Host-parasite interaction between crustaceans of six fish species from the Brazilian Amazon

. Host-parasite interactions between crustaceans and six fish species ( Psectrogaster falcata , Ageneiosus ucayalensis , Acestrorhynchus falcirostris , Hemiodus unimaculatus , Serrasalmus gibbus and Geophagus proximus ) from a reservoir in eastern Amazon, northern Brazil, were investigated. Eight hundred and seventy-eight parasites belonging to three crustacean species, Excorallana berbicensis, Argulus chicomendesi and Ergasilus turucuyus , which parasitized the hosts’ mouth, gills and tegument, were collected from 295 fish and examined. High infestation levels were caused by E. berbicensis on the body surface of the hosts. Excorallana berbicensis showed aggregate dispersion, except in S. gibbus , while E. turucuyus showed random dispersion in A. falcirostris . The host’s sex did not influence infestation by E. berbicensis , and high parasitism failed to affect the body conditions of the fish. In the case of some hosts, rainfall rates, temperature, dissolved oxygen levels and water pH affected the prevalence and abundance of E. berbicensis , the dominant parasite species. Results revealed that the environment and life-style of the hosts were determining factors in infestations by parasites. Current assay is the first report on E. berbicensis for the six hosts, as well as on A. chicomendesi for G. proximus and P. falcata . a precipitação pluviométrica, temperatura, níveis de oxigênio dissolvido e pH da água. Os resultados indicaram que o ambiente e hábito de vida dos hospedeiros foram fatores determinantes nas infestações parasitárias encontradas. Esse é o primeiro relato de E. berbicensis para esses seis hospedeiros, A. chicomendesi para G . proximus e P. falcata e E. turucuyus para H. unimaculatus . Palavras-chave: condição corporal, ectoparasitos, ambiente, peixes de água doce, infestação.

The host-parasite interactions between crustaceans and six fish species of a reservoir from eastern Amazon, northern Brazil, were investigated.The environmental variables that affect this interaction will also be assessed.

Study area
Current analysis was conducted in the reservoir of the Coaracy Nunes Hydroelectric Power Plant (Figure 1) on the middle Araguari river, municipality of Ferreira Gomes, state of Amapá, northern Brazil (00º54'11.8"N;51º15'35.5"W).The reservoir has a drainage area of 23.5 km 2 with a mean depth of 15 m.Around the reservoir there are two riverine communities (Paredão and Caldeirão) coupled to agricultural areas and private properties for leisure and recreation (Sá-Oliveira et al., 2013).The reservoir has areas with few aquatic macrophytes, mainly Eichornia crassipes and Eleocharis sp., and a large amount of decaying arboreal vegetation due to non-deforestation.

Hosts fish
Hemiodus unimaculatus is a non-migratory fish that occurs in the undisturbed zones of swamps or in the lower parts of stream.The species is omnivorous, mainly consuming algae, detritus and other aquatic invertebrates.Mature at 11 cm of length, the fish's spawning is limited during the rainy season.P. falcata is a non-migratory and detritivorous benthopelagic fish, feeding on detritus and microorganisms associated with the substrate.Its maximum length is approximately 17.0 cm.G. proximus is a non-migratory and omnivorous benthopelagic fish, mainly feeding on plants, mollusks, insects and other aquatic invertebrates.
Its maximum length is approximately 23.0 cm. A. ucayalensis is a nonmigratory benthopelagic fish occurring in the undisturbed zones of swamps or the lower sections of streams.It is a carnivorous species, consuming fish, insects and other aquatic invertebrates.
Its maximum length is approximately 29.0 cm. A. falcirostris is a piscivorous fish, but young specimens may also consume shrimps.Its maximum length is approximately 40 cm and spawning occurs with greater intensity at the end of the dry season and at the beginning of the flood season.S. gibbus is a piscivorous fish with benthopelagic and nonmigratory habits.Its maximum length is approximately 21.0 cm (Froese & Pauly, 2016;Santos, Mérona, Juras, & Jégu, 2004).
Water temperature, pH, electrical conductivity and water dissolved oxygen were obtained with a multi-parameter meter at each of the six sampling sites (YSI 85, USA).A pHmeter (YSI 60, USA) determined pH.Whereas turbidity was obtained by a microprocessed Plus II turbidimeter, transparency was obtained with a Secchi disk.Rainfall rates were retrieved from the National System of Environmental Data (Sinda-INPE) of the Coaracy Nunes Hydro-Meteorological Station (Table 1).
Simple gillnets of different meshes (20, 30, 40, 50, 60 mm), with 100 m in length, were used.Permanence time of each network was 12 hours, with inspections at every two hours.Standard length in centimeters and total weight in grams was obtained for each fish.Its sex was microscopically identified according to methodology by Vazzoler (1996).After collection, the fish were packed in a plastic drum, with 10% formalin, and transported to the Laboratory of Ichthyology and Limnology of the Universidade Federal do Amapá (Macapá, Brazil).Current study was developed following the principles by the Brazilian College of Animal Experimentation (Cobea).

Parasitological analysis
Mouth, tegument and fins of each fish were examined on the spot, immediately after capture, to verify the presence of crustacean ectoparasites.

Samplinh sites
The collected gills were fixed in 5% formalin and examined with a stereomicroscope for the collection and counting of parasites.The crustaceans' species collected were kept in 70% glycerin-alcohol and prepared for identification according to methods described by Eiras, Takemoto, & Pavanelli (2006).The ecological terms used were those recommended by Bush, Lafferty, Lotz, & Shostak (1997).
The dominance frequency (percentage of infracommunities in which the species is numerically dominant) was calculated according to Rohde, Hayward, & Heap (1995).Dispersion index (ID) and Poluin Discrepancy Index (D) were assessed with Quantitative Parasitology 3.0 software to detect the distribution pattern of each infracommunity of parasites (Rózsa, Reiczigel, & Majoros, 2000) with prevalence ≥ 10% and hosts with samples > 30 specimens.ID significance for each infracommunity was tested with d-statistic (Ludwig & Reynolds, 1988).

Data analysis
Standard length and weight data determined the relative condition factor (Kn), comparing parasitized and non-parasitized fish (Le-Cren, 1951) by test t.Spearman's correlation coefficient (rs) was used to determine possible correlations of parasites abundance with length and Kn of hosts.G-test determined the sex effect of the hosts in the prevalence of parasites, and Mann-Whitney test (U), with normal approach of Z, was employed to compare the abundance of species of parasites between male and female hosts (Zar, 2010).
Spearman coefficient (rs) determined possible correlations of prevalence and abundance of parasites in fish species with rainfall rates, water temperature, electrical conductivity, dissolved oxygen, pH, turbidity and transparency using the data of each collection.
Non-parasitized specimens predominated in the examined host species whilst none of the host species was parasitized by more than two species of crustacean ectoparasites (Figure 2).
Crustacean parasites were collected at a wide variety of sites in the hosts, such as mouth, gills, pectoral, pelvic, dorsal, anal, caudal fins and tegument of the hosts.The prevalence of parasites in sites of infestation was different, but prevalence was low in S. gibbus.Among the infestation sites, high prevalence was found predominantly on the body surface of the five host species, except G. proximus (Figure 3).Prevalence and abundance of E. berbicensis was not influenced by the host' sex (Table 4).
There were no significant differences for Kn of parasitized and non-parasitized fish in A. ucayalensis, H. unimaculatus, S. gibbus, G. proximus, A. falcirostris and P. falcata (Table 5).
In the case of A. falcirostris, the abundance of E. berbicensis showed a negative correlation with Kn (rs = -0.259;p = 0.042) of host and a positive correlation with length (rs = 0.258; p = 0.042).For P. falcata, abundance of E. berbicensis correlated with Kn (rs = -0.293;p = 0.018), with no correlation The prevalence of E. berbicensis was positively correlated with rainfall levels and water temperature for A. falcirostris.In the case of A. ucayalensis, G. proximus and P. falcata, there was also a positive correlation of the prevalence of Corallanidae with water oxygen, temperature and pH levels.Abundance of E. berbicensis was negatively correlated with the oxygen levels for A. ucayalensis whilst the abundance of E. berbicensis was positively correlated with water temperature and pH with those of G. proximus and P. falcata, respectively.No correlation of prevalence and abundance of these limnological parameters was found for S. gibbus and H. unimaculatus (Table 6).
Although Excorallana Stebbing, 1904 are Corallanidae that occur predominantly in marine environments substrates (Delaney, 1989), they have also been reported parasitizing a few species of marine Teleostei and Chondrichthyes (Williams-Jr & Bunkley-Williams, 1994;Semmens et al., 2006).E. berbicensis, the sole representative of freshwater corallanids, originally described in 1918 by Boone in the zooplankton from Berbice river (British Guiana), parasitized all the hosts analyzed in current study.However, the first reports on E. berbicensis in freshwater fish were given in 1925 and 1936 by Van Name, who detected the parasite in the gills and tegument of L. grossidens from British Guiana.Later, in 1969, Monod reported the same corallanid species on gills of the shark N. brevirostris collected in freshwater areas of French Guiana, in the Amazon region (Stone & Head, 1989).Subsequently, the occurrence of E. berbicensis was reported in A. inermis of the Crustacean Collection of the National Institute of Amazonian Researches (Thatcher, 1995), probably originating from the state of Pará, in the eastern Amazon region, Brazil.Therefore, current analysis is the second epidemiological study of E. berbicensis for freshwater fish species.
In the case of reservoirs from the southeastern region of Brazil, Ergasilus sp. and non-identified Cymothoidae were crustaceans parasitizing A. fasciatus, H. affinis, L. castaneus and H. malabaricus (Paraguassú & Luque 2007).Ergasilus sp. and nonidentified Vaigamidae were the crustaceans parasitizing I. labrosus (Moreira, Ito, Takemoto & Pavanelli, 2005) and A. osteomystax (Tavernari et al., 2009), respectively.In 12 species of hosts from a reservoir in Iran, only Cyprinus carpio, Barbus lacerta and Capoeta trutta were parasitized, and parasitic crustaceans community was constituted only by Lernaea cyprinacea and Tracheliastes polycolpus (Bozorgnia, Youssefi, Barzegar, Hosseinifard, & Ebrahimpour, 2012).However, the low richness of the crustacean species in hosts analyzed in current study was similar to other freshwater fish species from different localities in Brazil (Acácio et al., 2012;Moreira et al., 2005;Paraguassú & Luque 2007).Infestations of ectoparasites crustaceans may be influenced by various biotic and abiotic factors (Guidelli et al., 2009;Tavares-Dias et al., 2015), but the fish population density may also be a determining factor in hosts' parasites richness in ecosystems with homogeneous physical and chemical characteristics such as the reservoir studied (Takemoto et al., 2009).
This first study on the crustacean ectoparasites of A. falcirostris, A. ucayalensis, G. proximus, H. unimaculatus, P. falcata and S. gibbus showed greater parasitism of E. berbicensis, followed by low infestation of E. turucuyus and A. chicomendesi.Such results indicate a low parasitic specificity of E. berbicensis.However, in Pygocentrus nattereri of the Araguaia river (Brazil), infestations were caused by Argulus sp., D. carvalhoi, Braga patagonica, Amphira branchialis and Asotana sp., predominantly by argulid species (Carvalho, Arruda, & Del-Claro, 2004).Moreover, highest infestation of E. berbicensis occurred in G. proximus and P. falcata, and the lowest in S. gibbus.The highest parasitism of E. turucuyus occurred in A. falcirostris.G. proximus and P. falcata are fish with a sluggish behavior and this feature may have facilitated the establishment of E. berbicensis; contrastingly, S. gibbus is a fish of active behavior.Mamani, Hamel, & Van Damme (2004) argued that the sluggish fish lifestyle could make them more vulnerable to infectious forms of crustacean ectoparasites in the larval stage.
Excorallana berbicensis was collected from the tegument, mouth and gills of A. falcirostris, A. ucayalensis, G. proximus, H. unimaculatus, P. falcata and S. gibbus, predominantly on the tegument of these hosts.In general, Excorallana spp.infests gills, mouth and nasal cavity of marine teleost species (Delaney, 1984;Williams-Jr & Bunkley-Williams, 1994;Semmens et al., 2006).However, it was observed that E. berbicensis has a low restriction to site for infestation and settled mainly on the hosts' tegument which is the largest area available for fixation.The life history of Excorallana species is little known since the marine species of these ectoparasites seem to emerge from the criptofauna for temporarily parasitize fish, microcrustaceans, ascidians, sponges and mollusks (Delaney, 1989).Guzman, Obando, Brusca, & Delane (1988) reported that the females of E. tricornis occidentalis were found associated to substrates during the reproduction period.
Parasitic copepod species have a direct and short lifecycle.Many species find their hosts by close relationship with fish attacking them during their life cycle (Piasecki & Avenant-Oldewage, 2008;Tavares-Dias et al., 2015).E. turucuyus were found only on gills of H. unimaculatus and A. falcirostris and showed similar and low infestation level for the two hosts.However, such infestation levels were lowest than those reported for A. falcatus and A. falcirostris from the Pacaás Novos river, in western Amazon, Brazil (Malta & Varella, 1996).Only females are parasites among the species of Ergasilidae, releasing infective forms that freely swim until they find a fish to infect; the males live freely in the zooplankton (Malta & Varella, 1996;Piasecki & Avenant-Oldewage, 2008).
The infestation of E. berbicensis in A. ucayalensis, H. unimaculatus, S. gibbus, G. proximus, A. falcirostris and P. falcata showed an aggregated dispersion, a typical pattern for parasitic crustacean species of freshwater fish (Hoshino, Hoshino, & Tavares-Dias, 2014;Tavares-Dias et al., 2015).In contrast, there was a random dispersion of E. turucuyus in A. falcirostris and of E. berbicensis in S. gibbus.Such overdispersion may be related to diverse factors, including environmental, ecophysiological and immunological conditions and variations in the parasites' exposure time of host fish, as well as other factors (Poulin, 1993;Mamani et al., 2004;Tavares-Dias et al., 2015;Walker et al., 2008).However, random dispersion pattern may be related to a reduced opportunity to colonize this host (Guidelli et al., 2003).
A. chicomendesi was found only on the tegument of G. proximus and P. falcatus, with low and similar levels of infestation.In the case of P. nattereri and S. marginatus from the Paraguay river, Brazil (Fontana et al., 2012) and in the case of S. rhombeus from the Solimões river, Brazil (Acácio et al., 2012), low levels of infestation of the same argulid species were also reported.Therefore, A. chicomendesi has low parasitic specificity and causes low parasitism in wild fish populations, although high infestations are known in farmed fish (Acácio et al., 2012).Since aquatic macrophytes are substrates for the reproduction of argulid species (Piasecki & Avenant-Oldewage, 2008), their low abundance may be related to the reduced abundance of these plants in the reservoir under analysis.This is the first record of A. chicomendesi for G. proximus and P. falcata.
The host's sex did not influence the infestation levels of E. berbicensis in the fish under analysis.Guidelli et al., (2009) did not find any difference in the infestation levels by Gamispatulus schizodontis for males and females of Leporinus lacustris from the Paraná basin river (Brazil).In spite of the fact that high testosterone levels have been implied in the immunosuppression of males which favors parasitism when compared with that of females (Poulin, 1996), studies have not confirmed this information.Thereby, Tavares, and Luque (2004) argued that differences in parasitic infestation levels between fish sex might be related especially with ecological and behavioral differences between males and females.
Several parasites may have a deleterious effect on hosts, with consequences on body conditions.Thereby, the condition factor of wild and farmed fish populations, a quantitative measure of hosts welfare (Fontana et al., 2012;Guidelli et al., 2009;Hoshino, Hoshino, & Tavares-Dias, 2014), may be successfully employed as a tool to detect such negative effects of parasitism.However, in the hosts under analysis, Kn was not affected by parasitic infestation levels.Guidelli et al., (2009) reported similar results for L. lacustris infested by G. schizodontis.In contrast, in the case of P. nattereri and S. maculatus infested by D. bidentata and Dolops sp.(Fontana et al., 2012) and for Astyanax intermedius infested by Paracymothoa astyanaxi, Kn decreased due to parasitic infestation levels (Gomiero, Souza, & Braga, 2012).
Wild fish usually coexist in equilibrium with parasites within the environment, but this balance in the parasite-host-environment interactions, when broken by environmental changes, may negatively affect hosts and, consequently, increase their susceptibility to infections by parasitic crustaceans (Carvalho et al., 2003;Fontana et al., 2012;Guidelli et al., 2009;Morley, 2007).Changes in environmental quality have a relevant role in the parasitic infestations of ectoparasites crustaceans.In this study, rainfall and water temperature rates increased the infestation of E. berbicensis on A. falcirostris.Similarly, temperature increase favored the infestation of E. berbicensis on G. proximus, as well as the water pH increase in P. falcata.However, dissolved oxygen levels increased the prevalence of E. berbicensis in A. ucayalensis, but decreased its abundance.Pech, Aguirre-Macedo, Lewis, and Vidal-Martínez (2010) also reported that rainfall influenced the infestation levels by Argulus sp. and Ergasilus sp. on Cichlasoma urophthalmus from the Yucatan Peninsula (Mexico).

Conclusions
High parasitism did not affect the body conditions of A. ucayalensis, H. unimaculatus, S. gibbus, G. proximus, A. falcirostris and P. falcata, with a high abundance of E. berbicensis and low abundance of E. turucuyus and A. chicomendesi.In current study, the aggregation of these hosts may have facilitated E. berbicensis infestations.Results show that the hosts' size and sex were factors that did not influence parasitic infestation levels, although other characteristics of the environment had a strong influence.Since environmental factors influenced infestation levels by E. berbicensis, further studies on the seasonal variation of these ectoparasites may be more conclusive.

Figure 1 .
Figure 1.Collection area in the Coaracy Nunes HPP reservoir in eastern Amazon (Brazil).

Figure 3 .
Figure 3. Prevalence of crustaceans parasitizing different sites on the six fish species in a reservoir of the Coaracy Nunes HPP dam in eastern Amazon, northern Brazil.with hosts' length (rs = 0.130; p = 0.302).Abundance of E. berbicensis showed no correlation with Kn (rs = -0.333;p = 0.245) and length (rs = 0.179; p = 0.542) of G. proximus, as well as with length (rs = -0.171,p = 0.209) and Kn (rs = 0.121; p = 0.375) of H. unimaculatus.The abundance of E. berbicensis showed no correlation with length (rs = -0.079;p = 0.536) and Kn (rs = -0.167;p = 0.192) of A. ucayalensis, as well as with length (rs = -0.298;p = 0.078) and Kn (rs = 0.209; p = 0.220) of S. gibbus.In the case of A. falcirostris, there was no correlation of abundance of E. turucuyus with length (rs = -0.017;p = 0.896) and Kn (rs = -0.138;p = 0.286) of hosts.The prevalence of E. berbicensis was positively correlated with rainfall levels and water temperature for A. falcirostris.In the case of A. ucayalensis, G. proximus and P. falcata, there was also a positive correlation of the prevalence of Corallanidae with water oxygen, temperature and pH levels.Abundance of E. berbicensis was negatively correlated with the oxygen levels for A. ucayalensis whilst the abundance of E. berbicensis was positively correlated with water temperature and pH with those of G. proximus and P. falcata, respectively.No

Table 1 .
Physical and chemical parameters of water in the reservoir of the Coaracy Nunes HPP dam in eastern Amazon, northern Brazil, at the collection sites of crustacean species parasitizing six fish.
. Further, 30.6% of the 62 specimens of A. falcirostris examined were parasitized by E. berbicensis and E. turucuyus; 28.6% of the 56 specimens of H. unimaculatus examined were parasitized by E. berbicensis and E. turucuyus; 34.9% of the 63 specimens of A. ucayalensis examined were parasitized by E. berbicensis only; 40 and 42.9% of the 65 specimens of P. falcata and 14 specimens of G. proximus respectively were parasitized by A. chicomendesi and E. berbicensis; 19.4% of the 36 specimens of S. gibbus examined were parasitized by E. berbicensis only.

Table 2 .
Parasitological indexes of crustacean species parasitizing six fish in a reservoir of the Coaracy Nunes HPP dam in eastern Amazon, northern Brazil.P: prevalence, MA: mean abundance, MI: mean intensity, TNP: total number of parasites.

Table 3 .
Index of dispersion (ID), d-statistic and discrepancy index (D) for crustacean species parasitizing six fish species in a reservoir of the Coaracy Nunes HPP dam in eastern Amazon, northern Brazil.Species richness of crustaceans parasitizing six fish in a reservoir of the Coaracy Nunes HPP dam in eastern Amazon, northern Brazil.

Table 4 .
Effect of sex on the prevalence and abundance of Excorallana berbicensis in six fish species of a reservoir of the Coaracy Nunes HPP dam in eastern Amazon, northern Brazil.G: test-G; Z: Mann-Whitney with approach in Z.

Table 6 .
Spearman's correlation coefficient (rs) of prevalence and abundance of Excorallana berbicensis with water physical and chemical parameters for six fish species in a reservoir of the Coaracy Nunes HPP dam in eastern Amazon, northern Brazil.*p< 0.05,** p < 0.001.