MORPHOLOGICAL CHARACTERS AND HYGIENIC-SANITARY SIGNIFICANCE OF Tentacularia coryphaenae IN Coryphaena hippurus FROM 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) 3Laboratório de Helmintos Parasitos de Peixes, Instituto Oswaldo Cruz, FIOCRUZ, Av. Brasil, 4365, Manguinhos, 21040-900 Rio de Janeiro, RJ, Brasil. CARACTERES MORFOLÓGICOS E SIGNIFICÂNCIA HIGIÊNICO SANITÁRIA DE Tentacularia coryphaenae EM Coryphaena hippurus DO BRASIL


INTRODUCTION
The Yacht Club of Rio de Janeiro (ICRJ), sponsors sport fishing tournaments during which several species of oceanic fish are captured.These fish include economically valuable species with desirable meat, and so they are not only considered trophies, but are also sent to ICRJ's own fish market.One of these species is the dolphinfish, Coryphaena hippurus Linneaus, 1758.The species C. hippurus is cosmopolitan, which has a cosmopolitan distribution throughout, tropical and subtropical seas (GIBBS and COLLETTE, 1959;MENEZES and FIGUEIREDO, 1980).In Brazil, the dolphinfish is highly valued for food and is an important constituent of recreational and commercial fisheries (GIBBS and COLLETTE, 1959;DITTY et al., 1994;PALOMINO et al., 1998).
The maintenance of hygienic-sanitary conditions at the ICRJ fish market has been a concern.For example, larvae of trypanorhynch cestodes have been found in the musculature of dolphinfish at the ICRJ fish market, which usually produce a repugnant appearance resulting losses in sales.Cestodes of the order Trypanorhyncha possess worldwide distribution, and occur in marine animals of tropical and subtropical regions (PALM, 2004).
A s a d u l t s t h e s e c e s t o d e s i n h a b i t t h e gastrointestinal tract of elasmobranchs, while their larval forms are found in several species of teleost and elasmobranch fish, and a variety of marine invertebrates (CAMPBELL and BEVERIDGE, 1994).According to the Brazilian legislation that deals with fish and their derivative products, any fish with a repugnant appearance is considered improper for consumption, as in any musculature possessing massive parasite infection, whether or not it may affect the health of consumers (BRASIL, 2007).Furthermore, the presence of trypanorhynch larvae in fish is considered disconcerting since they can pose a risk of allergic reactions in humans (MATTOS et al., 2013).
The aims of this investigation were to: (1) identify the species of trypanorhynch cestodes parasitizing C. hippurus caught during sport fishing tournaments in the state of Rio de Janeiro by means of morphological and morphometric analyses using optical and scanning electron microscopy of specimens retrieved from the site of infection; (2) demonstrate the hygienic-sanitary significance of these parasites to collective human health; and (3) provide baseline data for subsequent investigations.

METHODS
In January and November 2014, seven specimens of dolphinfish, C. hippurus (85-100 cm total length and 6.5-7 kg) caught by fishermen during ocean sport fishing of the coast of the municipality of Cabo Frio, state of Rio de Janeiro, Brazil were brought to ICRJ and sent to its fish market.Fish species were identified according to MENEZES and FIGUEIREDO (1980).
The fish were then necropsied and filleted, and their organs separated into plastic bags, labeled and transported on ice in isothermic boxes for examination in the Laboratory of Helminth Parasites of Vertebrates, Oswaldo Cruz Institute, Oswaldo Cruz Foundation (FIOCRUZ), Rio de Janeiro.The three dolphinfish captured in January 2014 possessed plerocercoids of trypanorhynch cestodes parasitizing musculature, stomach, liver serosa and mesentery.The other four dolphinfish, caught in November 2014, had only their musculature examined, because their commercialization was prohibited by the massive presence of trypanorhynch plerocercoids.
In the laboratory, retrieved cestodes were placed in Petri dishes containing destiled water and maintained in a refrigerator for at least 24h to permit the relaxation of scolices and extrovertion tentacles.Some of the larvae were fixed in cold AFA, stained with Langeron's carmine, differentiated in 0.5% hydrochloric acid-ethanol solution, dehydrated in a series of increasing alcohol concentrations and clarified with beach-wood creosote.Ten specimens were then mounted whole preserved in Canada balsam and measured, while the other specimens were preserved in 70ºGL ethanol (KNOFF and GOMES, 2012).
The specimens were observed with an Olympus BX-41 bright-field microscope, and images were obtained using a Cannon Power Shot A640 digital camera coupled to Zeiss Axiophot microscope.Topographical descriptions of surface of scolices was performed using scanning electron microcopy (SEM).For SEM the cestodes were processed as described by LOPES TORRES et al. (2013), which envolved, in short, fixation in ethanol 70ºGL, dehydration in an acetone series, CO 2 critical-point drying and gold coating (20 nm deposition).Images were made using a JEOL LV 6940 SEM under 30kV acceleration voltage.
Voucher specimens were deposited in the Helminthological Collection of the Oswaldo Cruz Institute (CHIOC), FIOCRUZ, Rio de Janeiro, RJ, Brazil.

RESULTS
The three specimens of C. hippurus collected in January 2014 were parasitized by a total of 108 plerocercoids of trypanorhynch cestodes in the abdominal musculature, mesentery, stomach and liver serosa.The four hosts collected in November 2014 were parasitized by a total of 96 plerocercoids of the same species.These fish were removed from commercialization and discarded by the ICRJ fish market due to the massive infections in the abdominal musculature and the severe hemorrhage caused by the large number of cestodes (Figure 1 a-c).All plerocercoids were found alive and demonstrated high motility. CestodaTrypanorhyncha
In Brazil, T. coryphaenae has been reported parasitizing a variety of marine teleost hosts, including the dolphinfish and elasmobranch fishes.All these teleosteans were collected from the coastal waters of the state of Rio de Janeiro.SILVA and SÃO CLEMENTE (2001) examined 596 fillets from 41 individuals of C. hippurus (size range of 65-148 cm total length) from the same area and found T. coryphaenae present in 25 fillets; 1 infected fillet from the 65-106 cm size class, and 24 infected fillets from the 107-148 cm size class.
A total of 56 parasites were found, for a prevalence of 0.47% and an intensity of infection of 1 parasite per fillet in the smaller size class, and a prevalence of 6.25% and an intensity of infection of 2.29 parasites per fillet in the larger size class; however, the authors did not provide information about the number of parasitized fish and the mean intensity of infection, Moreover, SÃO CLEMENTE (personal information) did not observe massive infections by this species in the musculature.
Tentacularia coryphaenae has been reported from the mesentery of Genypterus brasiliensis with a prevalence of 1.35 and an intensity of 1 specimen per individual (SÃO CLEMENTE et al., 2004); in Katsuwonus pelamis L., 1758, with a prevalence of 92.9% and mean intensity of 36.3 and a range of infection of 1-110 plerocercoids per fish in the abdominal and body musculature (AMATO et al., 1990), and in another case with a prevalence of 66.7%, mean intensity of 5.8 and range of infection of 1-17 parasites in the musculature (ALVES and LUQUE, 2006); in Lophius gastrophysus Miranda-Ribeiro, 1915, with a prevalence of 2.3% and an intensity of 1 parasite in the musculature (SÃO CLEMENTE et al., 2007); in Scomberomorus cavalla (Cuvier, 1829), with a prevalence of 3% and an intensity of 2 parasites in the mesentery (DIAS et al., 2011).
Among elasmobranchs, T. coryphaenae has been reported parasitizing species off the coast of a variety of different Brazilian states, including being found in the stomach and spiral valve of a Carcharinus longimanus Poey, 1861, in Recife, state of Pernambuco, without information about the number of parasites collected (REGO, 1977), and from the spiral valve of Prionace glauca (L., 1758), C. longimanus and C. obscurus (Lesueur, 1818) collected in the state of Santa Catarina with prevalences of 16.7%, 50% and 100% and mean intensities of 0.2, 0.5 and 12 parasites per fish, respectively (KNOFF et al., 2002(KNOFF et al., , 2004)).Comparing the data of the present study with data for teleost species of commercial importance studied along the Brazilian coast, the specimens of C. hippurus studied herein (the previous report of C. hippurus did not make mention of a massive infection) and K. pelamis had highest indices of parasitism.The finding may be due to the influence of different physico-chemical factors and biological interactions experienced by the fish parasite communities of the different environments.
The species T. coryphaenae is known to exhibit low host specificity during its final and intermediate hosts.It is distributed worldwide in tropical and subtropical seas, and inhabit widely migrating scombroids and large pelagic predatory fish as second intermediate hosts.Adults of T. coryphaenae have been recorded from 11 different final host species, and is known from over 80 species of smaller and larger pelagic or oceanic fish and squids (PALM, 2004).
The findings of the present study corroborate previous reports and CHIOC examined material that the dolphinfish C. equiselis and C. hippurus, and tunas K. pelamis and T. albacares, as well as other pelagic and cosmopolitan migratory teleosts known are intermediate hosts in Brazil and throughout the world, and reinforce the hypothesis that these fish are acting as bridges for transfer of T. coryphaenae throughout marine food webs and among oceans (PALM et al., 2007).
Hepatoxylon trichiuri (Holten, 1802) Bosc, 1811, is another trypanorhynch species that parasitizes C. hippurus in Brazil (SÃO CLEMENTE et al., 2001), with a prevalence of 4.9% and a mean intensity of 1 parasite per fish in the liver and stomach serosas, however, none of the fish of the present study were parasitized with this species.AMATO et al. (1990) suggested the removal of abdominal musculature as method for exporting K. pelamis free of these trypanorhynch metacestodes, however, this procedure does not seem viable in the present study, because, of widespread distribution of large numbers of metacestodes in muscular fascia.This observation was also reported for another trypanorhynch species, Myxonybelinia sp., a parasite of Lophius gastrophysus Miranda-Ribeiro, 1915, collected off the coast of the state of Rio de Janeiro (SÃO CLEMENTE et al. , 2007).The present study represents the first report of T. coryphaenae causing severe hemorrhage in C. hippurus, which has never even been referred to in previous studies (SILVA and SÃO CLEMENTE, 2001).

CONCLUSIONS
The present study found T. coryphaenae plerocercoids to have significant hygienicsanitary imporytance for C. hippurus, mainly because of its massive presence in the musculature and the resulting severe hemorrhage.These findings indicate that commerce with this fish species is impracticable in the ICRJ fishmarket due to the repugnant appearance of parasitized individuals.
The possibility of causing hypersensitivity reactions in humans illustrates the importance of inspecting fish for these trypanorhynch parasites.Furthermore, the results of this study suggest that in the future immunological studies, with a murine model, into these plerocercoids will be subject are needed for evaluating the risk of causing allergic reactions in humans.

Figure 1 .
Figure 1.Tentacularia coryphaenae plerocercoid infection in Coryphaena hippurus.a. Fillets of the abdominal musculature of hosts collected in January 2014; arrows indicate the presence of the cestode parasites.b-c.Musculature of hosts collected in November 2014; arrows indicate the large number of cestodes causing severe hemorrhage.Bars a = 2.5 cm; b-c = 3 cm.

Figure 3 .
Figure 3. Tentacularia coryphaenae plerocercoid in Coryphaena hippurus under scanning electron microscopy.a. Detail of anterior region of scolex, showing tentacles and bothrial region.b.Detail of basal region in bilateral symmetry, with internal (I) and external surfaces (E) showing an unarmed inverted V-shaped pattern, and the bothrial surface (B) showing ascending rows of hooks (trident-like) in a V-shaped pattern beginning in the metabasal region.c.Detail of the metabasal region with uncinate hooks with rotational symmetry and a quincunxial arrangement on the bothrial surface.Bars a = 200 µm; b = 50 µm; c = 20 µm.