First study on communities of parasites in Triportheus rotundatus , a Characidae fish from the Amazon River system (Brazil)

This study was the first investigation on the parasites of Triportheus rotundatus , a Characiformes fish from the Amazon, in Brazil. All the fish collected (100%) in a tributary from the Amazon River system were infected by one or more parasite species. The mean species richness of parasites was 4.9 ± 0.9, the Brillouin index was 0.39 ± 0.16, the evenness was 0.24 ± 0.09 and the Berger-Parker dominance was 0.81 ± 0.13. A total of 1316 metazoan parasites were collected, including Anacanthorus pithophallus , Anacanthorus furculus , Ancistrohaptor sp. (Dactylogyridae), Genarchella genarchella (Derogenidae), Posthodiplostomum sp. (Diplostomidae), Procamallanus ( Spirocamallanus ) inopinatus (Camallanidae), Echinorhynchus paranensis (Echinorhynchidae) and Ergasilus sp. (Ergasilidae), but monogenoideans were the dominant parasites. These parasites presented an aggregate dispersion pattern, except for P. ( S. ) inopinatus , which showed a random dispersion pattern. The body conditions of the hosts were not affected by the parasitism levels. This first report of these parasites for T. rotundatus indicates that the presence of ectoparasites and endoparasites was due to hosts behavior and availability of infective stages in the environment, and this was discussed.


Introduction
The Amazon basin is a center of diversity for most groups of Neotropical fish, that is to say, it is an area of high species richness, due to its large extension of floodplains, which are important habitats for native fish, providing feeding and nursery zones.Conservative estimates suggest there are about 3,000 fish species in this basin (ALBERT & REIS, 2011;JUNK, 2013;FROESE & PAULY, 2016).In the Amazonian estuary, there are 243 fish species, of which 23 are endemic species.Many of these fish are important for trade and economy in the Amazon, and constitute the main source of food for human populations in the region.Moreover, Communities of parasites in Triportheus rotundatus this basin has diverse tributaries draining its water levels, which vary enormously during the year (ALBERT & REIS, 2011), including the Igarapé Fortaleza hydrographic basin.
In the Amazon River system, in the region of the state of Amapá (Northern Brazil), there is the Igarapé Fortaleza hydrographic basin, an important tributary of this river.The Igarapé Fortaleza basin, located at the estuarine coastal sector, is characterized for having a river system with extensive floodplains, which is drained by fresh water and connected to a main watercourse, influenced by high rainfalls and tides (every 12 hours) from the Amazonas River (TAVARES-DIAS et al., 2013).This tributary of the Amazon River system harbors more than 80 species of freshwater fish (GAMA & HALBOTH, 2004), including Characiformes species of the genus Triportheus Cope, 1872, which are popularly known as freshwater sardines and represents an important resource for artisanal fishing and subsistence of human population in the region.
Triportheus spp.are Characidae with a geographic wide distribution in Bolivia, Colombia, Peru, Argentina, Ecuador, Venezuela and Brazil (SANTOS et al., 2006;FROESE & PAULY, 2016).Currently, 18 species of Triportheus Cope, 1872 are known, including Triportheus rotundatus Jardine, 1841 (MALABARBA, 2004;FROESE & PAULY, 2016), the fish species that is the focus of the present study.The species of Triportheus inhabit most of the major river drainages of South America, and constitutes an important element in both commercial and subsistence fisheries in the Amazon basin (MALABARBA, 2004), its reproduction occurs during the rainy season in the Amazon River system (FROESE & PAULY, 2016).Triportheus rotundatus is a benthopelagic fish with an omnivorous diet, fed on fruits, seeds and insects that float on the water surface, besides microcrustaceans (PEREIRA et al., 2011;FROESE & PAULY, 2016;SUÇUARANA et al., 2016).However, studies on the biology of T. rotundatus are reduced, mostly those regarding its parasitic fauna.
Studies on parasitic fauna aspects should be directed to T. rotundatus, due to the importance of knowledge on several factors that could influence the diversity and structure of the parasite infracommunities in fish populations (MOREIRA et al., 2009;LAGRUE et al., 2011;TAVARES-DIAS et al., 2013;COSTA-PEREIRA et al., 2014;OLIVEIRA et al., 2016).Moreover, the knowledge about parasites community infracommunities and their relationship with the host fish is of great importance, because the parasites also play a key role in ecosystems by regulating the abundance or density of natural fish populations, thus stabilizing food web and host community structures (MOREIRA et al., 2009;MORLEY, 2012;TAVARES-DIAS et al., 2013;OLIVEIRA et al., 2016).However, the ecological knowledge of parasites in Neotropical environments is yet very limited.

Fish and parasite sampling
Thirty-two T. rotundatus (14.7 ±1.3 cm and 39.4 ± 10.8 g) were collected in the Igarapé Fortaleza basin, in the state of Amapá, eastern Amazon region, northern Brazil (Figure 1), in the period from December 2012 to August 2013 for parasitological analysis.The fish were caught with different nets and transported in box with ice to the Health Laboratory of Aquatic Organisms of Embrapa Amapá, Macapá, Amapá state (Brazil).The fish collected were weighed (g) and measured for total length (cm).The present work was developed according to the principles adopted by the Brazilian College of Animal Experiments (COBEA), with the authorization from Ethics Committee in the Use of Animal of the Embrapa Amapá (#004 -CEUA/CPAFAP) and ICMBio (# 23276-1).

Parasite collection and analysis procedures
Each individual was macroscopically evaluated regarding body surface, mouth, eyes, opercula and gills.The gills were removed to collect ectoparasites.The gastrointestinal tract was removed and examined to collect endoparasites.All the parasites were collected, fixed, quantified and stained for identification (EIRAS et al., 2006).The parasitological terms adopted were those recommended by Bush et al. (1997).
For the parasite community, the species richness, the Brillouin diversity index, evenness in association with the diversity index, and the Berger-Parker dominance index and the dominance frequency (percentage of the infracommunities in which a parasite species is numerically dominant) (ROHDE et al., 1995;MAGURRAN, 2004) were calculated using the Diversity software (Pisces Conservation Ltd., UK).The index of dispersion (ID), and the index of discrepancy of Poulin (D) were calculated using the Quantitative Parasitology 3.0 software to detect the distribution pattern of the infracommunity parasites (RÓZSA et al., 2000) for species with prevalence > 10%.The ID significance for each infracommunity was tested using the d-statistics (LUDWIG & REYNOLDS, 1988).
Fish data on weight (g) and total length (cm) were used to calculate the relative condition factor (Kn) of hosts, which was compared to the standard value (Kn = 1.00) using the t-test.Body weight (g) and total length (cm) were used to calculate the relative condition factor (Kn) of fish using the length-weight relationship (W = aL b ) after a logarithmic transformation of length and weight and subsequent adjustment of two straight lines, obtaining lny = lnA + Blnx (LE CREN, 1951).The Spearman correlation coefficient (rs) was used to determine possible correlations of parasite abundance with the length and weight, as well as with the species richness and the Brillouin diversity of the hosts (ZAR, 2010).

Results
All examined fish (100%) were parasitized by one or more metazoan species, and 1,316 parasites were collected.Anacanthorus pithophallus Kritsky, Boeger & Van Every, 1992; Anacanthorus furculus Kritsky, Boeger & Van Every, 1992 1). Monogenoideans were the dominant parasite species, and although these parasites were not possible to count by species, the predominance was de A. furculus and A. pithophallus.None protozoan parasite was found.Parasites had an aggregate dispersion pattern, except for P. (S.) inopinatus, which presented random dispersion pattern (Table 2).
The species richness of parasites, the Brillouin diversity (HB) and evenness (E) were low (Table 3).Species richness of parasites (rs = 0.243, p= 0.180) and HB (rs = 0.273, p= 0.130) did not show any significant correlation with total host length.Hosts parasitized by four to five parasite species were predominant (Figure 2).
The equation of weight (W)-length (L) relationship for this host was Wt = 0.1493Lt 2.0653 , r 2 = 0.710), with negative allometric, indicating greater increase in body weight than in size.The condition factor (Kn = 0.999, t = -0.0041,p = 0.997) of the parasitized fish did not differ from the standard value (Kn = 1.000), thus indicating that the parasitism did not impair host body condition.

Discussion
In T. rotundatus, the parasitic community was composed of 3 species of Monogenoidea, 2 Digenea, 1 Nematoda, 1 Acanthocephala and 1 Copepoda, with a dominance of monogenoideans (A.pithophallus, A. furculus and Ancistrohaptor sp.).Moreover, low species richness, low Brillouin diversity, low evenness, a high diversity of endoparasites species, and overdispersion of parasites characterized this parasitic community.Many parasite species commonly show overdispersion in different host fish (COSTA-PEREIRA et al., 2014;TAVARES-DIAS et al., 2015;OLIVEIRA et al., 2016), due to processes that produce variability in host exposure, host acceptability by the parasite and host immune response.For Triportheus nematurus from Pantanal Matogrossense (Brazil) 1 species of Monogenoidea, 3 Nematoda and 1 Copepoda were reported, being Anacanthorus sp. the main component and predominant in helminth species with overdispersion (COSTA-PEREIRA et al., 2014).For Triportheus curtus Garman, 1890 and Triportheus elongatus Spix & Agassiz, 1829 from same region of this study 3 species of Protozoa, 1 Monogenoidea, 1 Argulidae, 2 Digenea and 2 Nematoda were reported, being I. multifiliis the predominant parasite species (OLIVEIRA et al., 2016).However, we found a higher parasitic prevalence and abundance in T. rotundatus when compared to those observed by Costa-Pereira et al. (2014) in T. nematurus.Such differences for this fish species with a similar mode of life may be attributed to the environment and the opportunities of these hosts found infective stages of parasites with complex life cycle.
Monogenoideans are parasites that are highly host specific when compared to other groups of parasites.Studies suggest that the distribution of monogenoideans on their fish hosts is strongly influenced by evolutionary history, both between and within fish orders (KRITSKY et al., 1992;BRAGA et al., 2014).The gills of T. rotundatus were infected by A. pithophallus, A. furculus and Ancistrohaptor sp., parasites whose abundance presented a negative correlation with the size of the hosts.Such monogenoidean species has been also recorded in other Triportheus species from Brazil (KRITSKY et al., 1992;COHEN et al., 2013;OLIVEIRA et al., 2016).However, while Ancistrohaptor spp.seems restricted to Triportheus spp.(COHEN et al., 2013;BRAGA et al., 2014), Anacanthorus spp.presents a widespread distribution among freshwater fish species, once that they are also found in diverse Characiformes species of fish from several families (BRAGA et al., 2014).
Characteristics of the habitat may facilitate the transmission and establishment of fish parasites (MOREIRA et al., 2009;COSTA-PEREIRA et al., 2014;BITTENCOURT et al., 2014;OLIVEIRA et al., 2016).The abundance and diversity of the invertebrate fauna are also key components in the formation of a parasite community in fish populations (MOREIRA et al., 2009;MORLEY, 2012;TAVARES-DIAS et al., 2015).Moreover, environments with abundance of aquatic vegetation (e.g.macrophytes), as the ecosystem of this study (THOMAZ et al., 2004), may influence the abundance of parasites (MOREIRA et al., 2009;MORLEY, 2012), because its floodplains areas are widely used for shelter and feeding by many fish species.Consequently, this increases the possibility of the fish finding different infective stages of the parasite species in the environment.Therefore, these factors favored the transmission of G. genarchella, Posthodiplostomum sp., P. (S.) inopinatus and E. paranensis in T. rotundatus.Most of these endoparasites present a wide distribution in freshwater fish from Brazil and have different macroinvertebrates as intermediate hosts.Procamallanus (S.) inopinatus has chironomid species as intermediate hosts (MOREIRA et al., 2009), while digeneans G. genarchella and Posthodiplostomum sp. have mollusk species as intermediate hosts and aquatic fish-eating birds as definitive host.Concerning E. paranensis, it was described by the first time on Triportheus paranensis Günther, 1874 from Mato-Grosso, Brazil (MACHADO, 1959).Recently, this acanthocephalan has also been reported infecting Pygocentrus nattereri Kner, 1860 (Serrasalmidae) from Negro River, at Pantanal of the Mato Grosso do Sul (Brazil), as well as Chaetobranchus flavescens Heckel, 1840 and Chaetobranchopsis orbicularis Steindachner, 1875 (Cichlidae) from the region of this study (BITTENCOURT et al., 2014).However, as there are few reports on the occurrence of E. paranensis in fish, little is known about its biology and life cycle.
Concerning Ergasilus sp., these copepods occurred only on the gills of two specimens of T. rotundatus, because the water dynamic of the Igarapé Fortaleza basin seems to hinder the encounter of the parasitic crustacean species with host fish, once these ectoparasites need swimming to infect its host fish.Similarly, Costa-Pereira et al. ( 2014) also reported low prevalence and abundance of Ergasilus sp. in T. nematurus.Moreover, some parasitic crustacean species are host and site-specific, especially in relation to fish in particular habitats and life styles, while other parasites frequently have no preference.There is a dominance of Ergasilidae, mainly Ergasilus Nordmann, 1832, among the parasitic crustaceans in freshwater fish from Brazil.However, Ergasilus spp.have infested species of Characidae, Pimelodidae, Anostomidae and Cichlidae from the Amazon (TAVARES-DIAS et al., 2015;TABORDA et al., 2016).Twenty-two freshwater species of Ergasilus are known from the gills of Neotropical fish (TABORDA et al., 2016).
In summary, for T. rotundatus the ectoparasites community consisted of species with high prevalence and abundance, while the endoparasites community presented low prevalence and abundance, which did not affected the body condition of the hosts.The presence of endoparasites with a complex life cycle indicates that in this environment, the diet of T. rotundatus consists mostly of chironomids, mollusks and microcrustaceans.Thus, T. rotundatus is an intermediate or paratenic host for G. genarchella, Posthodiplostomum and E. paranensis, and a definitive host for P. (S.) inopinatus.The size of the host had little influence on parasite communities, once they had influence only on monogenoideans.Finally, the behavior and availability of infective stages, which intermediate hosts for endoparasites, were factors structuring the communities of parasites in this Amazonian host.

Figure 1 .
Figure 1.Collection locality of Triportheus rotundatus in tributary from Amazon River system, Northern Brazil.
P: Prevalence, MI: Mean intensity, MA: Mean abundance, TNP: Total number of parasites, SI: Site of infection, FD: Frequency of dominance.

Table 2 .
Index of dispersion (ID), d-statistic and discrepancy index (D) for the parasite infracommunities of Triportheus rotundatus from Amazon River system (Brazil).

Table 3 .
Parameters of diversity for Triportheus rotundatus from Amazon River system (Brazil).
Figure 2. Species richness of parasites in Triportheus rotundatus from Amazon River system (Brazil).