Helminth parasites in two populations of Astronotus ocellatus (Cichliformes: Cichlidae) from the eastern Amazon, Northern Brazil

May 31, 2019 Abstract This study compared the structure of helminth parasite communities in two populations of Astronotus ocellatus from two localities in Pará State, eastern Amazon (Brazil). Hosts from the Tapajós River were infected by Posthodiplostomum sp. metacercarie, larvae of Contracaecum sp. and Pseudoproleptus sp., with predominance of Contracaecum sp. Hosts from Iara Lake were infected by Procamallanus spiculastriatus , Proteocephalus sp. and Acanthocephala gen. sp., with predominance of P. spiculastriatus . Parasites had an aggregated dispersion and there were differences in Shannon diversity index and the evenness between both host populations, because the parasite component community showed no similarity. The existence of variation in infracommunity and community of parasites for A. ocellatus from different localities indicates the presence of an uneven distribution in terms of species and density of parasites, as well as of intermediate hosts in


Introduction
Astronotus ocellatus Agassiz, 1831 is a benthopelagic Amazonian cichlid that prefers lentic environments, finding protection under submerged branches and trunks.It is an omnivorous species, although its diet is heavily composed of fish, crustaceans, gastropods
Palavras-chave: Amazônia, peixe de água doce, Apaiari, parasito, nematoda, diversidade.(FRANCOVÁ & ONDRAČKOVÁ, 2011;MARCOGLIESE et al., 2016;TAVARES-DIAS & NEVES, 2017;OLIVEIRA et al., 2017).For A. ocellatus from the Brazil, a total of 22 parasite species has been estimated; in addition some composition and structural patterns were detected in the parasite community, such as dominance of monogenean species, dominance of ectoparasite species with crustacean infracommunities more diverse than other ectoparasites, overdispersion of ectoparasites and endoparasites, interspecific associations in the ectoparasite infracommunities, specifically a combination of generalist and specialist parasites that may contribute to host population control and correlation between parasite abundance and host total body size at the infracommunity level (TAVARES-DIAS & NEVES, 2017).However, this is probably an underestimation of the actual diversity, due to differences in species richness of parasites for different environments, since Pinheiro et al. (2018) has recently described a new species of nematode for A. ocellatus from the eastern Amazon, Procamallanus spiculastriatus.
In each wild host population, the structure and composition of the parasite communities are constituted from a set of parasite species available in the environment (FRANCOVÁ & ONDRAČKOVÁ, 2011;BITTENCOURT et al., 2014;MARCOGLIESE et al., 2016;OLIVEIRA et al., 2017;TAVARES-DIAS & NEVES, 2017), influenced by many local factors.As a result, for the same fish species living in different environments, the component community and richness of parasites may or may not be mainly linked to environmental and climate conditions, host size, intensity of food capture by host, structure of the local food web, distance from geographic localities and availability of intermediate hosts infecting the fish populations (FRANCOVÁ & ONDRAČKOVÁ, 2011;MARCOGLIESE et al., 2016;OLIVEIRA et al., 2017;TAVARES-DIAS & NEVES, 2017).In addition, the type of parasite life cycle also plays an important role in environment-host-parasite interactions.The question is thus whether the component community and species richness of parasites in A. ocellatus populations can vary in two localities in the Brazilian Amazon.The aim of this study was to compare the structure of helminth parasite communities in two populations of A. ocellatus from the eastern Amazon, northern Brazil.
The Tapajós River basin in region of the Pará state has an area of 221,992.98 km 2 (Figure 1), and presents typical fauna and vegetation of the Amazon region, which has clear waters and extensive floodplain areas.However, this area of the Tapajós River basin is suffering from the increase of human population and environmental pressures due to the accelerated rate of devastation of forest areas and widespread changes in aquatic systems caused by pollution, silting, barrages and mining activities.
Iara Lake is located in the middle of the "Jardim Zoobotânico da Amazônia, Bosque Rodrigues Alves" (Figure 2), in the urban space of the municipality of Belém, Pará state.The "Bosque Rodrigues Alves" has a 15 ha area consisting of a remnant of primary dryland forest that shelters species of fauna and flora of the Amazon.Iara Lake is an artificial lake that has around 1500 m 3 and is populated by different species of fish, including A. ocellatus (LUZ et al., 2012).All the runoff from the rains in the "Bosque Rodrigues Alves" is discharged into this lake, which has no presence of aquatic vegetation.

Procedures of collection and analysis of parasites
The fish were transported in an isothermic box with ice to the "Laboratório de Histologia e Embriologia Animal", "Instituto da Saúde e Produção Animal", "Universidade Federal Rural da Amazônia", "Campus Belém" for necropsy.After biometric analyses, the animals were necropsied for helminths.The gills were examined for the presence of ectoparasites, and the digestive tract (oesophagus, stomach, caeca and intestine), for the presence of endoparasites.The gastrointestinal tract of each fish was isolated in Petri dishes containing NaCl (0.9%) solution and analyzed using a stereomicroscope (LEICA-ES2).The collection, fixation, counting, preparation and staining of parasites for identification followed previous recommendations of Eiras et al. (2006).

Data analysis
The ecological terms (prevalence and mean abundance) used followed previous recommendations of Bush et al. (1997).The following descriptors for the parasite community were calculated: species richness, Shannon diversity index (H), evenness (E) in association with diversity index, and dominance frequency (percentage of infracommunities in which a parasite species is numerically dominant) (ROHDE et al., 1995;MAGURRAN, 2004), using the Diversity software (Pisces Conservation Ltd., UK).The Shapiro-Wilk test was used to determine whether parasite abundance data followed a normal distribution pattern.Differences of length and weight of hosts between years and rainy and dry seasons were evaluated using the Mann-Whitney test (ZAR, 2010).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% (BUSH et al., 1990).The significance of ID, for each infracommunity, was tested using the d-statistics (LUDWIG & REYNOLDS, 1988).
Differences in similarity of the parasite component communities were tested using the analysis of similarities (ANOSIM), between the localities.ANOSIM was based on ranked matrixes, generated from Jaccard index and Bray-Curtis distance (MAGURRAN, 2004), with 10,000 permutations.

Results
All parasite species in both localities were found in intestine of hosts and only metacercariae of Posthodiplostomum sp.(Diplostomidae) were found in gills.In addition, in cists of Nematoda and Cestoda were also found 37.5% of fish of Tapajós River, which were not quantified.
The results showed that 85.0% of the Tapajós River fish and 65.0% of the fish of Lake Iara had parasites of one or more species of helminth.The component communities of parasites of both localities presented differentiated characteristics (Table 3).Thus, there was no similarity in comparing the component communities of both localities (Jaccard index: R = 0.31, p = 0.0001 and (Bray-Curtis index, R = 0.27, p = 0.0001).
The size of the hosts of the Tapajós River was smaller than that of Iara Lake.However, host weight, species richness of parasites, Shannon diversity and evenness were similar for hosts of both localities (Table 4).
In the Tapajós River there was a predominance of hosts parasitized by one species of helminths, while in Iara Lake there was a predominance of hosts parasitized by 0 to 2 species (Figure 3).
Table 1.Helminth parasites in two populations of Astronotus ocellatus in eastern Amazon (Brazil).

Discussion
As parasite community structure is influenced by abiotic factors within a particular habitat, the parasite community structure of a same host species may vary between populations depending on its geographical occurrence and differences in environments (FRANCOVÁ & ONDRAČKOVÁ, 2011;MARCOGLIESE et al., 2016;OLIVEIRA et al., 2017).Although the environment of the artificial Iara Lake is a reduced environment lacking aquatic vegetation, the parasite community of A. ocellatus was characterized by 1 species of Nematoda, 1 Cestoda and 1 Acanthocephala, while the Tapajós River community of parasites was characterized by 1 species of Digenea and 2 Nematoda.The Tapajós River is a highly complex system and its regulation is a process that affects the integrity of the river-floodplain system.However, Tapajós River basin is suffering from the increase of human population and environmental pressures due to the accelerated rate of devastation of forest areas and widespread changes in aquatic systems caused by pollution, silting, barrages and mining activities.Thus, a dissimilarity between the component communities of both localities was expected.Therefore, in the localities of this study, A. ocellatus plays an important role in the life cycle and distribution of these endoparasites.In addition, environmental differences are good predictors of dissimilarity between component communities of parasites between both the populations of A. ocellatus.
We observe also differences in parasites component communities of A. ocellatus and Shannon diversity index and evenness between the both localities that might have been caused by different factors linked to host size, geographic distance between host populations, and environmental characteristics, such as water quality, among other factors.In addition, hosts from the Tapajós River had only species of generalist parasites, i.e. they infect many fish species in eastern Amazon (BITTENCOURT et al., 2014;OLIVEIRA et al., 2017;TAVARES-DIAS & NEVES, 2017).In addition, the absence or presence of these endoparasite species in hosts of both localities may be mainly related to differences in host diet, absence or unviability of infective stages and viability of adequate intermediate hosts between both environments (MARCOGLIESE et al., 2016;TAVARES-DIAS & NEVES, 2017;OLIVEIRA et al., 2017).
The infracommunities of parasites of a same species of host from different localities can vary depending on its geographical distance and on differences in habitat and parasite abundance, which can be influenced by biotic (e.g.presence of infective stages and ingested food preys, etc.) and abiotic (e.g.environment quality, temperature, precipitation levels, etc.) factors (FRANCOVÁ & ONDRAČKOVÁ, 2011;MARCOGLIESE et al., 2016;OLIVEIRA et al., 2017).In A. ocellatus, the helminths of hosts from the Tapajós River were Posthodiplostomum sp., Contracaecum sp. and Pseudoproleptus sp., with dominance of Contracaecum sp., while helminths of hosts from the Iara Lake were P. spiculastriatus, Proteocephalus sp. and Acanthocephala gen.sp., with dominance of P. spiculastriatus.Parasites had an overdispersion, a common distribution pattern in A. ocellatus (TAVARES-DIAS & NEVES, 2017).Furthermore, as the population of A. ocellatus from the Iara Lake consisted of larger (older) individuals than the population of hosts from the Tapajós River, it usually could ingest more infective larvae, either directly from the intermediate host or from paratenic hosts, with the increased volume of food consumed by larger fish.Therefore, A. ocellatus are ingesting infective stages, either in artificial and natural environment.
Life cycles of the Posthodiplostomum species involve two intermediate hosts, a snail and a fish species, besides a definitive host, a piscivorous bird species (RITOSSA et al., 2013).Contracaecum species use several species of fish as intermediate or paratenic hosts due to their lack of parasitic host specificity, but in general are found in fish-eating birds and piscivorous fish in the adult stages.The fish that act as intermediate hosts acquire this parasite by predation on smaller fish, which in turn become infected by ingesting copepods, gastropods, mollusks, coelenterates or ctenophores that contain infective forms, or when they directly acquire the larvae of Contracaecum sp. in their free life stage (OLIVEIRA et al., 2017;PINHEIRO et al., 2019).Metacercariae of Posthodiplostomum sp. and Contracaecum sp.larvae are frequent parasites in fish species from the eastern Amazon because they use several species of fish  MELO et al., 2011).However, P. spiculastriatus, a nematode recently described in A. ocellatus from Iara Lake (PINHEIRO et al., 2018), still has an unknown life cycle.Procercoids of Proteocephalus sp. were found in only A. ocellatus from Iara Lake.However, only Proteocephalus gibsoni Rego & Pavanelli 1990 is known to infect A. ocellatus from the Amazon (TAVARES-DIAS & NEVES, 2017).Planktonic crustaceansdiaptomid or cyclopoid copepods serve as intermediate hosts of the Proteocephalus species.Metacestodes or plerocercoids develop in the body cavity of these crustaceans and the definitive host, a fish, becomes infected directly after consuming them (SCHOLZ, 1999).Undetermined cystacanths of acanthocephalans were also found in A. ocellatus from Iara Lake.Acanthocephalans are obligatory endoparasites that utilize arthropods and vertebrates to complete their complex life cycle; thus, adult parasites are found in a variety of vertebrates, including fish, birds and mammals (SCHMIDT & HUGGHINS, 1973;KENNEDY, 2006).
In summary, communities of helminths in A. ocellatus of both localities were characterized by moderate prevalence and low abundance with low parasite species richness, low diversity, low evenness and overdispersion.Lastly, the community of helminth parasites in both populations of A. ocellatus were not similar as expected, and parasite diversity was the factor causing differences in the parasitic community.

Figure 1 .
Figure 1.Collection site of Astronotus ocellatus in Tapajós River, in eastern Amazon (Brazil).

Figure 2 .
Figure 2. Collection site of Astronotus ocellatus in Iara Lake, in eastern Amazon (Brazil).

Table 2 .
Dispersion index (ID), statistic-d and discrepancy index (D) for the parasites infracommunities in Astronotus ocellatus in eastern Amazon (Brazil).Figure 3. Species richness of helminth parasites in Astronotus ocellatus in eastern Amazon (Brazil).

Table 3 .
Component communities of helminth parasites in two populations of Astronotus ocellatus in eastern Amazon (Brazil).

Table 4 .
(PINHEIRO et al., 2019)14;OLIVEIRA et al., 2017;arasites communities in two populations of Astronotus ocellatus in eastern Amazon (Brazil).Mann-Whitney test as hosts due to their lack of parasitic host specificity, including A. ocellatus(BITTENCOURT et al., 2014;OLIVEIRA et al., 2017; TAVARES-DIAS & NEVES, 2017).Contracaecum sp., a parasite with zoonotic potential for humans(PINHEIRO et al., 2019), was present in A. ocellatus from the Tapajós River in higher prevalence and low abundance, while Posthodiplostomum sp. had a low prevalence and abundance.In addition, A. ocellatus population had larvae of Pseudoproleptus sp., which have been reported in the cichlid Satanoperca jurupari, a paratenic host of this nematode species because the prawn Macrobrachium amazonicum is a first intermediate host in the Amazon River delta ( U: