Infection by Henneguya sp . ( Myxozoa ) in the bone tissue of the gill filaments of the Amazonian catfish Hypophthalmus marginatus ( Siluriformes )

This study describes aspects of the infection caused by the myxosporean genus Henneguya, which forms cysts in the bony portion of the gill filaments of Hypophthalmus marginatus. Specimens of this catfish were acquired dead from artisanal fishermen near the town of Cametá, state of Pará, northern Brazil, between July 2011 and May 2012. They were transported in refrigerated containers to the Carlos Azevedo Research Laboratory at the Federal Rural University of Amazonia, in Belém, where analyses were performed. After confirmation of parasitism by the genus Henneguya, observation were made using optical and differential interference contrast (DIC) microscopy. The histological technique of embedment in paraffin was used. Ziehl-Neelsen staining was applied to the histological sections. Necropsy analyses on specimens of H. marginatus showed that 80% of them (40/50) had cysts of whitish coloration inside the bony portion of the gill filaments, filled with Henneguya spores. The present study found inflammatory infiltrate in the vicinity of the cysts. Furthermore, the special Ziehl-Neelsen staining technique made it possible to mark the Henneguya sp. cysts in the bone tissue and in spore isolates in the gill tissue structure. The descriptions of these histopathological findings show that this parasite is very invasive and causes damage to its host tissues.


Introduction
The Amazon basin is home to the largest and most diverse fish fauna in the world, with over 1500 recorded species (MONTAG et al., 2008).Several species among these fish are valued in the international market, as is the case of a flatfish known as the Amazonian catfish (CUTRIM & BATISTA, 2005).
The species Hypophthalmus marginatus Valenciennes, 1840, is known in Brazil as mapará and stands out as a flatfish of great commercial importance in the Amazon basin, and in the markets in that region (COSTA et al., 2010).It is a midsized catfish (order Siluriformes), in the Pimelodidae family, found in rivers throughout the Amazonian basin (MARTINS et al., 2011).They are rheophilic fish and depend on the natural river flow to perform their reproductive functions (CARVALHO, 1980).They differ from other members of the Pimelodidae in their feeding habits and their location in the water column: whereas most species in this family have demersal carnivorous habits, maparás have pelagic planctophagous habits (CUTRIM & BATISTA, 2005).Because of the great acceptance of the species H. marginatus in consumer markets, studies are being conducted to demonstrate the potential of this species for aquaculture (COSTA et al., 2010).
The parasite fauna of H. marginatus has been little explored and may include parasite species that have not yet been described in the literature.Parasitic infections very frequently affect aquatic animals, causing profound structural and physiological changes and increasing the mortality rate (TAVARES-DIAS et al., 2014).Many species of economic interest can be infested by parasites and other biological agents.Myxosporeans are prominent among these parasites: these endoparasitic organisms are responsible for myxosporidiosis, a disease that affects freshwater and marine fish in many different geographical areas (AZEVEDO et al., 2009).Myxosporeans taxonomically belong to the phylum Myxozoa Grassé, 1970, and more than 2,200 species have been described.They are mostly fish parasites, both in the natural environment and in farming systems, and some species are responsible for diseases that generate high rates of mortality around the world (LOM & DYKOVÁ, 2006).The genus Henneguya has worldwide distribution and is considered to be one of the largest and most important groups belonging to the phylum Myxozoa, with about 190 species described around the world (EIRAS & ADRIANO, 2012) and more than 39 species in South America (AZEVEDO et al., 2009).
The gills are a very complex organ and a great number of species of myxosporeans are found parasitizing this organ.These parasites produce cysts in various locations within this organ, and the infection can be characterized as three types: lamellar, filamental and arch (in the gill arches) (MOLNÁR, 2002).
The objective of this work was to provide a histopathological description of infection due to Henneguya sp. in the bony portion of the gill filaments of H. marginatus.

Materials and Methods
Fifty specimens of the catfish H. marginatus, of length 30 cm (range: 28 -34) and weight 75 g (range: 72 -78), were acquired dead from artisanal fishermen near the town of Cametá (2° 14' S, 49° 29' W), in the northern Brazilian state of Pará, between July 2011 and May 2012.The specimens were transported in refrigerated containers to the Carlos Azevedo Research Laboratory at the Federal Rural University of Amazonia, in Belém.The fish were necropsied and the gill region was observed under a stereomicroscope appropriate for parasitological studies.For fresh examination, small tissue fragments containing suspected parasitism were placed between slides and cover slips together with a drop of water, for observation under an optical microscope and a differential interference contrast (DIC) microscope (Nomarski).For the histological procedure, small fragments (0.5 cm) of the parasitized tissue extracted from the gill region of the fish specimens were fixed in Davidson's solution (neutral-buffered formalin, glacial acetic acid, 95% ethanol and distilled water) for 24 hours and were then processed for embedding in paraffin.Sections were cut and stained with Ziehl-Neelsen (LUNA, 1968), and these were mounted on slides with a coverslip and were photographed under a Zeiss Primo Star optical microscope equipped with the Zeiss AxioCam ERc 5s photographic camera and the AxioVision 5.1 software.

Results and Discussion
The necroscopic analysis on H. marginatus specimens revealed that 80% of the hosts (40/50) presented cysts of whitish coloration, inside the bony portion of the gill filament (Figure 1).From fresh examination under the optical microscope, it could be seen that when the cysts burst, they were full of mature spores exhibiting the diagnostic traits of spores of the genus Henneguya (Figures 2-4): myxospores ellipsoidal, bilaterally symmetrical with a bifurcated caudal process extending from the posterior end (LOM & DYKOVÁ, 2006).
In other species of Siluriformes, the prevalence rates of parasitism in the gill region caused by species of the genus Henneguya have been observed to be lower than those described in the present study.Naldoni et al. (2011) reported that the prevalence of parasitism by Henneguya eirasi in Pseudoplatystoma spp. was 17.1%; Adriano et al. (2012) reported that the prevalence of H. multiplasmodialis in Pseudoplatystoma corruscans was 3.3% and in Pseudoplatystoma reticulatum, 2.6%; and El-Mansy & Bashtar (2002) described parasitism by Henneguya suprabranchiae with a prevalence of 35.8%.
The histopathological analyses revealed the presence of diffuse necrosis of the lamellae and several gill filaments.This was associated with necrotic foci over large numbers of cysts in the bone tissue of the gill filaments.The site of the parasitic infection caused by Henneguya sp. has usually been observed in the intra Infection by Henneguya sp. in bone tissue and inter-lamellar region of the gill filaments (FEIJÓ et al., 2008;NALDONI et al., 2009;YE et al., 2012).There have not been any previous reports of infection located in the bone tissue of the gill filament.On the other hand, there have been descriptions of parasitism by Henneguya in cartilaginous tissue.Batueva et al. (2013) reported the presence of Henneguya cerebralis infection in the cranial cartilage of Thymallus arcticus nigrescens; Yokoyama et al. (2012) reported Henneguya cartilaginis infection in the cranial cartilage of Oncorhynchus masou masou; and El-Mansy & Bashtar (2002) reported parasitism by Henneguya suprabranchiae in the hyaline cartilage of the gills of the catfish Clarias gariepinus.
Ziehl-Neelsen staining revealed Henneguya sp.cysts in the bone tissue of the gill filaments (Figures 5-8), and parasite spores were released from the necrotic foci (Figures 6 and 8).Intense inflammatory infiltrate consisting of heterophils, lymphocytes and macrophages around cysts and spores was also observed.The gill filaments parasitized by this myxosporeans had become crooked and deformed, such that the cartilaginous parts had withered due to pressure caused by the development of plasmodium (Figure 7 and 8).Deformities in the gill filaments brought about by parasites of the genus Henneguya have often been described in the literature (ADRIANO et al., 2005;NALDONI et al., 2009;CAMPOS et al., 2011;YE et al., 2012;AZEVEDO et al., 2014).El-Mansy & Bashtar (2002) obtained findings similar to those described in the present study, through studying parasitism by Henneguya suprabranchiae.This parasitism induced atrophy of the hyaline cartilage, resulting from continued growth of the plasmodium.The high intensity of infection also led to desquamation and necrosis of the tissue surrounding the suprabranchial body.
Although reports of inflammatory responses in the absence of Henneguya spp.infection are fairly common (ADRIANO et al., 2005;FEIJÓ et al., 2008), the present study found inflammatory infiltrates in the vicinity of the cysts.Furthermore, the special Ziehl-Neelsen staining techniques (LUNA, 1968) made it possible to mark the presence of Henneguya cysts in bone tissue and spore isolates in the gill tissue structure.The descriptions of these histopathological findings show that this parasite is very invasive and causes damage to its host tissues.
Given the commercial importance of H. marginatus and its high potential for aquaculture, the results obtained from the present study are highly relevant.Thus, they need to be disseminated since they contribute towards characterizing the parasitological profile of the host species.Moreover, this study establishes that research aimed at standardizing the methods for controlling and preventing water-borne diseases is necessary.