . . . .| ...... MN699444_Adult1 A T A C T A T C C G C A A T G C G T T G T T T C A A G A T T T T G T C T T G T T T A G C G C G C G G A A C T T T C G G C G A G C A G T G A C G C T G C G A G C A C T A A G G T G C G A G C G C G C G C T C C T T G C T T A G G T T G A T T G C A MN699447_Adult4 ...... MN699445_Adult2 ...... MN736715_Larva1MN699446_Adult3 ...... MN736716_Larva2MN699447_Adult4 ...... MN736715_Larva1 ...... MN736716_Larva2 ...... 250...... 260...... 270...... 280...... 290...... 300...... 310...... 320...... 330...... 340...... 350. . 360 . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | MN699444_Adult1 A A C A G T C G250C A G C C A C A T 260G C T C G T A A T G270A G G C G A T G A280T G G C C G T G A290A G G T G T T G C 300T T G A C G A T G C310A A C T T C C A G320C A T G T G T T G 330A A G G T G G A T G340T T T T C A A A T350A T G G T T G G A360A G A G C T G C A T MN699445_Adult2 ...... | ...... | ...... | ...... | ...... |...... | ...... | ...... |...... | ...... | ...... |...... | ...... | ...... | ...... | ...... |...... | ...... | ...... |...... | ...... | ...... |...... | ...... | ...... MN699446_Adult3MN699444_Adult1 A.A.C.A .G .T C. G. C. A. G. C. C. A. C.A.T.G .C .T C. G. T. A. A. T. G. A.G.G.C.G .A .T G. A. T. G. G. C. C.G. T.G.A .A .G G. .T G. T. T. G. C. T. T.G.A.C .G .A .T G. C. A. A. C. T. T.C.C.A .G .C .A .T G. T. G. T. T. G. A.A.G.G.T .G G. A. T. G. T. T. T. T. C.A.A.A .T .A .T G. G. T. T. G. G. A. A.G.A.G .C .T G. C. A. T...... MN699447_Adult4MN699445_Adult2 ...... MN736715_Larva1MN699446_Adult3 ...... MN736716_Larva2MN699447_Adult4 ...... MN736715_Larva1 ...... MN736716_Larva2 ...... 370 380 390 400 410 420 430 440 450 460 . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . 370 380 390 400 410 420 430 440 450 460 MN699444_Adult1 .C.G.G. A| C. G. C. G. A| T. .T .A .G |G.G.G. C. A| G. C. G. T. C| A. .A .T .C|G.G. T. T. T| T. C. G. T. |T G. .A .T. T| T. T. G. A. C| C. T. C. A. G| .C.T.C. A| G. T. C. G. T| G. A. .A .T |A.C.C. C. G| C. T. G. A. A| .T .T .T .A|A.G. C. A. T| A. T. A. A. |T .T .A .A.G| C. G. G. A. G| G. A A A A G A A A MN699445_Adult2MN699444_Adult1 C.G.G.A .C G. C. G. A. T. T. A. G. G.G.G.C.A .G .C G. T. C. A. A. T. C.G.G.T.T .T .T .C G. T. T. G. A. T. T. T. T.G.A .C .C .T C. A. G. C. T. C. A.G. T.C.G .T G. A. A. T. A. C. C. C. G.C.T.G.A .A .T .T T. A. A. G. C. A. T.A.T.A .A .T .T A. A. G. C. G. G. A.G.G.A.A .A .A G. A. A. A...... MN699446_Adult3MN699445_Adult2 ...... MN699447_Adult4MN699446_Adult3 ...... MN736715_Larva1MN699447_Adult4 ...... MN736716_Larva2MN736715_Larva1 ...... MN736716_Larva2 ......

Figure 1. Nucleotide sequence alignment of the nDNA ITS2 obtained from Sulcascaris sulcata larvae Figure 1.1. Nucleotide Nucleotide sequence sequence alignment alignment of the of thenDNA nDNA ITS2 ITS2obtained obtained from Sulcascarisfrom Sulcascaris sulcata sulcatalarvae larvae (Accession No. MN736715, MN736716) in comparison with those previously identified as S. sulcata (Accession No. No. MN736715, MN736715, MN7 MN736716)36716) in comparisonin comparison with with those those previously previously identified identified as S. sulcata as S. sulcata adultsadults (Accession (Accession(Accession No.No. No. MN699444,MN699444, MN699444, MN699445, MN699445, MN699445, MN699446, MN699446, MN699446, MN699447). MN699447). MN699447). Dots Dots indicateDots indicate indicate identity identity identity with with with the consensusthe consensusconsensus sequence. sequence. sequence.

10 20 30 40 50 60 70 80 90 100 110 120 130 . . . . | . . . . | 10. . . . | . . . . | . 20. . . | . . . . | . .30. . | . . . . | . . .40. | . . . . | . . . 50. | . . . . | . . . . 60| . . . . | . . . . | 70. . . . | . . . . | .80. . . | . . . . | . .90. . | . . . . | . . 100. . | . . . . | . . . 110. | . . . . | . . . .120| 130 HQ328505_Adult1 T.T .T G. G. G| G. T. T. T. T| G. T.C.T.T |T .T G. T. T. T| C. T. A. T. T|A.T .A .T .T |T G. T. C. T. A| C. T. T.A.T |T G. .T T. T. A| G. G. T. G.G| T.T.T .T .A |T T. T. T. A. A| A. A.G. T.A |A .A .A A. A. A| T. T. G. A. G| T.A.T .C .A G| .T T. T. G. G| G. G. A.G. T|T .A .C .T T. T| G. T. A. G. A|G. T.T.T .T |T C. C. T. A. C| T. T. T. A.A|T .T .T .T A. G| . . . . | . . . . | HQ328505_Adult1MN991205_Adult2 .T. T. T. G. G. G. G. T. .T .T .T .G.T.C. T. T. T. T. G. T. T. .T .C .T .A.T. T. A. T. A. T. T. T. G. .T .C .T .A.C. T. T. A. T. T. G. T. T. .T .A TG.G.T.G.G. T. T. T. T. A. .T .T .T .T.A.A. A. A. G. T. A. A. A. A. .A .A .A.T. T.G. A. G. T. A. T. C. A. G. .T.T.T.G.G. G. G. A. G. T. .T .A .C .T.T.T.G. T. A. G. A. G. T. .T .T .T .T.C.C. T. A. C. T. T T A A T T T T A G MN991205_Adult2MN991206_Adult3 ...... T...... T...... MN991206_Adult3MN991207_Adult4 ...... T...... T...... MN991207_Adult4MN991208_Larva1 ...... T...... T...... MN991208_Larva1MN991209_Larva2 ...... T...... T...... MN991209_Larva2 ...... T ...... 140 150 160 170 180 190 200 210 220 230 240 250 260 . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | HQ328505_Adult1 T G A T A C A G A T 140A A T C C C T T C T T150T A A G T C T T T T A160T A T T A T T A T G G170T T T A A T A A A T 180T T G G A T A G T A A190T T T A A C T A T T A200A G G T T A C T G G T210C A T C A A T G A T A220T T G A A G T T A T 230G A G T T T A G T G A240T A T C C C T G G T T250T 260 ...... MN991205_Adult2 . . . . . |...... | . . . . . |...... | . . . . . | . . . . . |...... | . . . . . |...... | . . . . . | . . . . . |...... | . . . . . |...... | . . . . . | . . . . . |...... | . . . . . | ...... | . . . . . | . . . . . |...... | . . . . . | . . . . . |. | | HQ328505_Adult1MN991206_Adult3 .T.G. A. T. A. C. A. G. A. .T .A .A.T.C.C. C. T. T. C. T. T. .T .A .A .G.T.C. T. T. T. T. A. T. A. .T .T .A .T.T.A. T. G. G. T. T. T. A. A. .T .A.A.A. T. T. T. G. G. A. T. A. G. .T .A.A. T. T. T. A. A. C. T. A. .T .T .A .A.G.G. T. T. A. C. T. G. G. .T .C .A .T.C.A. A. T. G. A. T. A. .T .T G. .A.A.G. T. T. A. T. G. A. G. .T .T .T .A.G. T.G. A. T. A. T C C C T G G T T T MN991205_Adult2MN991207_Adult4 ...... MN991206_Adult3MN991208_Larva1 ...... T...... MN991207_Adult4MN991209_Larva2 ...... T...... MN991208_Larva1 ...... T ...... MN991209_Larva2 ...... 270. . . . . T . . . 280...... 290...... 300...... 310...... 320...... 330...... 340...... 350...... 360...... 370...... 380...... 390...... | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | HQ328505_Adult1 A G A A T T T G A C T C T T A T A T A A A A T C T T T A G A C C A G T T A A A T T T A G G A G A G C C C C G T T T G T T G G A A G T A G A T A A T C G T T G T G T T G T T C C T T G T G A T A C A A A T G T G C G T T T T T G T A T T A C T T C G G G G G A T G T C 270 280 290 300 310 320 330 340 350 360 370 380 390 MN991205_Adult2 ...... |...... | ...... |...... | ...... | ...... |...... | ...... |...... | ...... | ...... |...... | ...... |...... | ...... | ...... |...... | ...... | ...... | ...... | ...... |...... | ...... | ...... |. . . . . | . . . . | HQ328505_Adult1MN991206_Adult3 .A.G. A. A. T. T. T. G. A. .C .T .C.T.T.A. T. A. T. A. A. A. A. .T .C.T.T. T. A. G. A. C. C. A. G. .T .T .A .A.A. T. T. T. A. G. G. A. G. A. G. .C.C.C.C.G. T. T. T. G. T. .T G. .G .A.A.G. T. A. G. A. T. A. A. .T .C .G.T.T.G. T. G. T. T. G. T. T. C. .C .T.T.G. T.G. A. T. A. C. A. A. .A .T .G.T.G.C.G. T. T. T. T. T. G. .T .A .T.T.A. C. T. T. C. G G G G G A T G T C MN991205_Adult2MN991207_Adult4 ...... A...... MN991208_Larva1 ...... MN991206_Adult3 ...... MN991209_Larva2 ...... MN991207_Adult4 ...... A ...... MN991208_Larva1 ...... 400 410 420 430 440 450 460 470 480 490 500 510 520 MN991209_Larva2 ...... | ...... | ...... | ...... | ...... |...... | ...... | ...... |...... | ...... | ...... |...... | ...... | ...... | ...... | ...... |...... | ...... | ...... |...... | ...... | ...... |...... | ...... | ...... | ...... | ...... HQ328505_Adult1 A T T C A T T C G T G G G C T T T A C C T T G T A T A G C T A T T A A A T T G G A T G C T A T G A G G G G G G T T T T A A G A A C T T T A T C T T A T A G T T T T C C T A T T A T T G G T G T G T T T T A T G G A C A A T G T T C T G A G A T T T G T G G T G C A A MN991205_Adult2 ...... 400...... 410...... 420...... 430...... 440...... 450...... 460...... 470...... 480...... 490...... 500...... 510. 520 MN991206_Adult3 ...... |...... | ...... |...... | ...... | ...... |...... | ...... |...... | ...... | ...... |...... | ...... |...... | ...... | ...... |. G...... | ...... | ...... | ...... | ...... |...... | ...... | ...... |. . . . . | . . . . | HQ328505_Adult1MN991207_Adult4 .A. T. T. C. A. T. T. C. G. .T .G .G.G.C. T. T. T. A. C. C. T. .T G. .T .A.T.A.G. C. T. A. T. T. A. A. .A .T .T.G.G. A. T. G. C. T. A. T. G. .A .G.G.G.G.G. G. T. T. T. T. A. .A .G .A.A.C. T. T. T. A. T. C. T. .T .A .T .A.G. T. T. T. T. C. C. T. A. .T .T .A .T.T.G.G. T. G. T. G. T. .T .T .T .A.T.G.G. A. C. A. A. T. G. .T .T .C.T.G.A.G. A. T. T. T G T G G T G C A A MN991205_Adult2MN991208_Larva1 ...... MN991206_Adult3MN991209_Larva2 ...... G...... MN991207_Adult4 ...... MN991208_Larva1 ...... 530...... 540...... 550...... 560...... 570...... MN991209_Larva2 ...... | ...... | ...... | ...... | ...... |...... | ...... | ...... |...... | ...... | ...... HQ328505_Adult1 A T C A T A G T T T T A T G C C T A T T G C T G T A G A G G T A A C T T T G T T G G A T A A T T T T A MN991205_Adult2 ...... C ...... MN991206_Adult3 ...... 530...... C . 540...... 550...... 560...... 570 MN991207_Adult4 ...... |...... | ...... |. .. .. C. . .| ...... | ...... |...... | ...... |...... | ...... | . HQ328505_Adult1MN991208_Larva1 .A. T. C. A. T. A. G. T. .T .T .T .A.T.G.C. C. T. A. T. T. G. C. .T .G .T.A.G. A. G. G. T. A. A. C. .T .T .T .G.T. T.G. G. A. T. A. A. T. T T T A MN991205_Adult2MN991209_Larva2 ...... C .. C...... MN991206_Adult3 ...... C ...... MN991207_Adult4 ...... C ...... MN991208_Larva1 ...... MN991209_Larva2 ...... C ...... FigureFigure 2. 2.Nucleotide Nucleotide sequence alignment alignment of the of themtDNA mtDNA cox2 cox2obtainedobtained from Sulcascaris from Sulcascaris sulcata larvae sulcata larvae (Accession(Accession No. No. MN991208,MN991208, MN991209) MN991209) in comparison in comparison with withthose thosepreviously previously identified identified as S. sulcata as S. sulcata Figure 2. Nucleotide sequence alignment of the mtDNA cox2 obtained from Sulcascaris sulcata larvae adultsadults (Accession (Accession No. No. HQ328505, HQ328505, MN991205; MN991205; MN991206; MN991206; MN991207). MN991207). Dots Dots indicate indicate identity identity with with the (Accession No. MN991208, MN991209) in comparison with those previously identified as S. sulcata consensusthe consensus sequence. sequence. adults (Accession No. HQ328505, MN991205; MN991206; MN991207). Dots indicate identity with 3.3. Discussion Discussionthe consensus sequence.

3. DiscussionTheThe Mediterranean Mediterranean mussel mussel has has been been intensively intensively studied studied for pathogens for pathogens in the in whole the Mediterranean whole – asMediterranean well as the Campanian as well as the coastal Campanian areas [coastal19–21 ],areas but [19 to date,21], but our to studydate, our describes study describes the first the finding of first The finding Mediterranean of a parasite nematode mussel has in this been bivalve intensively . Accordingstudied for to McEl pathogenswain et in al. the[22] , whole a parasite nematode in this bivalve species. According to McElwain et al. [22], parasitic nematodes Mediterraneanparasitic nematodes as well are asuncommon the Campanian in marine coastal bivalves. areas In [19mussels,–21], but the to only date data, our is studyby Lauckner describes the arefirst[23 uncommon], reporting finding of in in amarine North parasite bivalves.Atlantic nematode an In infectionmussels, in this by bivalve the an only anisakid species. data is larva by According Lauckner thought to to [23 McEl be], reportingPhocanemawain et al. in North[22], Atlanticparasitic(Pseudoterranova an infectionnematodes) decipiens by are an in anisakiduncommon a Northlarva Atlantic in thoughtmarine Mytilus bivalves. to edulis be Phocanema. In mussels,(Pseudoterranova the only data) decipiens is by Laucknerin a North Atlantic[23], InreportingMytilus the Mediterranean edulis in North. Sea, Atlantic the occurrence an infection of S. sulcata by an in anisakidloggerhead larva turtles thought seems to to be belimited Phocanema (toPseudoterranova Inits theeastern Mediterranean basin) decipiens and the Sea,Tyrrhenianin a theNorth occurrence SeaAtlantic [1,24] .Myti ofRecently,S.lus sulcata edulis we in.observed loggerhead that all turtles Sulcascaris seems positive to be limited tologgerhead its easternIn the turtlesMediterranean basin from and thethe Tyrrhenian Sea, the occurrence cam Seae from [1,24 of the]. S. Recently, coastal sulcata sites in we loggerhead located observed between turtles that Castel all seemsSulcascaris Volturno to be limited positive and the Naples Gulf (it includes also Monte di Procida), where all Mediterranean mussel farms and loggerheadto its eastern turtles basin from and the the Tyrrhenian Tyrrhenian cameSea [1, from24]. Recently, the coastal we sites observed located that between all Sulcascaris Castel positive Volturno wild bivalve beds, registered along the Campania coast are concentrated [1]. According to Berry and andloggerhead the Naples turtles Gulf from (it includes the Tyrrhenian also Monte cam die from Procida), the coastal where sites all Mediterraneanlocated between mussel Castel Volturno farms and Cannon [2], it is plausible to think that the farmed Mediterranean mussels may have been infected wildand bivalve the Naples beds, Gulf registered (it includes along also the Monte Campania di Procida) coast are, where concentrated all Mediterranean [1]. According mussel to farms Berry and and by filtering seawater contaminated with S. sulcata eggs and/or larvae laid with feces by an infected Cannonwildloggerhead bivalve [2], itturtle isbeds, plausible while registered feeding to think along on farm that the ropes theCampania farmedof Mediterr coast Mediterraneananean are concentrated mussels. mussels [1]. According may have to been Berry infected and byCannon filtering [2] seawater, it is plausible contaminated to think that with theS. sulcatafarmedeggs Mediterranean and/or larvae mussels laid withmay have feces been by an infected infected loggerheadby filtering turtle seawater while contaminated feeding on farm with ropes S. sulcata of Mediterranean eggs and/or larvae mussels. laid with feces by an infected loggerheadAdult individuals turtle while of feeding loggerhead on farm turtles ropes areof Mediterr frequentlyanean observed mussels. feeding on Mediterranean mussel farm ropes along the coast of Campania. Ingestion of large amounts of Mediterranean Pathogens 2020, 9, 118 4 of 8 mussels by loggerhead turtles was confirmed by our unpublished observations at the post-mortem examination of large individuals stranded along the coast of Campania. In those loggerhead turtles, the remains of Mediterranean mussels in the commonly occurred in the presence of S. sulcata individuals within the gastric lumen. This particular feeding habit, rarely observed in other Mediterranean basins [25–27], could favor the increase of the incidence of infection with S. sulcata in Mediterranean mussels along the coast of Campania. Regarding the site of infection, all larval forms of S. sulcata occurred within the foot of the Mediterranean mussels showing an encapsulation-type inflammatory response [28,29]. In Australian and American waters, fourth-stage larvae occurred most commonly within the adductor muscle and gonads of its molluscan hosts (Table1). The only report of S. sulcata larvae within the foot was in the bivalve Spisula solidissima from the Atlantic coast of United States where larvae were associated with necrosis and hemocytic infiltration [16]. It has been documented that under the influence of environmental changes and the associated degradation of preferred habitats, host switching by parasites may increase [30], and parasite larvae may adapt their tropism changing the intermediate host species [31]. In particular, in the absence of preferred intermediate hosts and rich presence of definitive hosts, other marine mollusks might play a prominent role in the parasite transmission [32], and parasites may adapt in different way to the new hosts. Most of the marine bivalves found as intermediate hosts in Australian and American waters are Pectinidae of commercial importance (Table1). Contrarily to that observed in other Mediterranean areas (for example the Adriatic Sea), Pectinidae mollusks along the Tyrrhenian coast are rare and do not represent usual food items for loggerhead turtles [33]. The significantly higher values of S. sulcata infection among loggerhead turtles feeding in Adriatic compared to Tyrrhenian have been attributed to the differences of regional habitats supporting higher abundance and diversity of mollusk intermediate hosts [1], and it is plausible to suppose that other intermediate hosts for S. sulcata may occur in other Mediterranean areas where this nematode is known to be widespread. It is unknown if the adductor muscles of examined Mediterranean mussels were also infected because routinely it has been left on the internal surface of the valves, and it has not been processed by histological examination [34]. Further studies to evaluate the occurrence of nematode larval forms within the adductor muscle are warranted to understand if the prevalence of infection for S. sulcata larvae in Mediterranean mussels from this basin was underestimated and if it was an occasional finding. Here, the molecular study based on the sequence analysis at the ITS2 region of rDNA and the mtDNA cox2 gene loci, allowed the first molecular identification of the larval nematodes as belonging to S. sulcata in Mediterranean mussels. In particular, sequences analysis of the mtDNA cox2 generally allows the identification of larval stages of anisakid nematodes included in the genera , Pseudoterranova, and [35]. Additionally, the same primers used for the anisakid parasites permitted, in recent years, the direct sequencing of several adult nematodes belonging to the species S. sulcata obtained from loggerhead turtles stranded along the Mediterranean coast (Mattiucci, unpublished data). As a consequence, the direct alignment of the mtDNA cox2, here obtained on the two samples with respect to the sequences of adult specimens, have permitted the identification of those ascaridoid larval stages as corresponding to S. sulcata. On the other hand, mtDNA cox2 allowed also the identification of Anisakis pegreffii larvae in human granuloma tissues, surgically removed and formalin-embedded in human cases of anisakiasis [35]. Analogously, the direct sequences analysis of the ITS2 region of rDNA was also previously performed on adults of S. sulcata (Marcer, unpublished data) allowing the identification of its larvae in the same DNA samples. However, in the case of ITS2 region of rDNA, being generally a conservative gene locus in anisakid nematodes [35], no genetic variation was observed between adult and larval specimens of S. sulcata, here examined (Figure1), whereas the mtDNA cox2 presented, at the intraspecific level, variation at non-diagnostic nucleotide positions (Figure2); this finding is in accordance with the high polymorphism generally detected at this gene locus at the intraspecific level in several heteroxenous anisakid nematodes, such as those observed in species of the Anisakis [35]. Pathogens 2020, 9, 118 5 of 8

This result represents the first report of S. sulcata invading tissues of Mediterranean mussels, and it strongly suggests the Mediterranean mussel as an intermediate host for S. sulcata in the Tyrrhenian Sea. The Mediterranean mussel is the most economically important shellfish produced and consumed in the Western Mediterranean. The Gulf of Naples in southern Italy is among the most important production sites of the Mediterranean mussel in Italy with about 4170 tons per year [36]. Infection of Mediterranean mussels by larval nematodes may cause economic losses due to the prohibition of its marketing. Experimental studies performed on fish, , and suggest that infection by S. sulcata may occur only in sea turtles as definitive hosts [2]; however since S. sulcata is a strictly related species to zoonotic anisakid parasites [35,37], the concern regarding public health should not be underestimated.

4. Materials and Methods

4.1. General Data From February to August 2018, a total of 363 individual Mediterranean mussels (>5 cm in length) were collected from a mussel farm located in Monte di Procida (Naples municipality), under the framework of a control program for protozoan surveillance in the Campania region of southern Italy. The farm was located in the Tyrrhenian Sea about 800 m offshore the Campanian coastline on the sand bottom at a depth of 30 m; the Mediterranean mussels were cultured on ropes suspended from floating buoys at a depth of 5–12 m.

4.2. Histological Procedures

Immediately after collection, the mussels were stored at 4 ◦C, and within 12 h they were processed by histological examination for pathogen detection [34]. Transverse sections of the mantle, gonad, digestive gland, gills, and foot were obtained in a single slice through the midbody, fixed in buffered 10% formalin, and sectioned at a thickness of 5 mm. Sections were stained with hematoxylin and eosin for histologic evaluation.

4.3. Anisakid DNA Extraction and Molecular Identification To confirm the identity of larval nematodes observed by histological analysis, we performed the extraction of nematodes’ DNA from microtome slices of five paraffin-embedded infected Mediterranean mussels. The removal of paraffin and extraction of DNA was carried out using NucleoSpin® Tissue Macherey–Nagel kit (GmbH&Co., Germany) according to the manufacturer’s instructions for paraffin embedded tissues. Amplification of the ITS2 region of rDNA was carried out on the samples using the primers D (50-GAGTCGATGAAGAACGCAG-30) and reverse B1 (50-GAATCCTGGTTAGTTTCTTTTCCT-30)[38] in a 50 µL reaction, comprising 5 µL DNA, 1.5 mM MgCl2, 0.2 mM dNTPs (MBI Fermentas, Germany), 1X PCR buffer, 0.3 µM each of forward and reverse primer, and 1 U Platinum Taq DNA Polymerase (Invitrogen). Molecular grade water was added up to the final volume. The mixture was amplified with an initial activation step at 94 ◦C for 2 min, followed by 35 cycles of denaturation at 94 ◦C for 30 s, annealing at 58 ◦C for 30 s, DNA extension at 72 ◦C for 35 s, and a final extension step of 72 ◦C for 5 min. For sequencing the mitochondrial cytochrome C oxidase subunit II (cox2) gene, PCR amplification was performed using the primers 211F (50-TTTTCTAGTTATATAGATTGRTTTYAT-30) and 210R (50-CACCAACTCTTAAAATTATC-30)[39,40]. PCRs were carried out in a 50 µL volume containing 30 pmol of each primer, MgCl 2.5 mM (Amersham Pharmacia Biotech. Inc., Piscataway, NJ), 1 PCR 2 × buffer (Amersham Pharmacia Biotech. Inc., Piscataway, NJ, USA), DMSO 0.08 mM, dNTPs 0.4 mM (Sigma-Aldrich, St. Louis, MO, USA), 5 U of Taq Polymerase (Amersham Pharmacia Biotech. Inc., Piscataway, NJ, USA), and 10 ng of total DNA. PCR temperature conditions were the following: 94 ◦C Pathogens 2020, 9, 118 6 of 8

for 3 min (initial denaturation), followed by 34 cycles at 94 ◦C for 3 min (denaturation), 46 ◦C for 60 s (annealing), 72 ◦C for 90 s (extension), followed by post-amplification at 72 ◦C for 10 min. The PCR products were resolved in 2% agarose gel (expected fragments length: 500 bp of ITS2 and 629 bp of mtDNA cox2 gene) and sequenced in both directions by Macrogen (Macrogen Europe, The Netherlands). The chromatograms were corrected using the software ChromasPro version 2.4.3 (Technelysium Pty Ltd, Australia). The consensus sequences were assembled with the program SeqMan available in the DNAstar package. The consensus sequences were compared with the non-redundant database available in the GenBank using the software BLASTn [41].

Author Contributions: Conceptualization: M.S.; molecular analysis: E.M., F.M., M.P., and S.M.; histopathological analysis: B.D.U.; data curation and writing—original draft preparation: M.S.; reviewing and editing: all authors. All authors have read and agreed to the published version of the manuscript. Funding: This research received no external funding. Conflicts of Interest: The authors declare no conflict of interest.

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