Helminth fauna of Astyanax fasciatus Cuvier , 1819 , in two distinct sites of the Taquari River , São Paulo State , Brazil

This study assessed the helminth fauna of Astyanax fasciatus in two distinct sites of the Taquari River, São Paulo State, with 30 individuals sampled in a lotic site and 30 in a lentic site, recording the monogeneans: Cacatuocotyle paranaensis, Characithecium costaricensis, Diaphorocleidus kabatai, Jainus sp., Notozothecium sp. and Gyrodactylus sp., the digenean Antorchis lintoni and no-identified metacercariae; the nematode Procamallanus (Spirocamallanus) inopinatus and noidentified larvae. The mean abundances of total monogeneans (U = 1053; p = 0.042) and C. costaricensis (U = 1107; p = 0.005) were higher in the lotic site. This difference may be due to the higher density of the host population in the lotic site, and the water transparence in lentic environments that prevents A. fasciatus to form shoals, precluding the exchange of parasites with direct cycle within a host population. This study is the first report of the helminth fauna of A. fasciatus in the Taquari River, with ten taxa recorded, and reports A. fasciatus as a new host for Notozothecium sp. and C. paranaensis.


Introduction
The genus Astyanax Baird & Girrard, 1854, include fishes known as "lambaris" or "piabas", and is one of the most rich and complex genus in terms of species within the family Characidae, occurring from the South of the United States to Argentina (Oyakawa, 2006).Astyanax spp.are relatively small (10 to 12 cm when adults), live in shoals, are restricted to freshwater, show morning habits, and good sights (Orsi, 2001).They are commercially appreciated, used for recreational fishing, and are potentially useful for pisciculture (Andrade et al., 1985).They dwell the benthopelagic area of rivers, show annual and long reproductive period, partial spawning, and are also used as ornamental fish due to their colorful traits (Orsi, 2001).
Astyanax fasciatus is popularly known as "lambari do rabo vermelho", found along Upper Paraná River basin, and there is a higher frequency of capture of this fish in tributaries; the species shows light silver body color, red fins, cuspidate teeth, short reproductive dislocation, external fertilization, no parental care, and omnivorous feeding habit (Duke Energy, 2008).The helminth fauna of A. fasciatus has been studied for a long time, and the literature shows records of ten different species of monogeneans, 11 digeneans, and 13 nematodes (Table 1).
The Taquari River is the second biggest tributary formed by Jurumirim dam (Henry and Nogueira, 1999) and is one of the main tributaries of the Paranapanema River that suffers the impacts of the reservoir.There is a strong local interest in artisanal fishing in the Taquari River, due to the great diversity and abundance of fish.To date, there is no record of parasitological studies in this river.Thus, this study aimed to assess the helminth fauna of A. fasciatus in the Taquari River, São Paulo, and to compare for the first time the helminth communities of this host from different habitat types: lentic and lotic sites.

Study area
Jurumirim dam is a hydric complex formed by a reservoir with 449 km 2 of surface and three main tributaries: Paranapanema, Veados and Taquari rivers.Taquari River is in confluence with the left bank of the Upper Paranapanema River, located in the Southeast of São Paulo State (23°15'11.9"S;49°12'34.2"W).The fluctuations of the water level are determined either by natural (rainy and dry seasons) or artificial events like the reservoir operation system (Luciano and Henry, 1998).
Astyanax fasciatus specimens were sampled in two sites: a lotic site located upstream in the main channel of the river (23°40'2.90"S;49°7'56.85"W)with wide riparian forest, swamps and macrophytes; and a lentic site located in the river mouth, flooded by Jurumirim dam located approximately 10 km away from the impoundment (23°17'2.80"S;49°12'6.90"W),with absence of riparian forest and macrophytes, banks occupied by pasture and agriculture, and sand extraction.

Field and laboratory procedures
The fish were sampled in November and December of 2011, using gill nets exposed by approximately 14 hours.The sample was composed of 60 fish: 30 specimens sampled in the lotic site and 30 in the lentic site, all specimens analyzed were adults.After sampling, the fish were individualized in plastic bags, frozen and taken to the laboratory to perform parasitological analyses.The standard length in centimeters (L s ) and total weight in grams (W t ) of the specimens were measured.

Parasitological analyses
The body, fins, nasal cavity, and the inner face of the operculum of the fish hosts were examined to find helminth ectoparasites.The gills were removed and washed using 53 and 75 micrometer sieves, placed in Petri plate and examined to find helminth parasites.Following the external analyses, a longitudinal incision in the ventral surface was made and all inner organs were removed and separated.The visceral cavity and all organs were examined using stereomicroscope to find helminth endoparasites.All collected helminthes were preserved in alcohol 70%.For species identification, the monogeneans were cleared with Hoyer or Grey & Wess to visualize the sclerotized structures, the digeneans were stained with carmine and cleared with creosote, and the nematodes were diaphonized with lactophenol (Eiras et al., 2006).
The parasites were identified based mainly on identification keys and reference guides and analyzed using the computerized system for image analysis Qwin Lite 3.1 (Leica).Representatives of the helminth species were deposited in the Coleção Helmintológica do Instituto de Biociências (CHIBB), UNESP, municipality of Botucatu, São Paulo State, Brazil.

Statistical analysis
The ecological terminology, prevalence, infection/ infestation mean intensity, and mean abundance were determined following Bush et al. (1997).The parasites were classified according to Bush and Holmes (1986) into core species (prevalence higher that 66.6%), secondary species (prevalence between 33.3% and 66.6%) and satellite species (prevalence less than 33%).
To determine the ecological attributes of infracommunities the following diversity indexes were calculated: Shannon-Winner: H´= -∑(pi.lnpi), used to measure the order or disorder in a system, attributing greater weight to rare species and relatively independent of sample size, in which: H' = species diversity; pi = proportion of species i in the community, and pi = (ni/N): ni = individuals number of i species and N = total number of individuals (Krebs, 1989;Begon et al., 2007); and the Berger-Parker index (d = Nmax/N) was used to measure the dominance of the species, in which Nmax = number of individuals of each abundance species, N = total number of individuals of all species (Krebs, 1989).The Shannon-Winner index was calculated and compared using the statistical program Past -Paleontological Statistics version 3.0 (P < 0.05).
Similarity analysis of parasite communities between the sites studied was carried out using Jaccard Coefficient that is based on the presence and absence of local species sampled, this index varies from 0 (dissimilar) to 1 (similar) and is calculated by the equation (Q = c / (a + b -c).100), in which Q = Jaccard similarity coefficient; c = number of common species between a and b; a = number of species sampled in a; b = number of species sampled in b (Krebs, 1989).
The index of discrepancy (D), described by Poulin (1993), was used to evaluate spatial distribution of parasites based on their abundance between lotic and lentic sites.This index varies from 0 to 1, and can be interpreted as: D = 0, all hosts harboring the same number of parasites; D = 1, all parasites found in a single host.This analysis was calculated according to Rózsa et al. (2000) using the software Quantitative Parasitology 3.0.
The comparison among prevalence of parasites in the lotic and lentic sites was calculated with Z-test.The infection/infestation mean intensity and mean abundance between the two sites were calculated with Mann-Whitney U test.The statistical comparisons were made using the software SigmaStat 3.1.The statistical significance level adopted was P < 0.05.A principal component analysis (PCA) was carried out to compare parasites communities in the lotic and lentic sites and this multivariate analysis was calculated with the software MVSP.

Parasites communities
A richness of ten helminth taxa was found in the A. fasciatus samples analyzed in this study.For the lotic site, 93.3% of the hosts analyzed were parasitized by at least one helminth species (overall prevalence = 93.3%).A total of 169 parasites were found, with six helminth parasite taxa, and a mean of 5.64 parasite/fish.The richness varied from 1 to 4 parasites and the mean richness was 2.3 parasites/fish (Table 2).
For the lentic site, 90% of the hosts analyzed were parasitized by at least one helminth species (overall prevalence = 90%).A total of 169 parasites were found, with eight helminth parasite taxa, and a mean of 5.4 parasites/fish.The richness varied from 1 to 5 parasites and the mean richness was 2.3 parasites/fish (Table 2).
The monogeneans C. costaricensis and D. kabatai were the species with higher prevalence in the lotic and lentic sites, considered core species.Nematode larvae were secondary species and the other taxa were satellite species.

Comparison between lotic and lentic sites
The mean abundance of total monogeneans (U = 1053; p = 0.042) and C. costaricensis (U = 1107; p = 0.005) were greater in the lotic site (see Figure 1).The mean richness was similar between both sites, however the richness was greater in the lentic stretch.The overall prevalence (z = -0.00467;p = 0.996) and the total numbers of helminth found (U = 991; p = 0,264) were similar between lotic and lentic sites.
The Jaccard index (SJ = 0.7) demonstrated similarity between the parasite communities of both sites.The Shannon-Wiener index (Lotic = 1.15;Lentic = 1.55; p < 0.001) demonstrated that in the lentic site the helminth communities show a diversity greater than in the lotic site.The Berger-Parker index demonstrated that D. kabatai was the dominant species in both sites.However C. costaricensis and nematode larvae demonstrated dominance indexes similar to D. kabatai in the lotic and lentic sites, respectively (Table 3).Most of the parasites species found in both sites showed aggregated distribution pattern.Only the species C. paranaensis, D. kabatai and no-identified nematode larvae showed median aggregation index (Table 3).
The multivariate analysis based on the component community of the helminth parasites of A. fasciatus allowed observing that there is not separation among the host specimens from lotic and lentic sites.Antorchis lintoni, nematode larvae, D. kabatai and C. costaricensis showed more importance in the analysis (see Figure 2).

Discussion
Most of the parasites found in this study corroborate previous records, however, there has not been any record of Notozothecium sp. and C. paranaensis in this host species, thus this study is the first record of these monogeneans in A. fasciatus.All the records represent a new geographic distribution for these parasites, since to date parasitological studies have not been conducted in the Taquari River, São Paulo State.
Berger-Parker index demonstrated that D. kabatai was the dominant species in both sites and C. costaricensis showed dominance index similar to D. kabatai.These monogeneans were first described parasitizing the gills of Astyanax spp.(Mendoza-Franco et al., 2009), and there is only one report of D. kabatai parasitizing A. altiparanae Garutti andBritsky, 2000 (Almeida andCohen, 2011).Thus, this study corroborates the literature and strongly suggests the specificity of these monogeneans with the genus Astyanax.
Paraguassu and Luque (2007) studied the helminth fauna of A. fasciatus and observed a predominance of endoparasites, with the nematode Rhabdochona acuminata Molin, 1860, as the main component.However, they did not record monogeneans and digeneans.Kloss (1966) analyzed the occurrence of helminth parasites in simpatric species of Astyanax, including A. fasciatus, and found species of adult digeneans and nematodes.Moreover, species of Procamallanus (Procamallanus) Baylis, 1923 have often been recorded in Astyanax spp.(Table 1).Therefore, this study does not correlate with previous reports, since monogeneans were the dominant taxa showing the highest prevalence, infection intensity and abundance.Nonetheless, the records of the digenean A. antorchis and the nematode P. (Spirocamallanus) inopinatus in this study corroborate Travassos et al. (1928) and Pinto and Noronha (1976) respectively.Dogiel (1961) states that monogenean are more frequently found in lentic environments, since it is easier for the free-swimming larval stages find the hosts, which does not correlate with this study, considering that a greater abundance of monogeneans was found in the lotic site.However, it must be considered that it is expected more chances of transmission of parasites with direct cycles in denser populations, due to the proximity of the individuals, as observed in farmed fishes (Franceschini et al., 2013) or in natural populations (Takemoto et al., 2005).The fish used in this study were sampled together with a fish ecology project that carried out eight sampling efforts in 2011 and 2012, and a greater amount of A. fasciatus was sampled in the lotic site during these two years (lotic n = 206; lentic n = 36), suggesting that A. fasciatus population is denser in the lotic site.Takemoto et al. (2005) studied the population density of different host species as the major determinant of endoparasite richness in floodplain fishes of the Upper Paraná River, Brazil.The authors elucidate that host species occurring at higher population density should harbor more species of parasites, and their result was a positive relationship between hosts density and parasites   (Roberts et al., 2002), which predicts that the larger or denser the populations are, the more parasites populations they will support.Hence, the authors suggest that host density may develop the function of maintaining parasite species richness.Therefore, the greater abundance of the monogenean C. costaricensis in the lotic site might be associated with the greater amount of individuals in the population.Additionally, it was observed that Astyanax spp.populations form schools and seek shelter in sites with abundance of macrophytes and organic matter in shoals close to the banks (Orsi et al., 2004).In these areas the effects of the water flow are attenuated (Champion and Tanner, 2000;Franklin et al. 2008), thus the currents might not be influencing the abundance of parasites in this host.The water transparence in lentic environments also prevents the populations of A. fasciatus of forming schools, which would make these fish more exposed to predators (Orsi et al., 2004;Tondato et al., 2010).Thus, the transmission of parasites with direct cycles from host to host would be hindered in lentic sites.

Species
This study contributes with the first characterization of the helminthfauna of A. fasciatus in the Taquari River, with ten different taxa recorded, broadening the knowledge of geographic distribution for these parasites, and also reports A. fasciatus as a new host record for the monogenean Notozothecium sp. and C. paranaensis.Moreover, the greater abundance of C. costaricensis in the lotic site may be due to the higher density of the host population, and also due to the water transparence in lentic environments that prevents A. fasciatus to form shoals, precluding the exchange of parasites with direct cycle within a host population.

Table 3 .
Berger-Parker and Aggregation indexes for the helminth species of Astyanax fasciatus sampled in the lotic and lentic stretches of Taquari River, Jurumirim reservoir, São Paulo State, Brazil.