Communities of parasite metazoans in Piaractus brachypomus ( Pisces , Serrasalmidae ) in the lower Amazon River ( Brazil ) Comunidades de parasitos metazoários em Piaractus brachypomus ( Pisces , Serrasalmidae ) no baixo Rio Amazonas , Brasil

The aim of this study was to investigate the component community of parasite metazoans of Piaractus brachypomus in the lower Amazon River, northern Brazil. From 34 necropsied fish, 27,384 metazoan parasites were collected, such as Anacanthorus spathulatus, Mymarothecium viatorum and Notozothecium janauachensis (Monogenoidea); Spectatus spectatus and Contracaecum sp (Nematoda); Clinostomum marginatum and Dadaytrema oxycephala (Digenea); and Argulus carteri and Ergasilus sp. (Crustacea). The dominant species was S. spectatus followed by monogenoidean species, and there was aggregated dispersion of parasites, except for D. oxycephala and Contracaecum sp., which presented random dispersion. Positive correlation among the abundance of the three monogenoideans species were found, thus indicating that there was no competition between the species of these parasites on the gills of hosts. The abundances of some parasite species showed positive correlations with the size of the hosts, but the condition factor of the fish was not affected by the parasitism levels. It showed that this host had a metazoan community characterized by high species richness of metazoans, low evenness and high diversity of parasites, with prevalence of endoparasites, including larval stages. This was the first record of C. marginatum, A. carteri, Ergasilus sp. and Contracaecum sp. for P. brachypomus.


Introduction
The lower Amazon River region is covered by a wide variety of both terrestrial and aquatic vegetation, such herbaceous plants, shrubs and trees.Remarkable seasonal variations in the spatial and temporal distribution of flooding over these vegetation types are observed, caused by large annual variations in water levels (the "flood pulse").Flood magnitude and periodicity are the main drivers of productivity, biodiversity and biogeochemistry in the Amazon basin (ARNESEN et al., 2013).In this region, the climate is typical of tropical rainforest, with maximum rainfall rates of December to May, and minimum of June to November.For this reason, the periods of drainage and flood promotes transitions in the wetland system among vegetation, land and water, thereby creating a very dynamic ecosystem.Although there is great diversity of fish species in this region, there are few studies on the parasitic fauna of these fish.
Piaractus brachypomus Cuvier, 1818 is an important Amazonian Serrasalmidae that is known as pirapitinga in Brazil.This fish has wide distribution in the Orinoco and Amazon River system, including the Madeira, Tocantins and Araguaia rivers in Brazil (MÉRONA, 1987;ISAAC et al., 1996;MÉRONA et al., 2010;FERREIRA et al., 2011;QUEIROZ et al., 2013).Through intercontinental translocation, this Amazonian fish can now also be found in Poland (BOEGER et al., 2002) and Croatia (ĆALETA et al., 2011), probably released into the environment by aquarists.This large-sized pelagic fish can reach up to 80 cm in length and has an omnivorous diet, feeding mostly on fruits, seeds and, occasionally, insects and macroinvertebrates (FERREIRA et al., 1998;SANTOS et al., 2006;SOARES et al., 2011;QUEIROZ et al., 2013).Piaractus brachypomus is a migratory fish that forms schools during its reproductive period, have external fertilization and total spawning, which occurs during the period of the Amazon floods (SOARES et al., 2011;SANTOS et al., 2006).
Brazilian fish farms produced 9858.7 tons of P. brachypomus in 2011 (BRASIL, 2013), but there is an expectative of increase in the production (ROUBACH et al., 2015).This fish has also been farmed in the Colombian Amazon (VERJÁN et al., 2001) and Peruvian Amazon regions (GONZALES et al., 2009).In addition to the interest in this fish due to its good husbandry performance (BRABO et al., 2013), it has also been used in breeding with other serrasalmid species, such as Colossoma macropomum Cuvier, 1818 and Piaractus mesopotamicus Holmberg, 1887 (ROUBACH et al., 2015).However, little information on parasitic diversity in P. brachypomus is available.For P. brachypomus farmed in Colombia, infection by monogenoideans, myxosporidians, Piscinoodinium pillulare, Trichodina sp. and Ichthyophthirius multifiliis has been reported (VERJÁN et al., 2001).Mymarothecium viatorum has been described from P. brachypomus in the Oder River, Poland (BOEGER et al., 2002), and following was reported in this same host farmed in Brazil (COHEN & KOHN, 2005).For farmed P. brachypomus in northeastern Brazil, Cohen & Kohn (2009) registered occurrence of Anacanthorus penilabiatus and M. viatorum.
Studies on parasitic communities of wild fish populations increase understanding of the parasite-host-environment interactions, because parasites may be indicators of environmental conditions and of their hosts (BRASIL-SATO & SANTOS, 2003;GUIDELLI et al., 2003;TAVARES-DIAS et al., 2014;HOSHINO et al., 2014).In addition to interfering with the growth of the fish population, parasites can directly or indirectly affect fish farming production (TAVARES-DIAS et al., 2001a, b) and the fishing industry.Thus, the aim of the present study was to investigate the metazoans component community that parasitizes P. brachypomus in the lower Amazon River, northern Brazil.

Study area and fish collection
In October 2012, 34 specimens of P. brachypomus were collected lower in the Amazon River, near the city of Gurupá, in the state of Pará, Brazil (Figure 1), using nets (mesh 50-70 mm), for parasitological analysis.This study was developed in accordance

Parasite collection and analysis procedures
The fish were weighed (g) and their total length was measured (cm).They were then necropsied, and the mouth, gills, operculum, fins, viscera and gastrointestinal tract were examined for the presence of metazoan.The collection, fixation, preservation and preparation of the parasites for identification followed the recommendations of Eiras et al. (2006).
To analyze the parasite infracommunities, the ecological terms used were also those recommended by Bush et al. (1997).
The following descriptors for the parasite community were calculated: species richness; Brillouin diversity index (HB); evenness (E) in association with diversity index and Berger-Parker dominance index (d); and dominance frequency, i.e. the percentage of the infracommunities in which a given parasite species is numerically dominant (ROHDE et al., 1995;MAGURRAN, 2004), using the Diversity software (Pisces Conservation Ltd., UK).The dispersion index (ID) and discrepancy index (D) were calculated using the Quantitative Parasitology 3.0 software, in order to detect the distribution pattern of the parasite infracommunities (RÓZSA et al., 2000), for species with prevalence > 10%.The significance of the ID, for each infracommunity, was tested using the d-statistic (LUDWIG & REYNOLDS, 1988).
The fish weight (g) and total length (cm) data were used to calculate the relative condition factor (Kn) of the hosts, which was compared with standard value (Kn = 1.00), using the t test.Body weight (g) and total length (cm) were also used to calculate the length-weight relationship (W = aL b ) after logarithmic transformation of length (L) and weight (W).Subsequently, two straight lines were fitted to the data, thus obtaining lny = lnA + Blnx (LE-CREN, 1951).The Spearman correlation coefficient (rs) was used to determine possible correlations of parasite abundance with length and weight, and also with parasite species richness and the Brillouin diversity of the hosts; and correlations between monogenoidean species (ZAR, 2010).
Principal component analysis (PCA) was carried out to evaluate e the influences of body and diversity on the parasites communities of A. falcirostris and A. falcatus, and this was done using the Past-Paleontological Statistics software, version 3.0.
The parasites showed an aggregated dispersion pattern, except for infection by D. oxycephala in the gills of the hosts and Contracaecum sp., in the abdominal cavity, which presented random dispersion (Table 2).The metazoan community was dominated by species of endoparasites (Table 3).
The Brillouin diversity index and species richness of parasites were high, while the evenness and Berger-Parker dominance were low (Table 4).The hosts were predominantly infected by 4-6 species (Figure 2).The lengths of the hosts were not correlated with Brillouin diversity (rs = 0.140; p = 0.420) or with the richness of parasite species (rs = 0.092; p = 0.604).However, PCA analysis based on the body and diversity parameters showed that the two main factors together accounted for approximately 82.2% of the variance.The species richness, Brillouin index and evenness were the main factors relating to P. brachypomus (Figure 3).
The abundance of A. spathulatus showed a positive correlation with the abundance of M. viatorum (rs = 0.768; p = 0.0001) and the abundance of N. janauachensis (rs = 0.633; p = 0.0001), which also presented a positive correlation with the abundance of M. viatorum (rs = 0.520; p = 0.001).
For P. brachypomus, the equation that described the length-weight relationship was y = 0.0362x 2.8513 (r 2 = 0.975), thus showing that growth was allometrically negative, i.e. that there were greater increases in body mass than in size.The Kn (1.00 ± 0.02) of the hosts did not differ (t = 0.031; p = 0.975) from the standard (Kn = 1.00), and did not show any correlation (p = 0.05) with the abundance of parasites.However, the abundance of A. spathulatus, M. viatorum and D. oxycephala presented positive correlation with host length and weight (Table 5).

Discussion
Host fish feeding habits are among the most influential factors determining the endoparasites fauna composition, since many of the animals that play the role of intermediate host for parasites are also found in the diet of the final host (GUIDELLI et al., 2003;BRASIL-SATO & SANTOS, 2003;BULLARD & OVERSTREET, 2008;HOSHINO et al., 2014).For P. brachypomus, the metazoan component community was composed of three species of monogenoideans, two of digeneans, two of nematodes and two of crustaceans.Endoparasites species were dominant, thus reflecting the mode of life of this omnivorous host, which occupies a secondary level in the food chain.The parasite species presented an aggregated dispersion, which is a common distribution pattern for freshwater fish, because of the wide dimensions of the hosts' ecological niches, the genetic heterogeneity and the heterogeneity of exposure and susceptibility of the host population (DOBSON, 1990;POULIN, 2013;HOSHINO et al., 2014;TAVARES-DIAS et al., 2015).In contrast, infections by Contracaecum sp. and D. oxycephala showed random dispersion, which is a characteristic pattern of parasites with moderate or high pathogenicity and with reduced ability to colonize hosts (DOBSON, 1990;GUIDELLI et al., 2003).
Piaractus brachypomus was parasitized by A. spathulatus, M. viatorum and N. janauachensis, but the lowest infection levels in were caused by N. janauachensis, a natural monogenoidean of Colossoma macropomum gills (BELMONT-JÉGU et al., 2004).Therefore, this is the first report of N. janauachensis in P. brachypomus.Furthermore, it appears that there was no competition between these three monogenoidean species with moderate levels of infection, since the correlation between the abundance of these parasites    -MOYO et al., 2011).In contrast, the competition between Dactylogyrus carpathicus and Dactylogyrus malleus was due to high abundance and microhabitat preference for the gills of Barbus barbus (KADLEC et al., 2003).The abundance of A. spathulatus and M. viatorum increased with the growth of P. brachypomus, thus indicating that larger hosts harbor greater quantities of these parasites on the gills, due to the degree of specialization of this organ for fixation and the availability of infective forms of monogenoideans in larger hosts (LUQUE & CHAVES, 1999).Parasitic crustaceans generally have a low abundance of infection in wild fish populations (MALTA & VARELLA, 1983;TAVARES-DIAS et al., 2015), but they can cause great damage to farmed fish due to high abundance (TAVARES DIAS et al., 2015), which can be favored by aggregation of the fish.On the gills of P. brachypomus, only two specimens of Ergasilus sp. and one specimen of A. carteri were found.Twenty species of Ergasilus are known to parasitize freshwater fish in Brazil, but no species has been identified yet in P. brachypomus.However, A. carteri was previously only known to parasitize Hoplias malabaricus in Brazil and Paraguay (THATCHER, 2006;LUQUE et al., 2013).Therefore, the present study provides the first report of A. carteri in P. brachypomus.
Digeneans have wide geographic distribution and host range, given that they parasitize different vertebrate species, especially fish and fish-eating birds.The life cycles of these endoparasites usually include three hosts: mollusks, fish and birds (BRASIL- SATO & SANTOS, 2003;BULLARD & OVERSTREET, 2008).In fish, infection by digeneans occurs trophically when infective forms are ingested from infected mollusks (MORLEY, 2012), or through direct contact with the larval forms on vegetation (BRASIL-SATO & SANTOS, 2003;MORLEY, 2012).Dadaytrema oxycephala, a digenean species with distribution in South America (THATCHER, 1979;BRASIL-SATO & SANTOS, 2003;KOHN et al., 2007) was found in several organs (gills, abdominal cavity, pyloric caecum and intestine) of P. brachypomus, but the highest levels of infection occurred in the intestine.The abundance of this digenean increased with the growth of P. brachypomus, thus indicating that there was an accumulation of D. oxycephala in the host.Bellay et al. (2012) and Yamada et al. (2012) have reported similar finding for other parasite species.Metacercariae of C. marginatum were found in the gills of P. brachypomus, but at low levels of infection that seem be accidental.Therefore, P. brachypomus, a omnivorous fish   (SANTOS et al., 2006;SOARES et al., 2011;QUEIROZ et al., 2013), is the definitive host for D. oxycephala in the region of the present study.This was the first record of C. marginatum in P. brachypomus.Spectatus spectatus occurred in the intestine and abdominal cavity of P. brachypomus, but the highest intensity was found in the intestine.Thus, this nematode can migrate through the body of its hosts (DIAS et al., 2010).It has also been found to infect Myletes sp., P. mesopotamicus (Serrasalmidae) and Pterodoras granulosus (Doradidae) in different localities in South America (MORAVEC, 1998), but it occurs at highest abundance in P. mesopotamicus (SANTOS et al., 2003).Therefore, this high intensity of S. spectatus suggests that P. brachypomus is a secondary intermediate host for these endoparasites of unknown life cycle.In contrast, larvae of Contracaecum sp. were found at low infection levels in the intestine, pyloric cecum and abdominal cavity of P. brachypomus, similar to the results reported for Hemibrycon surinamensis of the Amazon River system (HOSHINO et al., 2014).Although this anisakid without parasitic specificity occurs in several fish freshwater in South America, this was the first record of Contracaecum sp. in P. brachypomus.
Finally, the metazoans community of P. brachypomus was characterized by high species richness of metazoans, low evenness uniformity and high diversity, which was not influenced by the size of the hosts except in relation to the monogenoidean community.The presence of endohelminth larvae suggests that the host is part of the diet of fish at the top of the food chain, and possibly part of the diet of aquatic mammals in the Amazon River.Furthermore, the body conditions of this host were not affected by the moderate parasitism.This first study on the diversity of metazoan parasites in P. brachypomus may provide support for intensive farming of this species in the Amazon region.

Figure 1 .
Figure 1.Collection locality of Piaractus brachypomus in the lower Amazon River, Northern Brazil.

Figure 2 .
Figure 2. Species richness of parasites for Piaractus brachypomus in the lower Amazon River, in Brazil.

Figure 3 .
Figure 3. Scatterplot scores from principal component analysis (PCA) on factors influencing the parasite communities of Piaractus brachypomus in the lower Amazon River, in Brazil.

Table 1 .
Parasites metazoans of Piaractus brachypomus in the lower Amazon River, in Brazil.
P: Prevalence; MI: Mean intensity; MA: Mean abundance; FD: Frequency of dominance; TNP: Total number of parasites; SI: Site of infection.

Table 2 .
Dispersion index (DI), d-statistic and discrepancy index (D) for the parasite infracommunities of Piaractus brachypomus in the lower Amazon River, in Brazil.

Table 3 .
Component community of the parasite metazoans of Piaractus brachypomus in the lower Amazon River, in Brazil.

Table 4 .
Diversity descriptors for communities of metazoans inPiaractus brachypomus in the lower Amazon River, in Brazil.

Table 5 .
Spearman correlation coefficient (rs) for metazoan species abundance in relation to total length, body weight and Kn of Piaractus brachypomus in the lower Amazon River, in Brazil.