Unusual Localization of Pennella Sp. in Swordfish (Xiphias gladius) Hearts

Simple Summary Copepods of the genus Pennella are parasites of marine aquatic organisms (e.g., cephalopods, pelagic fish, cetaceans). They can infest fish of economic interest, including tuna and swordfish. The infestation of Pennella sp. in fish creates problems of food safety and fish marketing. Generally, these copepods penetrate the host’s muscle, without damaging internal organs. Here, we report on Pennella sp. infestation in swordfish heart muscle. The severity of the cardiac lesions we observed suggests that parasites of the genus Pennella pose a concern for food safety as well as animal health. Abstract The genus Pennella comprises hematophagous parasites of marine aquatic species, including cephalopods, marine mammals, and pelagic fish. Nine species have been officially included in the genus Pennella plus another six species inquirendae. They are most often found in the host’s musculature, without penetrating internal organs. For the present study, 83 hearts from swordfish (Xiphias gladius) caught in the Mediterranean Sea were sampled and immediately fixed in formalin for histopathological analysis. In total, 10 (12.05%) hearts were found to be parasitized by copepods of the genus Pennella. Macroscopically, there was mild-to-severe fibrinous pericarditis with atrial wall thickening and multiple parasitic nodules. Histologically, the parasitic nodules were surrounded by an inflammatory-necrotizing reaction. Parasitic infestation by Pennella spp. is common in pelagic fish and in swordfish, in particular. Here, however, we report atypical cardiac localization. A future area of focus is the evaluation of cardiac Pennella spp. infestation by histopathology and genetic identification of the parasites.


Introduction
Pennella (family Pennellidae, order Siphonostomatoida) is a genus of mesoparasitic copepods that infect aquatic animals, including cephalopods, teleosts, and marine mammals [1,2]. Pennella species have a heterogenous life cycle: a brief planktonic phase with two naupliar stages, a copepodid stage in which males and pre-metamorphic females develop on the gills of Cephalopoda (e.g., cuttlefish and squids), a chalimus stage, and an adult stage. After mating on the first host, the inseminated females seek a second host (fish or marine mammal) to infest and produce eggs, which are then released into the environment [3]. Nine officially classified species (P. balaenoptera, P. benzi, P. diodontis, P. exocoeti, P. filosa, P. hawaiensis, P. instructa, P. makaira, P. sagitta) and six species inquirendae (P. elegans, P. longicauda, P. platycephalus, P. remorae, P. robusta, P. selaris) currently belong to this genus. In addition, other 29 species of Pennella not officially recognized are indicated in the literature [4,5]. For these species, the lack of an officially recognized nomenclature is due to single reports, incomplete information, or descriptions similar to species already known but with a different name (e.g., P. rubra is synonymous with the well-known P. filosa) [6].
Taxonomic descriptions are based on female adult morphology (overall length of the parasite, shape, size, and configuration of the cephalothoracic papillae, segmentation of the first and the second antenna, holdfast horn number, shape and configuration, structure of the abdominal plumes). Nonetheless, it is not always easy to obtain correct identification since adult females are hematophagous and embedded in the musculature of their definitive host [7,8]. The external portion (trunk, genital complex, abdominal plumes) can be easily seen outside the host, while the internal portion (cephalothorax, holdfast horns, neck) is embedded inside the host tissues. This peculiarity makes identification even more complex since the entire parasite needs to be collected for species recognition [5].
Although reports are scarce, pelagic species of the Mediterranean may acquire Pennella spp. infestation, especially swordfish [18,19]. Adult females of Pennella spp. are often found anchored to the fins and fin insertion points of swordfish; occasionally, they may also threaten vital functions by damaging internal organs: the heart, the aorta or other blood vessels, the ovaries, the intestines, and the stomach [18]. The literature on Pennella spp. infestation provides little to no information about the histopathological aspect of lesions but rather focuses more on the morphological description [4]. Here, we report on 10 cases of unusual Pennella sp. cardiac localization in swordfish.

Materials and Methods
The study sample was 83 hearts of swordfish fished from the Mediterranean Sea in 2018 for human consumption. After evisceration, the hearts were immediately collected and entirely fixed in 10% neutral buffered formalin (pH 7). A macroscopic external examination was conducted at the Department of Veterinary Sciences, University of Turin. The hearts were sectioned across the median longitudinal plane and representative samples were prepared. The sections were routinely embedded in paraffin wax blocks, sectioned to 5 µm thickness, mounted on glass slides, and stained with haematoxylin and eosin (HE) staining for histological evaluation. Additionally, Weigert-Van Gieson histochemical staining was performed on selected sections. The parasites were macroscopically identified according to published guidelines [4,5].

Results
At gross examination, a single tubular structure up to 2 cm in length attached to a head with multiple branching "horns" or anchors of up to 1 cm in length compatible with Pennella sp. was observed in 10/83 hearts (12.05%) (Figure 1a). Morphological identification to the species level could not be carried out because the parasite was only partially isolated from the heart. Furthermore, biomolecular identification of the parasite was not carried out because no frozen samples were available. On gross examination, mild-to-severe fibrinous pericarditis and serous atrophy of pericardial fat were noted in nine hearts, and no alterations of the pericardium or pericardial fat in one heart. There was marked atrial wall thickening, with single/multiple whitish, firm, irregular nodules from a few millimetres to 2 × 2.5 cm in diameter. The cutting surface of the nodules presented a necrotic centre surrounded by a voluminous fibrous capsule (Figure 1b). No macroscopic alterations of the atrioventricular valves were found. Histologically, there was a moderate, chronic necrotizing-inflammatory reaction, with lymphohistiocytic elements and heterophils infiltrating the myocardium around the parasite (Figure 1c,d). Weigert-Van Gieson staining revealed a thick fibrous capsule surrounding the parasitic nodules.

Discussion
Infestation with parasites of the genus Pennella is not a rare finding in the necropsy of pelagic fish. The genus Pennella may pose a serious threat to the swordfish (Xiphias gladius), which is one of the commercially most important species on European markets [18]. Previous studies have reported Pennella spp. in swordfish caught in the Atlantic Ocean [20][21][22] and in the Mediterranean Sea [23,24]. Here, we report on Pennella spp. infestation in the heart of 10/83 swordfish fished from the Mediterranean Sea.
Most previous reports of Pennella infestation have described subcutaneous parasitic nodules [13], whereas we noted cardiac localization with moderate chronic necrotizinginflammatory reaction around the parasitic nodules in the atrial walls. These copepods can occasionally penetrate deeper tissues after digging into the muscle, and sporadic cases have been reported [19]. Parasites, including Pennella spp., in commercial fish species have become an emergent food safety problem [7,[15][16][17][18]. Our report highlights that Pennella sp. can cause serious cardiac damage in swordfish.
Further studies on a wider range of fish species are needed to determine the prevalence of Pennella sp. in internal organs and to assess its impact on fish health. In addition, genetic identification should be carried out to determine whether an association exists between Pennella species infestation and internal organ lesions.