Morphological aspects of Clinostomidae metacercariae ( Trematoda : Digenea ) in Hoplerytrinus unitaeniatus and Hoplias malabaricus ( Pisces : Erythrinidae ) of the Neotropical region , Brazil

Species of fish of Marajó Island, State of Pará, Brazil, were examined to identify the trematodes parasitizing 102 Hoplerytrinus unitaeniatus (gold wolf fish) and 104 Hoplias malabaricus (thraira). Metacercariae of two species of trematodes, 170 specimens of Clinostomatopsis sorbens and 10 Ithyoclinostomum dimorphum were found and identified. The parasitary indices of C. sorbens from H. unitaeniatus and H. malabaricus, were 43.14% and 30.77% for prevalence, 2.52 and 1.84 for mean intensity, 1.09 and 0.57 for mean abundance and 1 to 9 and 1 to 7 for range of infection, respectively, on both fish the site of infection was the mesentery. The parasitary indices of I. dimorphum from H. unitaeniatus were 2.94% for prevalence, 2.66 for mean intensity, 0.08 for mean abundance, 1 to 4 for range of infection, and the sites of infection were the mesentery and the muscle. Metacercariae of I. dimorphum were collected in muscles of a specimen of H. malabaricus, with 0.96% of prevalence, intensity of infection of 2 parasites and 0.02 of abundance. New morphological data of external and internal structures are presented. This is the first record of metacercariae of C. sorbens and I. dimorphum in Amazonian fish.


INTRODUCTION
The genera Hoplerythrinus and Hoplias, have a wide distribution in the Neotropical region (Godoy 1975, Buckup 1999, Oyakawa 2003, Graça and Pavanelli 2007, Oyakawa and Mattox 2009).The Hoplerythrinus unitaeniatus occurs in Central and South America, and inhabits swamps and creeks with little current, as well as flooded savannas.The Hoplias malabaricus occurs in Central and South America from Costa Rica to Argentina, being found in most rivers basins.They constitute an important fishery resource, also used in aquaculture and as ornamental fish (Froese and Pauly 2012).http://dx.doi.org/10.1590/0001-3765201420130025733-744 RAIMUNDO N.M.BENIGNO et al.In Brazil, data on the fishery of these two species indicate their economic value related to the amount of fish obtained, and taking into account the internal acceptance of the product.Both erythrinids fish are important sources of protein for the Amazonian riverside populations and can represent up to 50% the diet of the Marajó Island communities (Marinho 2005, Murrieta et al. 2008).In the fish markets of Marajó Island these fish are usually sold whole making it to view parasites and because of the constant presence of larvae of these Clinostomidae helminths, the consumer tends to reject them during evisceration and filleting.H. unitaeniatus and H. malabaricus have been previously studied considering hygienicsanitary procedures regarding ichthyoparasitological approaches, mainly on anisakids and eustrongylids nematode species (Benigno et al. 2012).
The presence of parasites in fish products indicates a harmful sanitary problem not to be underestimated.Even considering that most of the parasitic agents are not pathogenic to humans, some species can be associated to serious diseases due to the ingestion of infected fish, caused by helminth larvae, and few species of clinostomid trematodes may rarely infect people (mainly associated to Clinostomum spp.), causing pharyngitis, laryngitis, laryngo-pharyngitis or eye infections, who have consumed raw fresh-water fish, in Japan, Korea, Thailand, India, and Israel (Williams and Jones 1994, Chung et al. 1995, Tiewchaloern et al. 1999).
Beyond the zoonotic importance of this group of parasites it is related to the disagreeable aspect they present to potential consumers of infected fishes that often are discharged either in processing facilities or during inspection procedures, causing economic losses.Reports of parasitism by a Clinostomidae trematode Clinostomum sp. in tilapia species, Oreochromis spp. in Zaire have often been disreputed or simply rejected by consumers because of parasitic worms (Kabunda and Sommerville 1984).
The metacercariae of Clinostomum sp., C. complanatum (Rudolphi 1814) and C. marginatum Rudolphi 1819 species usually involved with zoonosis in other countries have been reported in Brazilian freshwater fish, and H. malabaricus was included among these hosts (Dias et al. 2003a, 2006, Eiras et al. 2010).
This work aimed to study the digenetic trematodes clinostomids parasites of fishes collected in Lake Arari, Marajó Island, State of Pará, Brazil, analyzing morphological structures on the helminth species, and parasitological indexes related to prevalence, mean intensity, mean abundance, infection range, and sites of infection.

MATERIALS AND METHODS
From August to December 2009, were collected 102 specimens of gold wolf fish, Hoplerytrinus unitaeniatus (Spix and Agassis 1829) of weight CLINOSTOMIDAE METACERCARIAE IN ERYTHRINIDAE 107-376g and with a standard length of 15.4-25 cm, 104 specimens of thraira, Hoplias malabaricus (Bloch 1794), of weight 110-530g and with a standard length of 17.8-27.2cm, in the Arari Lake, Marajó Island, State of Pará (PA), Brazil (00°39'48" S, 49°10'30" W).The fish were kept in isothermic boxes with ice and transported to the Laboratório de Parasitologia Animal da Universidade Federal Rural da Amazônia, Campus Belém, PA.After tegumental surface was inspected, the specimens were necropsied, the organs were separated, and transfered to the Petri dishes with 0.65% NaCl solution and analyzed under stereoscopic microscope.The fish fillets were obtained by incision of musculature, from area close to the operculum to caudal fin, analyzed by candling table, and the parasites were collected.For morphologic and morphometric studies, whole mounts of the metacercariae were made according to Amato et al. (1991) and Eiras et al. (2006) methodology.Drawings were made with the aid of a drawing tube connected to a Olympus BX 41 brightfield microscope.For studies in scanning electron microscope (SEM), metacercariae samples were fixed in 2.5% glutaraldeid in a sodium cacodilate buffer solution 0.1 M, pH 7.4, submitted to six washings with the same buffer at intervals of 15 minutes and post-fixed in 1% osmium tetroxide, dehydrated in a graded ethanol series (20-100 °GL) for one hour each step, CO 2 critical-point dried, coated in gold (20-25 nm deposited) and examined, and images were obtained by digital aquisition system using a scanning electronic microscope LEO 1450 VP, under an accelerating voltage of 15 Kvolts in the Laboratório de Microscopia Eletrônica do Instituto Evandro Chagas (IEC), Belém, PA.The digenetic trematodes metacercariae were identified based on Kanev et al. (2002).On the description the terms forebody and hindbody followed sensu Manter (1970).Measurements were in milimetres (mm), with the range followed by means indicated in parentheses.Prevalence, intensity, mean intensity, abundance, and mean abundance were obtained in accordance with Bush et al. (1997), the range of infection and infection sites of each helminth species, were also presented.Voucher specimens were deposited in the Coleção Helmintológica do Instituto Oswaldo Cruz (CHIOC), State of Rio de Janeiro, Brazil.The studied metacercariae were compared with adult and metacercaria specimens of different states of Brazil deposited on CHIOC.

RESULTS
In both fish species analyzed, living and nonencysted clinostomid metacercariae specimens of two different species described below, were found.
For generic diagnose Kanev et al. (2002) was used and specific diagnose was based on descriptions of Lent and Freitas (1937) and Travassos et al. (1969), and the present paper adds details about external and internal structures, mainly on suckers, genital pore and terminal genitalia.
In Brazil that metacercaria was reported parasitizing the mesentery, musculature, heart, esophagus, cloaca, gills, opercula and fins (Pavanelli et al. 1990, Moreira 2000, Rodrigues 2010).In the present study, the prevalence indices were lower than those recorded in H. malabaricus by Pavanelli et al. (1990), Weiblen and Brandão (1992), Paraguassú and Luque (2007) and Rodrigues (2010), but the mean intensity of infection was very close to those recorded by Pavanelli et al. (1990) (1.53), Weiblen and Brandão (1992) (2.8), and Rodrigues (2010) (2.52), this may be related to different ecological factors of the collection locations.Dias et al. (2003b) by SEM described the oral sucker aperture of I. dimorphum as eliptical, which differs from the specimens studied in the present work, which showed apertures being triangular; but are in accordance with body surface with rounded sensory papillae, furrows and rings forming superficial annulations, dorsal and ventral, both in the forebody and hindbody (Figures 3a-b, 4a-d) and ventral sucker with subtriangular aperture (Figures 3a-b).

DISCUSSION
This is the first report of C. sorbens and I. dimorphum metacercariae in Amazonian fish species, adding morphological contributions on the external and internal structures mainly on suckers, genital pore, and terminal genitalia, which will be useful for future researches.The data obtained from this study on fish species caught in Arari Lake, Marajó Island confirm the role that these fish play in the life cycle of C. sorbens e I. dimorphum.
The presence of living and non-encysted clinostomid metacercariae species findings in this study on both fish species, brings out certain interesting facts concerning hygienic-sanitary, and about the potential hazard to human health, because other Clinostomatidae species have been reported to cause laryngitis, laryngo-pharigitis and in also an eye infection, or even have often been rejected by consumers because of their repugnance aspect when they are present on the musculature, viscera or abdominal cavity (Kabunda and Sommerville 1984, Williams and Jones 1994, Chung et al. 1995, Tiewchaloern et al. 1999).The previous species of clinostomids were involved in human infections belonging to other known species, we suggest further investigation concerning the role of these Brazilian clinostomids, because the visual analysis of the parasite species (visible and sometimes very large) of the contaminated fish specimens, take it into account that their discharge is recommended and foreseen CLINOSTOMIDAE METACERCARIAE IN ERYTHRINIDAE by the legislation of several countries including Brazil, Spain and France, among others (Brasil 2007).
Adults of I. dimorphum have been reported parasitizing birds, A. cocoi, from State of Pará and State of Mato Grosso and Paraná River Basin REMARKS