Morphological identification of Skrjabinisakis Mozgovoi, 1951 (Nematoda: Anisakidae) in Kogia sima (Cetacea: Kogiidae) from Brazilian waters

Abstract New morphological, morphometric and scanning electron microscopy data of a nematode of the family Anisakidae, recovered from a specimen of Kogia sima, a cetacean that died off the northern coast of Brazil, are presented in this paper. Morphological features such as the violin-shaped ventricle and short and equal spicules, as well as the distribution of post-cloacal papillae and specificity for the definitive host (Kogiidae cetaceans) demonstrate similarity to Skrjabinisakis paggiae. This research records Kogia sima and S. paggiae on the estuarine coast of Pará, northern Brazil.


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Skrjabinisakis in Brazilian waters The genus Anisakis had been grouped into two subgenera, Anisakis and Skrjabinisakis, based on the shape and length of the ventriculus and male spicules.Few authors have used these subgeneric names (Takano & Sata, 2022).Safonova et al. (2021) proposed the use of Skrjabinisakis as a genus name rather than a subgenus for Anisakis physeteris (Baylis, 1923), A. brevispiculata (Dollfus, 1966) and A. paggiae Mattiucci, Nascetti, Dailey, Webb, Barros, Cianchi & Bullini, 2005, and the resurrected generic status of Peritrachelius for A. typica.Takano & Sata (2022), in their studies based on molecular data, do not assign A. typica to Peritrachelius, since the species was nested in Anisakis s.s., with similar phylogenetic relationship for A. simplex s.s. and A. typica observed by cox1 sequences.Mostafa et al. (2020) characterizes A. simplex s.s., A. pegreffii and A. typica as non-monophyletic groups, therefore, the use of cox1 sequences may be inadequate for the reconstruction of relationships among Anisakis species.
Although the Brazilian literature includes several studies on the parasitic fauna in terrestrial mammals in Brazil (Vicente et al., 1997;Vieira et al., 2008;Pinto et al., 2011), these data are scarce for the Amazon region when related to helminthological studies in aquatic mammals.With that in mind, this study taxonomically describes the anisakid parasites, recovered post-mortem from a female pygmy sperm whale, found stranded on the northern coast of Pará State, Brazilian Amazon.

Materials and Methods
Nematodes were recovered during necropsy of a female dwarf sperm whale (Kogia sima) stranded on Humaitá beach (0°55'37"S; 48°17'12"W), municipality of Colares, state of Pará, northern Brazil, on 4 October 2018.Nematodes were fixed with 10% formaldehyde, stored at room temperature and examined using light microscopy and scanning electron microscopy following procedures described by Pinheiro et al. (2018).For light microscopy 18 females, 1 male and 6 fourth-stage larvae were examined and, for scanning electron microscopy 5 females and 1 male were observed.All measurements are presented in micrometers, unless otherwise indicated.The taxonomic classification of nematodes was in accordance with Safonova et al. (2021) and Takano & Sata (2022).

Results
A total of 42 nematodes (32 females, 4 males and 6 fourth-stage larvae -L4) were recovered from the intestine of a specimen of Kogia sima.The morphological and morphometric characteristics of the nematodes recovered from dwarf sperm whale are presented below, Figures 1, 2, 3 and in Table 1.
Males (based on 1 specimen): body 21 mm, maximum width at ventricule/intestinal junction 343.Nerve ring 371, measured to the anterior end.Muscular esophagus 2 mm long, 429 wide; the esophagus represents 9.5% of the total body length.Ventriculus 343 in length, 200 in width at level of constriction.Length of entire esophagus and ventriculus representing 11% of body length.Caudal papillae sessile, proximal and disposed in single row (Figure 2c): 1 median papilla; 1 pair of single proximal papillae (a) lateral to cloaca; 1 pair of double paracloacal papillae (b); 4 pairs of distal papillae (c, d, e, f), of which (a, c, f) are equal in size and larger than (d, e), and (d, e, f) are very close to each other (Figure 2d).The papillae all have the same circular structure surrounded by wrinkled Skrjabinisakis in Brazilian waters cuticle and provided with a small central knob.One pair of extremely small papilla-like phasmids situated more laterally and posterior to last pair of distal papillae (Figure 2d).Spicules short, stout, slightly equal and sclerotized 0.18 mm long, representing 0.85% of total body length (Figure 1d).Gubernaculum Absent.Tail rounded, 267 in length; distal extremity of tail rounded (Figure 1e) and 2 cuticular dilatation structures (Figure 2c).Three narrow denticulate caudal plates (plectanes) are present (Figure 2e).

Discussion
The nematode parasites from Kogia sima collected in the municipality of Colares, State of Pará, have similar characteristics to those of other species of the family Anisakidae and genus Skrjabinisakis.Based on the morphological features of the violin-shaped ventriculus, short and equal spicules and the distribution of caudal papillae in the male, the anisakid adult and L4 specimens were identified as S. paggiae.Although there are few articles related to parasitic helminths of cetaceans in Brazil, different researchers have sporadically attempted to learn about the biodiversity of whale parasites present along the Brazilian coast (see Yamaguti, 1959Yamaguti, , 1963Yamaguti, , 1971;;Travassos, 1965;Travassos et al., 1969;Vicente et al., 1997;Muniz-Pereira et al., 1999).Muniz-Pereira et al. (1999) presented the first checklist of helminths in cetaceans in Brazil.Later Vieira et al. (2008) and Muniz-Pereira et al. (2009), seeking a more comprehensive and integrated description of the parasitic helminth fauna of vertebrates (host, locality and geographical distribution), included records of parasitism in cetaceans present in the Helminthology Collection of the Instituto Oswaldo Cruz (CHIOC).
When proposing the description of Skrjabinisakis paggiae the authors associated the morphology of the ventricle (the adult ventriculus is short, never sigmoid and broader than long) and the spicules (the male spicules are short, stout and of similar length).These characteristics are observed in Skrjabinisakis physeteris and S. brevispiculata, which are parasites of cetaceans of the families Physeteridae and Kogiidae (Mattiucci & Nascetti, 2008).Skrjabinisakis paggiae and S. brevispiculata share the pygmy sperm whale as definitive hosts (Paggi et al., 1998;Mattiucci et al., 1986Mattiucci et al., , 1997Mattiucci et al., , 2001Mattiucci et al., , 2005;;Mattiucci & Nascetti, 2006, 2008;Safonova et al., 2021).This host preference allows anisakids to be used as biological indicators of their distribution and ultimate host abundance, since they closely follow the trophic relationships of their successive hosts (Mattiucci & Nascetti, 2008;Kuhn et al., 2011).
For Brazilian waters, Luque et al. (2010) presented a checklist that adds data to previous sparse publications but does not mention the occurrence of S. paggiae, only that of A. insignis, A. simplex, S. physeteris and P. typica, in addition to Anisakis sp., Contracaecum sp. and Pseudoterranova sp.along the Brazilian coast in different hosts.The same authors recorded S. physeteris, Anisakis sp. and Pseudoterranova sp. in Rio de Janeiro State, Ceará State and Pernambuco State (Fernando de Noronha Archipelago), parasitizing the stomach of pygmy sperm whales.This is the first record of S. paggiae on the northern coast of Brazil (brackish water), although not its first record for K. sima.Among anisakid parasites infecting stranded cetaceans, especially Kogiidae whales, Di Azevedo et al. (2017) recorded P. typica, A. ziphidarum, S. brevispiculata and S. paggiae, parasitizing K. sima on the northeast coast (marine environment) of the country.Skrjabinisakis paggiae was recorded by Di Azevedo et al. (2015) in dwarf sperm whales stranded on Barra das Moitas beach, municipality of Amontada in Ceará state, northeastern Brazil, with details obtained by scanning electron microscopy for this parasite.Di Azevedo et al. (2017) recorded parasitism by Skrjabinisakis paggiae infecting K. breviceps in the country.
Although in this study the specimens observed were recovered from the intestine, factors such as time of host death and deterioration can cause parasites to migrate between organs, thus modifying the usual site of infection, which for Skrjabinisakis paggiae is the stomach.Skrjabinisakis paggiae was described from the stomach of K. breviceps on the Atlantic coast of Florida (United States) based on three males and four females collected and on one male K. sima from the same locality (Mattiucci et al., 2005).Subsequently, its known geographic distribution was expanded Braz J Vet Parasitol 2023; 32(4): e013423
In this work, observations by light microscopy and scanning electron microscopy highlight important features for future taxonomic studies for Skrjabinisakis paggiae, such as anterior end morphology (denticles that may be related to fixation on host tissue, double papillae in the dorsal lip), location and morphology of the vulva (6−8 mm), in addition to the distribution of the postcloacal papillae (1 median papilla; 1 pair of adcloacal papillae; 1 pair of double paracloacal papillae; 4 pairs of distal papillae) in males, confirming the data observed in SEM by Di Azevedo et al. (2015) for Skrjabinisakis paggiae and in light microscopy by Mattiucci et al. (2005).The spicule size (0.18 mm) of this study showed equivalence to that in Mattiucci et al. (2009) (0.17−0.22 mm); Di Azevedo et al. (2015) (0.18−0.19 mm) and Shamsi et al. (2019) (0.18−0.25 mm) for S. paggiae.Additional morphometric comparisons between S. paggiae are presented in Table 1.
Due to the type of preservation of the material (in 10% formaldehyde) we do not present molecular data, although Di Azevedo et al. (2017) have provided molecular records based on cox2 mtDNA sequences, for the occurrence of an A. paggiae-like species, with a genetic distance between the A. paggiae-like specimen and S. paggiae of (0.06), which is the same as between A. simplex and A. pegreffii, defined as distinct species (see Mattiucci et al., 1997Mattiucci et al., , 2014) ) which are possibly being reported because they are different genera.According to phylogenic analyses, the branching order so far proposed for the Anisakis/Skrjabinisakis taxa showed that nematodes from the sperm whale and pygmy sperm whales (i.e., S. physeteris, S. brevispiculata and S. paggiae) always occupy a well-supported basal lineage (Mattiucci & Nascetti, 2008).Cipriani et al. (2022) when presenting the phylogeny of some Anisakids, based on cox2 mtDNA sequences, divided the species into three clades: Clade (I) comprises species of the A. simplex (s.s.) complex, A. pegreffii, A. berlandi; Clade (II) A. ziphidarum and A. nascettii and Clade (III) S. physeteris, S. brevispiculata, and S. paggiae, the last clade also being confirmed with the morphological data for the three species.

Conclusion
This research records S. paggiae parasitizing Kogia sima in brackish water of northern Brazil, adding clarifying taxonomic features in SEM for S. paggiae.

Table 1 .
Morphological and morphometric comparison of S. paggiae specimens collected from Kogiidae.

Morphological and morphometric comparison Skrjabinisakis paggiae Hosts K. sima Atlantic coast of Brazil (State of Pará) K. breviceps and K. sima, Atlantic coast of Florida K. sima Atlantic coast of Brazil (State of Ceará) Locality Sex Male Females with eggs Females without eggs L4 Male Female Male Female
L: length, W: width.Braz J Vet Parasitol 2023; 32(4): e013423 4/9 Skrjabinisakis in Brazilian waters