Parasitic fauna of Hyphessobrycon takasei and Hyphessobrycon amapaensis (Osteichthyes: Characidae), ornamental fish of two basins from Amapá state, Brazil

. This study compared the parasites fauna of Hyphessobrycon takasei and H. amapaensis from hydrographic basins in Amapá state, Brazil. Of the 42 H. takasei examined, 95.2% were parasitized by one or more species such as Dactylogyridae gen. sp., encysted metacercariae of digenean, adults and larvae of Procamallanus ( Spirocamallanus ) inopinatus and larvae of Camallanus sp. A total of 224 parasites were collected from H. takasei , and there was a dominance of nematode species and dispersion pattern aggregated of parasites. The species richness of parasites varied of 0 to 3 species, Brillouin index of 0-0.97 and evenness of 0-0.70. Of the 32 specimens of H. amapaensis examined, 28.1% were parasitized by Camallanus sp. larvae, which presented random dispersion pattern. For H. takasei , no correlation of host length with Brillouin diversity and species richness of parasites was found. The presence of these nematode species and metacercariae of digenean indicates that these fish may act as intermediate hosts. This is the first study to gather information about the parasite fauna of these endemic fish from the eastern Amazon, providing records that contribute new reports on occurrence of parasite species in new localities. This study on parasites of H. takasei and H. amapaensis may help prevent the introduction of parasites in other regions of the country, as well as in other countries due to aquarium fish. Since the ornamental fish exportation from the Brazil is largely dominated by wild Amazonian species, biosecurity export conditions should be require all exported fish to be inspected and certified to show no clinical signs of parasite and diseases. Lastly, this knowledge on the parasitic epidemiology may help improve the health and quality of these fish being exported from the Amazon region, reducing losses on all stages of this production chain.


Introduction
Aquarium fish is a popular pastime of growing interest, which has resulted in the expansion of trade in ornamental fish in more than 125 countries (Dey, 2016). Currently, it has been estimated that the global trade of more than 4500 species of freshwater ornamental fish and 1450 species of marine fish varied from 350 million to 1.5 billion fish, with a value of US$ 800 million to US$ 30 billion per year (Stevens, Croft, Paull, & Tyler, 2017). Therefore, the ornamental fish trade represents a consolidated market over the world.
The ornamental fish trade is one of the most important economic activities for thousands of riverine communities from Amazon region (Aguinaga et al., 2015;Ferreira, Passador, & Tavares-Dias, 2019). In Amazon, most ornamental freshwater fish species are captured by the extractive fishery in rivers and streams, and then commercialized by exporters. Subsequently, these fish are passed on to intermediary agents, who transfer them to exporters until they reach aquarium stores in various parts of the world. During this transport to the exporter, more than 70% of fish die due to various factors (Aguinaga et al., 2015), mainly due to inadequate handling and parasites infections.
In general, protozoan and metazoan species can cause many of the economic losses that occur in ornamental fish (Hoshino, Hoshino, & Tavares-Dias, 2018) due to damages histopathological (Santos et al., 2017). The exportation of ornamental fish may therefore represent a gateway for parasites in new environments (Alves, Luque, Paraguassú, & Marques, 2000;Thilakaratne, Rajapaksha, Hewakopara, Rajapakse, & Faizal, 2003;Trujillo-González, Becker, Vaughan, & Hutson, 2018) into country due to the translocation of hosts and parasites, as well as in other countries, due to the lack of care with sanitary management during this phase of commercialization. Therefore, knowledge about the parasites affecting ornamental fish is crucial to success in the ornamental fish trade. In addition, knowledge about epidemiology of parasites are important for the successful of implementation of treatment and prophylaxis programs, besides control and prevention strategies of diseases in ornamental fish (Santos et al., 2017;Hoshino et al., 2018;Ferreira et al., 2019). In addition, parasitic diseases reduce the quality of ornamental fish and affects the economic activity of exporting enterprises, leading also to credibility loss.
Species of the genus Hyphessobrycon, in general, are omnivorous fish, feeding mainly on insects, algae, detritus, vegetal fragments and microcrustaceans. They are ornamental fish found mainly in environments with aquatic macrophytes (Malabarba, Carvalho-Neto, Bertaco, Carvalho, Santos, & Artioli, 2013;Vieira et al., 2016). Hyphessobrycon takasei Géry, 1964 is a benthopelagic fish with distribution in the basins of the rivers Araguari and Oiapoque (State of Amapá, Brazil) and French Guiana (Lima et al., 2003;Froese & Pauly, 2019). Hyphessobrycon amapaensis Zarske & Géry, 1998 is also benthopelagic and endemic from the state of Amapá, northern Brazil (Froese & Pauly, 2019). Both species of ornamental fish, H. takasei and H. amapaensis, have great potential of use in aquaculture. Thus, the aim of this study was to compare the parasitic fauna of two congeneric species H. takasei and H. amapaensis both native of two basins from the Amapá state, Brazil.

Fish and locality of collection
In July 2016, 42 specimens of H. takasei were captured in stream from the Cachorrinho River, municipality of Pedra Branca do Amapari, Amapá state, in Brazil. In November 2017, 32 specimens of H. amapaensis were caught in a stream from the Cajari River, Amapá, in Brazil ( Figure 1). Fish of both species were captured using hand nets, sieves and/or trawl nets and transported in plastic bags containing dissolved oxygen to the Laboratory of Aquaculture and Fisheries from Embrapa Amapá, Macapá (Brazil), for parasitological analysis.
The rivers Cachorrinho and Cajari presents large areas of flood plain, with very peculiar characteristics, since they are strongly influenced by the high rainfall of the Amazon region. These are highly complex river systems and regulation of them is a process that affects the integrity of the river-plain flood system. The vegetation of these hydrographic basins is typical of savannah and forest of dryland (Queiroz, Silva, Reis, Lima, & Lima, 2011), and system of both basins has not signals of anthropic eutrophication. In stream from the Cajari River, the dissolved oxygen (6.6 ± 0.3 mg L -1 ), temperature (29.3 ± 0.1ºC) and pH (6.4 ± 0.3) were measured using a multiparameter probe (Horiba U52 model, Kyoto, Japan). In stream from the Cachorrinho River the dissolved oxygen (6.3 ± 0.2 mg L -1 ), temperature (30.1 ± 0.1ºC) and pH (6.2 ± 0.1) were measured.
This study was developed in accordance with the principles recommended by the Brazilian College of Animal Experimentation (Cobea) and with the authorization from Ethics Committee in the Use of Animals of the Embrapa Amapá (Number 005 -CEUA/CPAFAP) and authorization from collection of SISBio (No 60877-1).

Collection procedures and analyses of parasites
All fish were measured for total length (cm), and then necropsied for parasitological analysis. The mouth, opercula, gills and gastrointestinal tract were examined to collect the parasites (protozoans and metazoans). Gills were removed and analyzed with the aid of an optic microscope. To quantify metazoan parasites, each viscera was dissected separately and washed with sodium chloride solution (0.85%) and examined under a stereomicroscope. Previously described techniques were used to collect, count, fix, preserve, and stain the parasites for identification (Eiras, Takemoto, & Pavanelli, 2006).
To analyze the parasite infracommunities, the ecological terms used were those recommended by Bush, Lafferty, Lotz, and Shostak (1997). The following descriptors for the parasite community were calculated: the species richness (number of species), the Brillouin diversity index (HB), evenness (E) in association with diversity index and dominance frequency (percentage of the infracommunities in which a parasite species is numerically dominant) (Rohde, Hayward, & Heap, 1995;Magurran, 2004), using the Diversity software (Pisces Conservation Ltd., UK). The dispersion index (DI) and discrepancy index (D) were calculated using the software Quantitative Parasitology 3.0, in order to detect the distribution pattern of the parasite infracommunities (Rózsa, Reiczigel, & Majoros, 2000), for species with prevalence >10% (Bush, Aho, & Kennedy, 1990). The significance of DI, for each infracommunity, was tested using the d-statistics (Ludwig & Reynolds, 1988). The Spearman correlation coefficient (rs) was used to determine possible correlations of length with the species richness of parasites and Brillouin diversity index (Zar, 2010).
For H. takasei, Brillouin diversity index and evenness were low and the species richness of parasites was low for both host species (Table 3). In addition, correlation of host length with Brillouin diversity index (rs = -0.017, p = 0.917) and species richness of parasites (rs = 0.09, p = 0.953) was not observed. In H. takasei, there was a predominance of hosts parasitized by 1 and 2 species and in H. amapaensis the predominance was of non-parasitized hosts (Figure 2).  (Table 4). However, none of these species were found in H. takasei, which was parasitized by one species of Monogenea, one Digenea and two Nematoda, or in H. amapaensis that was parasitized only by one species of Nematoda. Therefore, there was a dominance of nematodes in H. takasei. For H. takasei, there was an aggregated dispersion of parasites, a common pattern in wild fish populations (Tavares-Dias, Oliveira, Gonçalves, & Silva, 2014;Oliveira, Gonçalves, Neves, Ferreira, & Tavares-Dias, 2017;Ferreira et al., 2019). In contrast, for H. amapaensis the parasite dispersion was random, a typical pattern of parasites with moderate or high pathogenicity, because these regulate the density of the host population (Moller 2006). Furthermore, low Brillouin diversity and low species richness of parasites was observed in H. amapaensis when compared to H. takasei, probably due to differences among the congeneric species.  Chemes & Takemoto (2011) Monogeneans are ectoparasites that have been used as indicators of environmental changes, since for many species the eutrophized environments and low oxygen dissolved level increases the reproduction and the levels of parasitism in the host fish (Mackenzie, 1995;Aguinaga et al., 2015;Santos et al, 2017). There is a complex relationship between these ectoparasites, environment and host, because initially the parasites try to establish themselves in the hosts while the resistance of the infection occurs through the defense mechanisms . In the gills of H. amapaensis no monogenean species was found, while in H. takasei a low level of infestation was observed, because the quality environmental was good. In contrast, infestation levels were lower than the reported for farmed Hyphessobrycon sp. (Thilakaratne et al., 2003) and wild H. copelandi (Tavares-Dias, Lemos, & Martins, 2010). Since only two specimens of Dactylogyridae gen. sp. were found in H. takasei, it was not possible to identify the species. However, Dactylogyrus spp., Gyrodactylus spp. and Urocleidoides sp. are the monogeneans known to infect species of Hyphessobrycon, and therefore are not known no species of these ectoparasites for Hyphessobrycon species (Table4).
The importation of fish ornamental may lead to the introduction of exotic parasites into native hosts. For example, Camallanus cotti Fujita, 1927, a native nematode from Japan was first recorded in Brazil infecting Poecilia reticulata Peters, 1859 (Alves et al., 2000), assumed to be introduced along with the introduction of the exotic poeciliid fish species. Fish may be intermediate, paratenic or definitive hosts for different species of nematodes (Molnár, Buchmann, & Székely, 2006;Acosta & Silva, 2015;Oliveira et al., 2017;Tavares-Dias et al., 2017;Hoshino et al., 2018). In general, larvae are more pathogenic than adult forms, since they may migrate through various host organs. Species of camallanids can feeding on blood causing anemia in the hosts. Consequently, high levels of infection by nematode larvae may be harmful to health of hosts (Molnár et al., 2006;Acosta & Silva, 2015), mainly for small ornamental fish as H. takasei and H. amapaensis. In H. takasei, there was higher level of infection by Procamallaunus (Spirocamallanus) inopinatus Travassos, Artigas and Pereira, 1928 and Camallanus sp. when compared to infection by Camallanus sp. in H. amapaensis. This is the first report of P. (S.) inopinatus and Camallanus sp. for H. takasei, as well as of Camallanus sp. for H. amapaensis.

Conclusion
Most of the parasites were found in the intestine of H. takasei and H. amapaensis, and both hosts had low richness and low parasites diversity. The presence of nematodes and digenean indicates that H. takasei is an intermediate and definitive host for these endoparasites. This is the first study to gather information about the parasite fauna of these endemic fish from the eastern Amazon, providing records that contribute new reports on occurrence of parasite species in new localities. Furthermore, these results may help in programs development of prophylaxis and treatment against diseases of parasites for these fish at aquarium.