Anisakid larva parasitizing Plagioscion squamosissimus in Marajó Bay and Tapajós River , state of Pará , Brazil

In November 2014 and May 2015, a total of 44 specimens of the South American silver croaker Plagioscion squamosissimus were collected: 30 in Marajó Bay and 14 in the Tapajós River, state of Pará, Brazil. The aim was to investigate the presence of anisakid nematodes and determine their parasitism indices and sites of infection, because of their importance regarding health inspection. Sixty-nine Anisakis sp. larvae were found; among them, 16 larvae in seven fish collected in Marajó Bay and 53 larvae in four fish in the Tapajós River. The parasitism indices of the nematodes collected from the fish in Marajó Bay comprised prevalence (P) of 23%, mean infection intensity (MI) of 2.28, mean abundance (MA) of 0.53, range of infection (RI) of 1-4, and infection site (IS) in the mesentery. The fish from the Tapajós River showed P = 28%, MI = 13.2, MA = 3.8, RI = 1-22, and IS = mesentery and intestine. To assist in taxonomic identification, images of the specimens obtained through optical microscopy with Nomarski’s interferential contrast system and scanning electron microscopy were used. This is the first record of Anisakis sp. parasitizing P. squamosissimus.


Introduction
Plagioscion squamosissimus (Heckel, 1840) is a sciaenid fish commonly known as the South American silver croaker.It lives in benthopelagic environments and, during its life cycle, it migrates from freshwater to brackish water.In the juvenile stage, it feeds on shellfish larvae and aquatic insects; and in the adult stage, it feeds on copepods.In South America, it occurs in many countries basins: Amazon, Orinoco, Paraná, Paraguay, and São Francisco River basins and rivers of Guianas (CASATTI, 2003).This fish is considered to be of high commercial importance in northern Brazil, i.e. in the lower Amazon region, including the city of Santarém, state of Pará, and on the coast of the state of Amapá and in indentations of the states of Maranhão and Pará (FERREIRA et al., 1998;CAMARGO & ISSAC, 2001;MELO et al., 2014).
Species belonging to the genus Anisakis (Dujardin, 1845) have mostly been found in many species of Brazilian marine fish (KNOFF et al., 2013;FONTENELLE et al., 2013).There is also one record in a fish species that inhabits freshwater and brackish water, P. squamosissimus, in the municipality of Colares, state of Pará (RODRIGUES et al., 2015).
A single case of anisakiasis has been reported in Brazil, in a man in the state of Mato Grosso, with the larvae located in the mucosa of the duodenum (CRUZ et al., 2010).
This study aimed to investigate the presence of larval stages of anisakid nematodes in P. squamosissimus collected in Marajó Bay, in the municipality of Belém, and in the Tapajós River, municipality of Santarém, both in the state of Pará, Brazil, and to determine their parasitism indices and infection sites.

Materials and Methods
A total of 44 specimens of the South American silver croaker, P. squamosissimus (Heckel, 1840) were purchased from professional fishermen in the state of Pará, Brazil.Among these, 30 silver croaker (measuring 30-32 cm in total length) had been caught in Marajó Bay, municipality of Belém (latitude 1° 0' 2" S, longitude 50° 12' 28" W) in November 2014; and 14 (measuring 23-29.5 cm in total length) in the Tapajós River, municipality of Santarém (latitude 02° 24' 54.6" S, longitude 54° 44' 18.6" W) in May 2015.The fish were transported in isothermal boxes with ice to the Carlos Azevedo Research Laboratory, Institute of Animal Health and Production, Federal Rural University of the Amazon Region, Belém, state of Pará, Brazil.The fish were identified in accordance with Casatti (2003).
After necropsying and filleting the fish, nematode larvae were found and placed in Petri dishes with 0.65% NaCl solution.The specimens were then fixed in alcohol, formalin, and acetic acid (AFA) (60 °C), preserved in a solution of 70 °GL ethanol plus 5% glycerin and clarified with Amman's lactophenol, as described by Knoff & Gomes (2012).
The taxonomic classification of nematodes was made in accordance with Fagerholm (1991).The larvae were identified as described by Felizardo et al. (2009) and Fontenelle et al. (2013).
The samples were analyzed by bright-field microscopy using Nomarski's differential interference contrast (DIC) apparatus.Images were obtained using a Canon digital camera (Power Shot A640) coupled to a Zeiss Axiophot microscope.For topographic characterization of the cuticular surface, four third-stage larvae of Anisakis sp. were analyzed using a scanning electron microscope (SEM).The material was processed as described by Lopes Torres et al. (2013).The samples fixed in 70% ethanol were dehydrated in an ethanol series (70º and 100º GL), with CO 2 critical point drying, coated in gold, examined and photographed using a SEM (JEOL SM-25 SII), under 15 kV acceleration voltage.
Morphometric analyses were performed using an Olympus BX41 microscope.Measurements were shown in millimeters (mm) with the averages in parentheses, unless otherwise indicated.The parasitism indices of prevalence, mean intensity and mean abundance were obtained as described by Bush et al. (1997).
Representative specimens of Anisakis sp. were deposited in the Helminthological Collection of the Oswaldo Cruz Institute (CHIOC), Rio de Janeiro, state of Rio de Janeiro, Brazil.

Results
Among the 44 specimens of P. squamosissimus analyzed, 11 were parasitized with 69 live third-stage larvae (L3) of Anisakis sp.: seven fish from Marajó Bay containing 16 L 3 ; and four fish from the Tapajós River containing 53 L3.Table 1 shows the prevalence The main features observed in the third-stage larvae were as follows: (1) anterior extremity with one dorsal and two poorly developed ventrolateral lips; (2) six cephalic papillae, one pair in the dorsal lip and one pair in each ventrolateral lip; (3) boring tooth below the oral aperture, between the two ventrolateral lips; (4) excretory pore opening beneath the boring tooth; (5) inconspicuous nerve ring; (6) ventriculus length greater than its width; (7) ventricular appendix and intestinal cecum absent; (8) two nearly spherical rectal glands; and ( 9) conical tail and mucron present.

Discussion and Conclusion
Plagioscion squamosissimus is a species of freshwater fish that also habits brackish water environments (CASATTI, 2003).In the present study, it was collected from two municipalities, Belém and Santarém, in the Amazon hydrographic basin, which are affected by direct influence from Atlantic ocean waters.The presence of larval stages of Anisakis sp.parasitizing P. squamosissimus confirms this environmental intersection of salinities, given the hosts used by the four larval stages within the life cycle of Anisakis species, within the eggs (L1-L3) and subsequently in intermediate and paratenic hosts (L3), and as pre-adults (L4) and adults in the cetacean final hosts (KLIMPEL & PALM, 2011).The present study expands the occurrence of Anisakis sp. in P. squamosissimus to two additional municipalities in the state of Pará (RODRIGUES et al., 2015).
The larvae of Anisakis sp.collected in the present study were morphologically similar to specimens of A. simplex (Rudolphi, 1809) and Anisakis sp.2013) and the present study are possibly related to different hosts or the influences of different ecoregions, given that the specimens examined were purchased in different biogeographic areas, i.e. temperate South America (state of Rio de Janeiro) and tropical Atlantic (state of Pará) (SPALDING et al., 2007).Melo et al. (2014) reported that anisakid larvae (without generic identification) were present as parasites in 100% of the P. squamosissimus specimens collected from the estuary of the Guamá River and the neighboring Guajará Bay, adjacent to the municipality of Belém, state of Pará.The prevalence in their study was around 75% higher than what was found in the present study for Anisakis sp.Rodrigues et al. (2015) recorded that the prevalence of Anisakis sp. in the same host was slightly lower (10%) than that of the present study.In the present study, the infection sites were the mesentery and intestine, and these sites have also cited by other authors.Melo et al. (2014) found larvae in the serosa surrounding the internal organs, in which they observed weak inflammatory reaction.
In both previous studies in the state of Pará involving anisakids, there was no indication of nematode specimens in a zoological collection.This made it impossible to determine the morphology and morphometry of their larvae in comparison with those of the present study.
The presence of live larvae parasitizing P. squamosissimus specimens caught in Marajó Bay and the Tapajós River, along with high prevalence, enables postmortem migration of these larvae into the musculature.Therefore, attention paid towards consumption of raw fish in exotic dishes needs to be increased, because such consumption can increase the risk of acquiring anisakiasis.It is also important to draw attention to this species, given that it can cause gastrointestinal disorders and allergic reactions even if consumed cooked (AUDICANA & KENNEDY, 2008).

Table 1 .
Prevalence (P), mean intensity (MI), mean abundance (MA), range of infection (RI), infection site (IS) and CHIOC deposit number of Anisakis sp.third-stage larvae collected from Plagioscion squamosissimus caught in Marajó Bay, municipality of Belém, in November 2014; and in the Tapajós River, municipality of Santarém, in May 2015; both locations in the state of Pará, Brazil.