NEMATODE AND CESTODE LARVAE OF HYGIENIC-SANITARY IMPORTANCE IN Lopholatilus villarii ( ACTINOPTERYGII ) IN THE STATE OF RIO DE JANEIRO , BRAZIL

1Laboratório de Inspeção e Tecnologia de Pescado, Faculdade de Medicina Veterinária, Universidade Federal Fluminense, Rua Vital Brazil Filho, 64, Vital Brazil, 24320-340 Niterói, RJ, Brasil. 2Laboratório de Helmintos Parasitos de Vertebrados, Instituto Oswaldo Cruz, FIOCRUZ, Av. Brasil, 4365, Manguinhos, 21040-900 Rio de Janeiro, RJ, Brasil. E-mail: knoffm@ioc.fiocruz.br (corresponding author) LARVAS DE NEMATOIDES E CESTOIDES DE IMPORTÂNCIA HIGIÊNICO-SANITÁRIA EM Lopholatilus villarii (ACTINOPTERYGII) NO ESTADO DO RIO DE JANEIRO, BRASIL

Cestodes of the order Trypanorhyncha are of hygienic importance because of their repugnant aspect, particularly when teleostean fish present massive infections in their musculature and organs that may make commercialization infeasible due to sanitary inspection and/or rejected by the consumer (AMATO et al., 1990;SÃO CLEMENTE et al., 2004,2007;FELIZARDO et al., 2010).This order possesses a great diversity of species with global distributions.As adults, these worms inhabit the stomach and intestine of elasmobranch fish, while the larval forms can infect a large number of teleostean fish and marine invertebrates in tropical and subtropical regions, but rarely freshwater fish and other vertebrates (CAMPBELL and BEVERIDGE, 1994;PALM, 2004).These cestodes cause significant economic losses.Furthermore, accidental infections of humans by larval trypanorhynchs have been reported due to the ingestion of raw fish meat, and although they do not present zoonotic potential, recent research reports that these cestodes can cause allergic disorders in humans because immunological hypersensitivity has been demonstrated for some species in studies using a murine model (RODERO and CUÉLLAR, 1999;VÁZQUEZ-LÓPEZ et al., 2001, 2002;GÒMEZ-MORALES et al., 2008;MATTOS et al., 2013).
The aim of this study was to report, for the first time, larvae of anisakid and raphidascaridid nematodes and trypanorhynch cestodes as parasites of L. villarii marketed in the municipality of Niterói, RJ, Brazil.Parasitological indices were calculated and infection sites presented, with comments on the hygienic-sanitary significance of these parasites.

METHODS
From August 2015 to September 2016, 31 specimens [total length 43-71 (52.7) cm; weight 0.970 -4.810 (1.925) kg] were acquired from fish markets in the municipality of Niterói, state of Rio de Janeiro, Brazil.The fish were transported in an isothermal box to the laboratory for necropsy, where they were identified according to FIGUEIREDO and MENEZES (1980).
Plerocerci of trypanorhynchs were transferred to distilled water and the cysts opened under a stereomicroscope with the aid of sharp needles to release the larvae, which were then refrigerated for at least 24h to permit the relaxation of scolices and tentacular extroversion.The larvae were then fixed in cold AFA, stained with Langeron´s carmine, clarified in beechwood creosote, and preserved as whole mounts on Canada balsam according to KNOFF and GOMES (2012).The classification of Trypanorhyncha followed PALM (2004), BEVERIDGE andJUSTINE (2007) andSCHAEFFNER andBEVERIDGE (2013).
Measurements were obtained by bright field microscopy using an Olympus BX 41 microscope.The samples were then analyzed by bright-field microscopy with a Zeiss Axiophot microscope using Nomarski's differential interference contrast (DIC) apparatus, and images obtained with a Canon digital camera (Power Shot A640).Some specimens were prepared for scanning electron microscopy (SEM) as described by LOPES TORRES et al. (2013).The samples fixed in 70% ethanol were dehydrated in an ethanol series (70º to 100º GL), CO 2 critical point dried, coated in gold, and then examined and photographed using a SEM (Jeol JSM-6390LV), under 15 kV acceleration voltage.Measurements were shown in millimeters (mm) with averages in parentheses, unless otherwise indicated.
The parasitological indices of prevalence, intensity, mean intensity, abundance and mean abundance were obtained as described by BUSH et al. (1997).Representative specimens of the parasites were deposited in the Helminthological Collection of the Oswaldo Cruz Institute (CHIOC), Rio de Janeiro, Brazil.

RESULTS
From the 31 specimens of L. villarii analyzed, 28 (90.The morphological and morphometric data of the helminths found are shown in Tables 1, 2 and 3.The parasitological indices of prevalence, intensity/ mean intensity and abundance/mean abundance, as well as the range of infection, sites of infection and the CHIOC deposit numbers, are shown in Table 4.
A l l l a r v a l s p e c i m e n s o f A n i s a k i s s p ., Terranova sp., Pseudoterranova sp. and various H. deardorffoverstreetorum were free, and some of H. deardorffoverstreetorum were causing granulomas in serosas.One Anisakis sp., Pseudoterranova sp., and various H. deardorffoverstreetorum were live with high motility.The unique H. deardorffoverstreetorum L4 was found live and parasitizing the intestinal serosa.Only one fish possessed one H. deardorffoverstreetorum L3 parasitizing the abdominal musculature.
The trypanorhynch cestode Otobothrium sp.exhibited tiny plerocerci with spherical blastocysts.Only one fish was parasitized with a large number of cestodes (232), which were located in the intestine serosa where they were becoming visible and producing a repugnant appearance (Figure 12).After extroversion, most plerocerci were alive and exhibited moderate motility.
The specimens of Terranova sp.(L 3 ) possessed larval tooth, conic and striated tail, absent mucron, elongated ventriculus (less than 7 times longer than wide) and intestinal cecum, all diagnostic characteristics of the genus.When compared to the specimens of other reports, one morphological difference was observed; the length of the intestinal cecum is approximately half the length of the ventriculus, while those Terranova sp.specimens from Engraulis anchiota Hubbs &Marini, 1935, from Argentina andUruguay (TIMI et al., 2001) and Paralichthys isosceles Jordan, 1890, P. patagonicus Jordan, 1889, Xystreurys rasile (Jordan, 1891) and Cynoscion guatucupa (Cuvier, 1830) from state of Rio de Janeiro, Brazil, had an intestinal cecum that was always almost twice the length of the ventriculus (FELIZARDO et al., 2009;FONTENELLE et al., 2013;FONSECA et al., 2016).
Pseudoterranova sp.(L 3 ) specimens possessed a dorsal lip bearing a pair of double papillae, two ventrolateral lips, a tiny boring tooth between the ventrolateral lips, an excretory pore at the base of the ventrolateral lips, a long ventriculus, absent ventricular appendix, intestinal cecum and a tail with a mucron, which were in agreement with the description of P. decipiens (Krabbe, 1878) (L 3 ) collected from Gadus morhua L., 1758 fillets marketed in Nova Scotia, Canada and Brazil, Thrysites atun (Euphrasen, 1791) from New Zealand and Trigla lucerna L., 1758, from the Mediterranean Sea, France (MCCLELLAND, 1980;HURST, 1984;PETTER and MAILLARD, 1988;MAFRA et al., 2015).Morphometrically, specimens of the present study had a body size approximately three times smaller than those reported by the aforementioned authors.
The majority of H. deardorffoverstreetorum were collected from the interior of small granulomas, as was also observed by FELIZARDO et al. (2009) and KURAIEM et al. (2016).
The H. deardorffoverstreetorum (L 4 ) specimen found in the present study is in agreement with the morphology and morphometry of Hysterothylacium sp.n° 2 and H. deardorffoverstreetorum (L 4 ) reported by PETTER and MAILLARD (1988) and FELIZARDO et al. (2009), respectively.These larvae possesses morphology distinguishable from that of L 3 , including having well-developed lips, posterior end with a caudal multi-spinous structure and mucron absent.

DISCUSSION
The parasitological indices of the anisakid and raphidascaridid nematodes were compared with those for other teleostean species from along the coast of the state of Rio de Janeiro reported since 2001.
T h e i n d i c e s f o r t h e s p e c i m e n s o f H . deardorffoverstreetorum were much higher than those for Hysterothylacium sp. from G. brasiliensis (KNOFF et al., 2007).Additionally, the range of infection was more than one and half to four times higher than those of Hysterothylacium sp. from Trachinotus carolinus (Linnaeus, 1766) and Chaetodipterus faber (Broussonet, 1782) (RIBEIRO et al., 2014) and T. lepturus (BORGES et al., 2012); and as high as those H. deardorffovestreetorum from P. isosceles (FELIZARDO et al., 2009), C. guatucupa (FONTENELLE et al., 2013), P. arenatus (KURAIEM et al., 2016), P. patagonicus and X. rasile (FONSECA et al., 2016).
was not found occurring with other trypanorhynch species.
Species of Otobothrium have been reported, and are now well known, for their ability to invade and inhabit fish meat, thus causing economic losses in teleostean fish with commercial importance (PALM et al., 1994).PALM et al. (2000) reported a massive infection by O. cysticum (=Otobothrium sp.) plerocerci in the musculature of Peprilus burti Fowler, 1944 andP. alepidotus (Linnaeus, 1766) in the Gulf of Mexico, where the species has varied in prevalence from 20% to 100% annually since 1970, with a range of infection of 1-3500 plerocerci per fish.In the present study, this trypanorhynch cestode was not found in the musculature.

CONCLUSIONS
This is the first record of anisakid and raphidascaridid nematodes and trypanorhynch cestodes in L. villarii.
The presence of the larvae of these nematodes in organ serosas should not be underestimated, since they are capable of migrating from the time of capture to commercialization, which can take a few days.This was the case for the unique H. deardorffoverstreetorum larva found in the abdominal musculature in the present study, which would be sufficient to cause an accidental human infection, through the ingestion of improperly processed meat of this fish.
In addition, antigens from these nematodes can be released into fish meat and cause allergic disorders.It is suggested that additional research be undertaken to evaluate the knowledge used for diagnosis of not only nematodes but also trypanorhynch cestodes.This research should address the potential risk of these helminths to humans and potential for control and prevention of the parasitic diseases they cause.
Although trypanorhynch cestodes have not been found in musculature, their massive presence in the intestinal serosa of the one fish collected produced a repugnant appearance, which leads to loss of the commercial value or complete rejection of the fish by the consumer.Thus, it is suggested that fish viscera be removed when marketed.Schmidt (Serviço de Produção e Tratamento de Imagens do Instituto Oswaldo Cruz, FIOCRUZ) for processing the figures; the Rudolf Barth Electron Microscopy Platform of the Oswaldo Cruz Institute / Fiocruz for producing the SEM images; and the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) for financial support.
38471a-c * Only the intensity is shown, because only one host was parasitized.IS = intestinal serosa.SS = stomach serosa.LS = liver serosa.AM = abdominal musculature.Table4.Parasitological indices of prevalence (P), intensity/mean intensity (I/MI), abundance/ mean abundance (A/MA), range of infection (RI) and sites of infection (SI), and numbers of deposit in the Helminthological Collection of the Oswaldo Cruz Institute (CHIOC) of the anisakid and raphidascaridid nematode larvae and Otobothrium sp.plerocercus cestode from Lopholatilus villarii marketed in the municipality of Niterói, state of Rio de Janeiro, Brazil.

Table 1 .
Inconspicuous in some specimens.b Distance to the anterior end.L = length.W = width.n = number of measured specimens.Morphological and morphometric data of third instar anisakid larvae collected from Lopholatilus villarii marketed in the municipality of Niterói, state of Rio de Janeiro, Brazil.
a a Inconspicuous in some specimens.b Distance to the anterior end.L = Length.W = width.n = number of measured specimens.

Table 2 .
Morphological and morphometric data of third and fourth instar larvae of Hysterothylacium deardorffoverstreetorum collected from Lopholatilus villarii marketed in the municipality of Niterói, state of Rio de Janeiro, Brazil.

Table 3 .
Morphological and morphometric data of Otobothrium sp.plerocercus collected from Lopholatilus villarii marketed in the municipality of Niterói, state of Rio de Janeiro, Brazil.