Nematode larvae infecting Priacanthus arenatus Cuvier , 1829 ( Pisces : Teleostei ) in Brazil

From July to December, 2013, thirty Priacanthus arenatus specimens commercialized in the cities of Niterói and Rio de Janeiro, State of Rio de Janeiro, were acquired. The fish were necropsied and filleted to investigate the presence of nematode larvae. Twenty fish (66.7%) out of the total were parasitized by nematode larvae. A total of 2024 larvae were collected; among them, 30 third-instar larvae of Anisakis sp. showed prevalence (P) = 20%, mean abundance (MA) = 1, and the mean intensity (MI) = 5, and infection sites (IS) = caecum, stomach, liver, and mesentery; and 1,994 third-instar larvae (1,757 encysted and 237 free) of Hysterothylacium deardorffoverstreetorum with P = 66.7%, MA = 66.5, and MI = 99.7, and IS = spleen, caecum, stomach, liver, mesentery, and abdominal muscle. This is the first study to report H. deardorffoverstreetorum and Anisakis sp. larvae parasitizing P. arenatus.


INTRODUCTION
Priacanthus arenatus Cuvier, 1829, Atlantic bigeye, occurs in the waters of the western Atlantic.It is a species of nocturnal habits, that lives on the coast in depths of about 130 meters that feeds on small fish, crustaceans, and polychaete (Figueiredo and Menezes 1980).
The nematodes of Anisakidae and Raphidascarididae families parasitize marine mammals and have teleost fish, crustaceans, and small cephalopod molluscs as intermediate hosts (Adams et al. 1997, Anderson 2000).Humans are an accidental host, acquiring the larvae by eating raw or undercooked, smoked, or insufficiently salted fish (Amato and Barros 1984).
Some species of parasites found in fishery products are capable of causing diseases if ingested, such as anisakidosis caused by nematodes larvae of Anisakidae (Adams et al. 1997, Klimpel andPalm 2011).Adult of Hysterothylacium Ward & Magath, 1917, belonging to the Raphidascarididae, has also identified as an accidental parasite of humans (Yagi et al. 1996).In 2010, a unique anisakidosis case was reported in Brazil, in a 73-years-old man in the of Barra do Garças city, State of Mato Grosso, with the larvae located in the duodenum mucosa (Cruz et al. 2010).BIANCA P. KURAIEM et al.
These parasites also have importance in seafood sanitary inspection by the repugnant aspect in fish with large infestation by these parasites.According to the Regulation of Industrial and Sanitary Inspection of Animal Products -RIISPOA (Regulamento da Inspeção Industrial e Sanitária de Produtos de Origem Animal) (Brasil 1997), Article 445, item 4, the seafood with massive muscle parasite infestation is considered inappropriate for human consumption.
This study aimed to investigate the parasitism of the nematode larvae present in P. arenatus commercialized in the cities of Niterói and Rio de Janeiro, State of Rio de Janeiro, Brazil; identify the helminth larvae taxonomy; establish the parasite indices as prevalence (P), mean abundance (MA), mean intensity (MI), and infection sites (IS); and evaluate the importance in detecting the presence of helminths in seafood for public health.

MATERIALS AND METHODS
From July to December, 2013, thirty P. arenatus specimens were acquired measuring 20-63 cm total length and weighing 400-3.600g from the fish markets in the cities of Niterói and Rio de Janeiro, State of Rio de Janeiro, Brazil.These fish were transported in isothermal boxes to the Laboratory of Inspection and Fish Technology of the Veterinary Medicine School of the Fluminense Federal University, where they were necropsied and filleted.The fish species was identified according to Figueiredo and Menezes (1980).Collected nematodes were fixed in hot ethanol 70 0 GL, formaldehyde, and acetic acid (AFA ) (65 °C) and subsequently stored in ethanol 70 0 GL 5% glycerinated, clarified with Aman's Lactophenol, according to Knoff and Gomes (2012).The used taxonomic classification for Anisakidade and Raphidascarididae was in accordance with Fagerholm (1991), and the larvae identification was based on Felizardo et al. (2009a) and Knoff et al. (2012).The larvae were observed using Olympus BX-41 brightfield microscope, and the measurements were obtained in millimeters (mm) with the averages shown in parentheses.The parasitic indices on the prevalence, mean intensity and mean abundance, were calculated according to Bush et al. (1997).Representative specimens of Anisakis sp. and Hysterothylacium deardorffoverstreetorum were deposited in the Helminthological Collection of the Oswaldo Cruz Institute (CHIOC).

RESULTS
Twenty fish (66.7%) among the 30 collected were parasitized by nematode larvae; in total, 2,024 parasites were collected.Thirty free larvae of Anisakis sp. and 1,994 H. deardorffoverstreetorum larvae [237 larvae were in free form and 1,757 larvae were inside of granulomas (Figure 1 a-e)] were collected from abdominal cavity.Most larvae were alive and showed high motility.
The morphological and morphometric data of third-instar larvae of the Anisakis sp. and H. deardorffoverstreetorum nematodes colleted in P. arenatus are presented in Table I.The parasitic indices of prevalence, mean abundance and mean intensity of infection, as well as the infection sites and the deposit number at CHIOC, are depicted in Table II 2 a-c) The main morphological characteristics observed in 15 third-instar larvae from P. arenatus were: cuticle with delicate transversal striations, most evident in the posterior portion; anterior end with a dorsal lip and two ventrolateral lips poorly developed; six cephalic papillae, a couple in the dorsal lip and a pair in each ventrolateral lip; larval tooth below the mouth opening, between the ventrolateral lips; excretory pore located below the larval tooth; ventricle longer than wide; ventricular appendix and intestinal caecum absent; esophagus approximately twice the ventricle size; two rounded rectal glands; conical tail and mucron present.Raphidascarididae (Hartwich, 1954) sensu Fagerholm, 1991Hysterothylacium Ward & Margath, 1917 Hysterothylacium deardorffoverstreetorum Knoff, Felizardo, Iñiguez, Maldonado Jr, Torres, Pinto & Gomes, 2012 (Figure 3 a-c)

TABLE II Parasitic indices of prevalence (P), mean intensity (MI) and mean abundance (MA), infection sites (IS), and number of deposit in the Helminthological
The main morphological characteristics observed in 15 third-instar larvae from P. arenatus were: cuticle with lateral extension along the body, devoid of basal extension; anterior end with a dorsal lip and two ventrolateral lips poorly developed; nine cephalic papillae, two pairs on the dorsal lip near a large papilla and one pair in each ventrolateral lip; larval tooth absent; excretory pore opening below the nervous ring, inconspicuous in some specimens; ventricle slightly spherical; ventricular appendix of similar length to the esophagus; intestinal caecum present; four subspherical rectal glands; conical tail and mucron present.

DISCUSSION
The third-instar larvae of Anisakis sp.found in this study showed morphological similarities with Ani sakis simplex larvae collected from Paralichthys isosceles Jordan, 1890 in Brazilian coast by Felizardo et al. (2009a); although slightly larger in size, it showed proportionately similar morphological structures.
The morphology and morphometry of the H. deardorffoverstreetorum third-instar larvae collected in P. arenatus were similar to those reported by Fontenelle et al. (2013) in Cynoscion guatucupa (Cuvier, 1830) from the State of Rio de Janeiro.These same larvae are similar to those described by Knoff et al. (2012) obtained from P. isosceles on the Brazilian coast, and also morphologically similar to those specimens previously described as Hysterothylacium sp.n. 2 (Petter and Maillard 1988), Hysterothylacium MD (Deardorff and Overstreet 1981), Hysterothylacium KB (Petter and Sey 1997), and Hysterothylacium sp.(Pereira Jr et al. 2004, Ribeiro et al. 2014).
In this study, granuloma formation was observed, due to the presence of H. deardorffover streetorum larvae, as reported by Felizardo et al. (2009b) in P. isosceles; however, in this study, it was also possible to macroscopically observe the presence of larvae in the surface of these granulomas.
This is the first study to report P. arenatus parasitism by H. deardorffoverstreetorum and Anisakis sp.larvae.
Anisakis sp.larvae have admittedly zoonotic potential; and H. deardorffoverstreetorum larvae can also be considered as having zoonotic potential according to some studies.Although most of these nematodes are present in the abdominal cavity and in the viscera, they are able to migrate into the fish muscle, so there is risk of ingestion by humans.It is highly recommended to educate the population about the dangers of eating raw fish, as well as its acquisition from questionable sources, due to the anisakidosis.Furthermore, there is a pressing need for the intensification of surveillance on seafoodbased food in addition to an effective application of reliable techniques by responsible agencies for inspection and processing, as suggested by Fontenelle et al. (2013) and Ribeiro et al. (2014).

ACKNOWLEDGMENTS
The authors would like to thank Heloisa Nogueira Diniz and Rodrigo Mexas (Serviço de Produção e Tratamento de Imagens do Instituto Oswaldo Cruz, FIOCRUZ) for processing the figures; and the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) and Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) for partial financial supports.

Figure 1 -
Figure 1 -Granulomas containing third-instar larvae of Hysterothylacium deardorffoverstreetorum on abdominal cavity of Priacanthus arenatus.a. Open abdominal cavity presenting infection with granulomas.b.Detail of the infection.c.Petri dish with newly collected granulomas.d.Larva getting off of the granuloma.e. Encysted larva.The scale bars in a and c = 2 cm, b = 1 cm, d = 0.5 cm and e = 0.2 cm.See the colors in the online version.

TABLE I Morphological and morphometric data of third-instar larvae of the Anisakis sp. and Hysterothylacium deardorffoverstreetorum nematodes collected from Priacanthus arenatus (Cuvier, 1829) commercialized in the State of Rio de Janeiro, Brazil.
* Inconspicuous in some specimens; ** Distance from anterior end; L = length; W = width; n = number of measured specimens.BIANCA P. KURAIEM et al.