First record of larvae of Hysterothylacium ( Nematoda : Anisakidae ) with zoonotic potential in the pirarucu Arapaima gigas ( Osteichthyes : Arapaimidae ) from South America

Third-stage larvae (L3) of Hysterothylacium sp. were collected by the first time in juveniles of pirarucu Arapaima gigas farmed in the Rio Preto da Eva, Amazonas state. Ninety-eight (98) out of 100 examined fish showed to be parasitized. Five hundred and ninety larvae of Hysterothylacium sp. were collected from the intestines, stomach and pyloric caeca. The mean intensity of parasite indexes was 6.02 (±5.75) ranging from 1 to 40 larvae per host and the mean abundance was 5.9 (±5.76). The A. gigas is the new host record for larvae of Hysterothylacium sp. in Brazil, and this is the first record of larvae of Hysterothylacium (Nematoda: Anisakidae) with zoonotic potential in the pirarucu from South America.


Introduction
Nematodes of the family Anisakidae parasitize fish, mammals, birds and reptiles (Moravec, 1998), and are agents of anisakiasis, a parasitic disease brought about by the consumption of raw fish infected with anisakid larvae.The first case of anisakiasis was reported in the Netherlands (Van Thiel et al., 1960).Thereafter, many cases have been reported in Japan, Korea, Netherlands and in Western Europe in regions where raw fish are consumed.In Korea, a removal of Anisakis Dujardin, 1845 larvae from the human oropharynx was reported as the first case in 1971 (Kim et al., 1971).Larvae of anisakid nematodes can be observed in the muscle or adhered to the internal organs of marine and freshwater fish.Most papers are limited to reports on their occurrence and/or description (Moravec, 1998).
Human infection is related to accidental ingestion of infectious larvae L 3 found in raw, under cooked, smoked and salted fish (Adams et al., 1997).Although the larvae do not achieve the maturity in humans, they can provoke severe symptoms when by invading the intestine wall and produce eosinophilic granuloma around the worms characterized by neutrophils infiltration (Oshima, 1972).However, the antigen exposition causes allergic reactions (Fernández et al., 2001).
Species of the genus Hysterothylacium are parasitic in both larval and adult stage and can be found parasitizing the visceral cavity, mesentery and intestine of marine and freshwater fishes (Khaleghzadeh-Ahangar et al., 2011).In natural conditions, they reach their maturity in the intestine of fish and mammals and have copepods, polychaeta and other invertebrates as intermediate hosts (Moravec, 1998).
Arapaima gigas (Schinz, 1822) (Osteichthyes: Arapaimidae) is the world's largest scale fish, may reach 3 m length, presents wide geographical distribution in the Amazonian region and occurs in the floodplain of the rivers Araguaia-Tocantins, Solimões-Amazonas and their effluents, Amazonas river in the Peruvian Andes, tributaries of the river systems Essequibo and Rupununi the Guiana (Imbiriba, 2001).It presents an excellent flesh, is commercialized fresh or sun-dried in fairs and markets (Imbiriba, 2001) and has been used for Japanese culinary as sushi, sashimi and ceviche (SEBRAE, 2009).
In Brazil, statistical data from FAO (2012) show A. gigas production of 10 ton with an estimated farm-gate of USD 130,000.00.Fingerlings production is low and represents one of the most important problems in A. gigas production.Its reproductive behavior and low female fecundity point out the A. gigas fingerlings production to be difficult and expensive (FAO, 2012).As the low production, the export volume is also prejudiced.Some assays of commercialization were made in international events in Europe and United States.The fillet of A. gigas is focused on the gourmet market, mainly restaurants where the prices range from US$ 20-25/kg in Europe and United States and USD 12-15/kg in South American cities (FAO, 2012).
This study is the first report of Hysterothylacium sp.L 3 larvae parasites on juveniles of A. gigas farmed in the Amazonia and South America.

Material and Methods
A total of 100 fish (14.5±2.1 cm total length and 32.6±16.4g weight) were captured in a fish farm in the Rio Preto da Eva, Amazonas state (2°41′55″S, 59°42′3″O).Fish and nematodes were processed according to Eiras et al. (2006).They were recovered from the intestine, stomach and the pyloric caecum were washed in 0.9% physiological saline, fixed in hot70% ethanol, clarified in phenol and examined using a Zeiss Axioscope 2 microscope equipped with a camera lucida.All measurements are in millimeters unless otherwise stated, and quoted as the ranges with means in parenthesis.
Nematode identification was based on morphology and morphometric parameters following Moravec (1998)

Results
Ninety eight percent of A. gigas were parasitized by L 3 larvae and the nematodes were identified as Hysterothylacium sp.(Figure 1).Three out of 98 infected fish presented ascitis, lesions and petechiae in the intestinal mucosa.One of them displayed a complete obstruction of the intestine.A total of 590 larvae of Hysterothylacium sp.were recovered from the intestine, stomach and pyloric caecum.The mean intensity was 6.02 (±5.75), and mean abundance 5.9 (±5.(2009) reported this anisakid from Gymnotus carapo Linnaeus, 1758 (Gymnotidae) and Leporinus friderici (Bloch, 1794) (Anostomidae) from upper Paraná River floodplain, Brazil.However, this genus is being referred to for the first time in a fish from the Amazonian Region.Moravec et al. (1993) reported Hysterothylacium sp.larvae from eight fish species of the Paraná River, Brazil.The morphology and measurements of the present material are similar to those described by them.Although many conspecific larvae present a mucron in the tail tip, the present nematode and that described by Moravec et al. (1993) do not bear this structure (Table 1).Hysterothylacium sp.larvae were also reported from the bivalve Diplodon suavidicus (Lea, 1856) (Mollusca, Unioniformes, Hyriidae) in Aripuanã River, Amazon, Brazil.Although these larvae were recovered from the same locality, the larvae found in D. suavidicus show to be larger-sized; while in the present study the larvae present an average total length of 2.531mm, those studied by Lopes et al. (2011) have an average total length of 19.9mm (Table 1).
Most registers of Hysterothylacium sp.larvae in Brazil are found in fish captured from the littoral of the state of Rio de Janeiro, Southeastern Brazil.Tavares and Luque (2006) related 44 fish species from Rio de Janeiro as hosts.However, Hysterothylacium sp.larvae were also reported from the intestine, intestinal caecum, visceral cavity, liver and spleen of Micropogonias furnieri (Desmarest, 1823) in the state of Rio Grande do Sul, Southern Brazil (Pereira et al., 2004), mesentery of Gymnotus spp.from

Discussion
Most registers of Hysterothylacium sp.larvae are from marine fish, but these larvae might be carried from the marine environment into freshwaters by some migratory Baía River, state of Mato Grosso do Sul, Central Brazil (Isaac et al., 2004) and in Scomberomorus brasiliensis Collette, Russo and Zavala-Camin, 1978 and Trichiurus lepturus Linnaeus, 1758 from the state of Rio Grande do Norte, Northeastern Brazil (Cavalcanti et al., 2012), showing its wide distribution throughout Brazil.
Although a prevalence of 98% was found in the present study, the mean intensity was similar to that found in Macrodon ancylodon (Bloch and Schneider, 1801) from the coastal zone of the state of Rio de Janeiro (Sabas and Luque, 2003).High prevalence (97.5%) was also reported in Prionotus punctatus (Bloch, 1793) from the municipality of Angra dos Reis, state of Rio de Janeiro (Bicudo et al., 2005) and the mean intensity and abundance were higher than those observed in this study.Conversely, Knoff et al. (2007) have related 27% prevalence of anisakids Anisakis sp., A. physeteris Baylis, 1923, A. simplex (Rudolphi, 1809), Pseudoterranova sp., P. decipiens (Krabbe, 1878), Hysterothylacium sp., Raphidascaris sp., Contracaecum sp. and Terranova sp. in Genypterus brasiliensis Regan, 1903 (Ophidiidae) examined from the marketed fish in the municipalities of Niterói and Rio de Janeiro state of Rio de Janeiro.
Similar lesions reported in this study in the intestinal mucosa were observed in Paralichthys isosceles Jordan, 1891 in which a total of Hysterothylacium sp.L 3 larvae showed 100% prevalence and mean intensity of 30.3 parasites per host (Felizardo et al., 2009).Lopes et al. (2011) documented the occurrence of Hysterothylacium larvae in the pericardic cavity of specimens of D. suavidicus (Lea, 1856) (Mollusca, Hyriidae) from Aripuanã River, state of Amazonas, Brazil, but there was no record of this larvae parasitizing fishes in the Amazon until now.In Brazil, despite the recovery of anisakid larvae from different hosts in marine and freshwater fishes, there are no reports relative to human anisakiasis.However, with the globalization of food habits and the increasing consumption of raw fish, epidemiological studies are needed to show fish parasitism by anisakid nematodes, especially in the Amazonian region.Arapaima gigas is parasitized by three species of anisakid nematodes and two of them acts as definitive host G. spinulosa and T. serrata. A. gigas is probably intermediate or parathenic host of Hysterothylacium sp.Although these larvae are not common in humans, their zoonotic potential should be highlighted.

Moravec et al. (1993) D. suavidicus Lopes et al. (2011)
distance from anterior end.b it comprises the ventriculus with ventricular appendix.c excretory pore anterior to the level of the nerve ring.*No data registered.
L length.a