Histopathology and autoecology of Didymocylindrus simplex ( Digenea : Didymozoidae ) , parasite of Katsuwonus pelamis ( Scombridae ) in the Southwestern Atlantic Ocean , off South America

Pathological alterations and autoecology of Didymocylindrus simplex in Katsuwonus pelamis (Linnaeus, 1758) in the Southwestern Atlantic Ocean off of South America are presented. This is the first record of the species in the region; the specimens are described. The pathologic changes observed most often were infiltration of lymphocytes and slight fibrosis of the connective tissue surrounding the parasite. The typical aggregated distribution pattern was observed for Didymocylindrus simplex Ishii, 1935. Statistical analysis demonstrated no correlation between the abundance and prevalence of parasites with the total length of the host. The sex of the host does not influence the prevalence and intensity of infection.

Digenean trematodes of the family Didymozoidae Monticelli, 1888, parasitizing marine fishes, predominantly Scombridae are widely documented in the literature.However, most publications correspond to taxonomic studies and only a few investigate the pathological reactions in the host and report data on the parasitological parameters of the species.
Continuing our studies of helminth parasites of scombrid fishes off the coast of Rio de Janeiro, Brazil, Southwestern Atlantic Ocean, Katsuwonus pelamis (Linnaeus, 1758) was found parasitized by Didymocylindrus simplex Ishii, 1935.Katsuwonus pelamis is a species of great importance in Brazil because of the large fish canning industry.It is an epipelagic, oceanic species with adults distributed roughly within the 15°C isotherm, whereas larvae are mostly restricted to waters with surface temperatures of at least 25°C.Food items of this species include fishes, crustaceans and molluscs and cannibalism is common (COLETTE & NAUEN 1983).Others studies about the helminthofauna of K. pelamis from the Brazilian coast were published by KOHN & JUSTO (2006, 2008) and JUSTO & KOHN (2005, 2012a, b).
This study describes, for the first time, the pathological alterations induced by D. simplex on K. pelamis.It also provides new geographical distribution records for didymozoid parasites of scombrid fish in South America, Southwestern Atlantic Ocean.The specimens collected are described.

MATERIAL AND METHODS
Sixty-one specimens of K. pelamis (26-73 cm in total body length and 1-8 kg in weight), including 30 males (49.2%) and 31 females (50.8%), were examined for helminths between January, 2004 andApril, 2011.Fishes were obtained by local fishermen from the coastal zone of the State of Rio de Janeiro, off Cabo Frio, Brazil (22°52'46"S, 42°01'07"W).Parasites were released from their cysts and fixed under cover glass pressure in AFA (alcohol 93%, formalin 5%, acetic acid 2%), stained in alcoholic-acid carmine, dehydrated in an alcohol series, cleared in methyl salicilate and mounted in Canada balsam.Analysis of the parameters of infection prevalence (%), mean intensity and range of infection were based on BUSH et al. (1997) and the confidence intervals (CI) were calculated assuming a binominal distribution, using the software Quantitative Parasitology 3.0 (RÓZSA et al. 2000).
The quotient between variance and mean parasite abundance (index of dispersion) was used to determine distribution patterns and was tested by the d statistical index (LUDWIG & REYNOLDS 1988).
The Spearman's rank correlation coefficient (r s ) was used to determine possible host-length correlations with parasite abundance.The Pearson's coefficient of correlation (r) was used to determine possible correlations between the host's total ABSTRACT.Pathological alterations and autoecology of Didymocylindrus simplex in Katsuwonus pelamis (Linnaeus, 1758) in the Southwestern Atlantic Ocean off of South America are presented.This is the first record of the species in the region; the specimens are described.The pathologic changes observed most often were infiltration of lymphocytes and slight fibrosis of the connective tissue surrounding the parasite.The typical aggregated distribution pattern was observed for Didymocylindrus simplex Ishii, 1935.Statistical analysis demonstrated no correlation between the abundance and prevalence of parasites with the total length of the host.The sex of the host does not influence the prevalence and intensity of infection.KEY WORDS.Didymozoids; histopathology; parasites of fish; Trematoda.
length and prevalence of parasites, with angular transformation of prevalence values; the Mann-Whitney's U Test with Z c normal approximation, to determine host sex effect on infection/infestation abundance of each parasite species (ZAR 1996).
A statistical significance level of p р 0.05 was adopted.Measurements are in micrometres, unless otherwise stated, with the mean in parentheses followed by the number of specimens measured in brackets, where applicable.Light micrographs were taken with a digital camera connected to a Nikon Eclipse E 800 microscope.Representative specimens were deposited in the Helminthological Collection of the Oswaldo Cruz Institute (CHIOC), Rio de Janeiro, Brazil.
For the histopathological analysis, gills of K. pelamis were removed, fixed in 10% formalin, decalcified with EDTA (tetrasodium ethylenediaminotetracetate) for three days to one month and prepared through a routine technique of dehydration in ethanol and then impregnation and embedding in paraffin.Sections of 5-10 µm were made with microtome.The sections were stained with hematoxylin and eosin (H&E) for general visualization of the affected tissues and organs (MICHALANY 1980).
Site: Encysted in pairs in the epidermis of gill lamellae under the basal membrane.
Didymocylindrus simplex were found parasitizing the epidermis of gill lamellae under the basal membrane of 24 of 61 specimens (39.3%) of K. pelamis with one to 28 cysts containing two similar hermaphroditic parasites with a total of 360 specimens.Didymocylindrus simplex showed a typical aggregated distribution pattern (DI = 21.2, d = 33.5).The Pearson's and Spearman's coefficients demonstrated no correlation between the abundance and prevalence of parasites and the total length of K. pelamis (r = -0.075,p = 0.626; r s = -0,047, p = 0.763).The mean abundance and prevalence were not influenced by the sex of the hosts (Z = -1.71,p = 0.087).
Histopathological analysis demonstrated that D. simplex infected the space between the basement membrane of the epithelium of the gill and the efferent artery of the primary gill lamellae (Fig. 4).A slight fibrosis of connective tissue surrounding the parasite was verified, formed by a few layers of collagenous tissue and fibroblasts and a slight inflammatory infiltrate with lymphocytes and granulocytic cells such as monocytes and neutrophils.Adjacent to the gill arch, in the place where the encystment of the parasite occurs, a fibrotic reaction with some blood vessels was observed, between the gill arch and the parasite.Besides that, a slight inflammatory infiltrate was observed adjacent to the fibrotic reaction (Figs 4 and 5).

DISCUSSION
Katsuwonus pelamis from the coast of Rio de Janeiro were highly parasitized by D. simplex.The specimens of this Digenea are described for the first time from off the Atlantic coast of ZOOLOGIA 30 (3): 312-316, June, 2013 South America and histopathological studies are presented.No correlation was observed between the abundance and prevalence of the parasites and the total length of the hosts.According to LUQUE & ALVES (2001), correlations between total length of the host and prevalence and abundance of the parasite is a pattern widely observed in marine fishes off the coast of Rio de Janeiro.However, this pattern cannot be generalized because in many host-parasite systems the correlations are positive but weak and non-significant (SAAD-FARES & COMBES 1992, POULIN 2000).
In our results we observed that the sex of the host does not influence the prevalence and intensity of infection.According to LUQUE et al. (1996), the absence of correlations in parasite prevalence and abundance with the sex of the fish host is a widely documented pattern, and interpreted as a consequence of the absence of sexual differences in some biological aspects of the fish.
According to FEIST & LONGHAW (2008), the gill is a primary target organ and pathogenicity usually depends on the intensity of infection where the presence of large number of parasites or cysts compromises respiratory functions.Most studies of histopathology of fishes associated with parasitism by didymozoids in gills arches, operculum, dermi and palate have demonstrated that the general components of the host defense responses are basically similar in all hosts, being restricted to the affected area, and do not appear to harm the fish (LESTER 1980, EIRAS & REGO 1987, ABDUL-SALAM & SREELATHA 1992, PEREIRA 1992a,b, 1994, CRUZ-LACIERDA et al. 2001, MLADINEO 2006, DI MAIO & MLADINEO 2008, JUSTO et al. 2008, TUDKAEW et al. 2008).
In this study, no gross histopathological alterations were found in the gills of K. pelamis parasitized by D. simplex; we observed that the parasite occupies a space between the basement membrane of the epithelium and the efferent artery and causes a typical chronic inflammatory reaction in the host, characterized by cellular infiltration (leucocytes) and connective tissue fibrosis.These results are consistent with those of PERERA (1992a, b) and MLADINEO (2006), who observed that the first response in the development of the parasitic cyst is the organization of a connective tissue envelope that firmly surrounds the cyst in different species of didymozoids.The envelope consists of few layers of collagenous tissue, with abundant fibrocytes and fibroblasts on the periphery, indicating the continuous activity of the process.For these authors, the absence of a strong inflammatory response was reflected in the absence of gross pathological signs on the gills.This suggests that didymozoids do not elicit a strong cellular response in the gills, but only the demarcation of the cyst by a proportionally thick capsule containing an abundance of connective elements in the invaded tissue.
Histopathological studies by MARINO et al. (2003), with the cysts of Unitubulotestis sardae (MacCallum, 1916), showed fusion of secondary lamellae in the regions near the cyst and severe damage in the tissue where many cysts were present.The damage consisted of an infiltration of lymphocytes, plasmacells and eosinophil granulocytes, as well as the loss of   (1980) andPERERA (1992a, b), neither of the didymozoids secreted a cyst wall and the only species found so far within a true cyst is the highly modified Kollikeria filicollis (Rudolphi, 1819), which was enclosed in a multi-layered wall of fibrous tissue with sheets of fibers and blood capillaries penetrating between the coils, so that the whole surface of the worm was in intimate contact with its host.On the other hand, in a study with didymozoid parasites, PERERA (1994) noted that they appeared to be surrounded by a true capsule.
According to WILLIAMS (1959), in K. filicollis the capsules are a reaction of the host tissue against secretions from some of the subcuticular gland cells of the female worm, and the inner layer of the capsule is of parasitic origin, derived from discharge products of subcuticular gland cells.
On the other hand, ABDUL-SALAM & SREELATHA (1992) reported that the cyst of Gonapodasmius epinepheli is made up of a capsule composed predominantly by connective tissue of host origin.They found that the capsule was formed by three layers, which was not observed in this study.LESTER (1980) also observed that light microscope studies on host defense response to didymozoids have indicated a typical chronic inflammatory reaction characterized by cellular infiltration and extensive fibrosis.PERERA (1992a) observed that the response of the host consisted mainly of a stretching of the lateral epithelium and the formation of a layer of columnar epithelial cells, and that this layer was observed above the basement membrane of the mature capsules.The formation of this layer may be due to a reaction of the host tissue to the parasite, and this type of epithelium was not observed in this study.
The results of the present study indicate that D. simplex does not pose risks to the hosts, since the changes observed in it were restricted only to the region where the parasite was inserted, and this is perhaps a reflection of the low degree of intensity of the parasite in the fish.