Helminth endoparasites of endemic ﬁ sh Pygocentrus piraya (Characiformes, Serrasalmidae) from Três Marias reservoir, Minas Gerais, Brazil

: Ten endoparasite species found in Pygocentrus piraya , which is a piranha species native to São Francisco river, were collected from 108 fi sh caught in Três Marias reservoir in 2004 and 2005, namely: Digenea - Austrodiplostomum sp. (metacercariae); Eucestoda – Proteocephalidae gen. sp. (plerocercoids); and Nematoda - Procamallanus ( Spirocamallanus ) inopinatus , Cystidicoloides fi scheri and Capillostrongyloides sentinosa (adults); Spinitectus rodolphiheringi (juvenile); Hysterothylacium sp., Contracaecum sp. Type 1 , Spiroxys sp. and Goezia sp. (larvae). In helminth fauna, P. (S.) inopinatus has shown higher prevalence and dominance. Fish sex has infl uenced the prevalence of Hysterothylacium sp., which was higher in female specimens. Longer total length of fi sh has positively infl uenced the abundance of C . sentinosa and Hysterothylacium sp.. The rainy season has favored parasitic indices, mainly P. (S.) inopinatus abundance, as well C . sentinosa and Hysterothylacium sp. prevalence and abundance. There were co-occurrences between adult and some larval nematodes. Endemic piranhas, as well as fi nal hosts, are important intermediate and/or paratenic hosts, given the relevant number of larvae (proteocephalideans and nematodes) of heteroxene cycle found in them. Proteocephalidae gen. sp., C . sentinosa , Hysterothylacium sp., Contracaecum sp. Type 1 , Goezia sp. and S . rodolphiheringi are new records parasitizing P. piraya in São Francisco basin.


INTRODUCTION
São Francisco river basin covers an area of approximately 645,067 km 2 ; it is the largest basin fully inserted in the Brazilian territory and the third largest basin in Brazil. This basin is of paramount importance to water supply in the semiarid region, as well as to electric power production (Kohler 2003). Três Marias reservoir is located in the upper portion of São Francisco basin, the upper São Francisco, and it was built to regulate the fl ow of São Francisco river (Cachapuz 2006).
Serrasalmidae comprises 98 fi sh species and 16 current genera. Pygocentrus encompasses four valid species, namely: Pygocentrus cariba (Humboldt, 1821); Pygocentrus nattereri Kner, 1858;Pygocentrus palometa Valenciennes, 1850; and Pygocentrus piraya (Cuvier, 1819) (Froese & Pauly 2019). The distribution of this family is limited to the Neotropical region. The incidence of individuals belonging to this family in other places results from their introduction in these environments. Pygocentrus spp. has the ability to adapt to new environments, mainly to lakes and reservoirs (Agostinho et al. 2007). Pygocentrus piraya, commonly known as piranha, is endemic to São Francisco river basin (Fink 1993, Reis et al. 2003 and abundant in Três Marias reservoir; it is carnivorous and prefers to feed on other fish (Britski et al. 1988).
Piranhas are neotropical fish capable of attacking bigger animals (Agostinho et al. 1997); their mutilating and opportunistic habit enables them to attack several fish species living in captivity or in natural environments (Sazima & Machado 1990), as well as fish caught in fishing nets (Agostinho & Marques 2001). The fact that P. piraya is a predatory species at the top of the food web reveals the key role played by this species in controlling populations of other fish species in the ecosystem. However, it is described as a species capable of causing harm to local fishermen, because it damages their fishing nets (Trindade & Jucá-Chagas 2008).
The aim of the present study was to identify species composing the endoparasite community found in piranha P. piraya in Três Marias reservoir, upper São Francisco river, state of Minas Gerais, Brazil.

MATERIALS AND METHODS
For investigation of metazoan endoparasites, 108 specimens of P. piraya were caught in the Três Marias reservoir (18º 12' 59" S, 45º 17' 34" W), upper São Francisco river, Três Marias municipality, state of Minas Gerais, in the period between July and August, 2004 (dry period) and January, 2004, December, 2004and January, 2005 ), where they were subsequently necropsied. During fish necropsies, their total length, weight and sex were recorded on forms. The procedure for fixing and preparing temporary or permanent slides of specimens parasites followed a standardized methodology (Amato et al. 1991).
Digenea metacercariae was identified and classified according to Niewiadomska (2002). The classification of Eucestoda plerocercoids followed Chervy (2002) and Chambrier et al. (2017). Specimens of Nematoda were identified and classified according to Moravec (1998) and through specialized scientific articles.
Voucher specimens of P. piraya were deposited in the Museu de Zoologia of the Universidade de São Paulo (MZUSP), São Paulo, Nº 95149. Voucher specimens of parasites of P. piraya were deposited in the Coleção Helmintológica do Instituto Oswaldo Cruz (CHIOC), Rio de Janeiro and/or in the Academia de Ciências da República Tcheca (ASCR), according to the numbering presented in the results section.
Statistical tests were applied to analyze parasitic infrapopulations and infracommunities in accordance of Bush et al. (1990Bush et al. ( , 1997. The parasite species were classified within the community according to their importan ce value (IV) (Bush & Holmes 1986). Species dominance in communities was tested using Simpson's index (C) (Stone & Pence 1978). The frequency of dominance, the shared frequency of dominance and the mean relative dominance of each parasite species were calculated according to Rohde et al. (1995). The ratio between the mean parasite variance and abundance (dispersion index -DI) was calculated for each parasite species in order to determinate their distribution pattern. Their significance was tested using the statistical d-test (d>1.96) (Ludwig & Reynolds 1988).
Student's t test was used to check for possible differences between the weights and sizes of hosts in relation to their sex. The chisquare test (X 2 ) was used to determine the influence of sex and the period of collection on the prevalence of parasites. Spearman's correlation coefficient by ranks (r s ) was used to evaluate possible correlations between the host's total length and intensity, abundance, richness and parasite diversity. Pearson's correlation coefficient (r) was used to assess the correlation between parasite prevalence in relation to the hosts' size classes, as estimated by Sturges's formula (Sturges 1926). The Mann Whitney test (U) was used to assess the possible differences between intensity, abundance, richness and parasite diversity in relation to sex and period of collection of hosts.
The community descriptors of parasitism that were calculated included the following: mean parasite abundance, mean parasite richness, Shannon-Wiener's parasite diversity (H') and Hill's equivalence modified (E). The DivEs software was used to calculate these indexes (Rodrigues 2019). Berger-Parker's numerical dominance (Dbp), as based on parasite infracommunities, was calculated from the respective abundances (Magurran 1988). The level of significance for all tests was p<0.05 (Zar 1996).

RESULTS
The total number of 108 P. piraya specimens were collected in Três Marias reservoir: 56 of them were male and recorded mean total length 18.1±4.4 cm (11.1 to 30.5 cm) and mean weight 169.8±180.1 g (21.0 to 835.0 g), whereas 52 were female and recorded mean total length 21.4±6.0 cm (13.0 to 34.0 cm) and mean weight 323.4±308.4 g (40.0 to 1225.0 g). In the dry season, 53 piranha specimens were collected: 31 were male and recorded mean total length 16.8±5.1 cm (11.1 to 30.5 cm) and mean weight 160.8±224.5 g (21.0 to 835.0 g); whereas 22 were female and recorded mean total length 18.8±6.2 cm (13.2 to 32.0 cm) and mean weight 244.2±320.0 g (48.9 to 967.0 g). In the rainy season, 55 piranha specimens were collected: 25 of them were male and recorded mean total length 19.5±3.0 cm (14.5 to 23.6 cm) and mean weight 181±105.3 g (46 to 390 g), whereas 30 were female and recorded mean total length 23.2±5.2 cm (13.0 to 34.0 cm) and mean weight 381.5±291.3 cm (40.0 to 1225.0g).
Female specimens were larger and heavier than males both in the integral collection (t= 3.25, p= 0.001; t= 3.19, p= 0.002, respectively) and in the rainy season (t= 3.09, p= 0.003; t= 3.26, p= 0.002, respectively), whereas the dry season did not present difference between the size and weight of male and female hosts (t=1.27, p= 0.20; t= 1.11, p= 0.27, respectively).

Component endoparasite community
Not all fish in the analyzed sample were parasitized (n= 6). Ten metazoan helminth species were found; which totaled 737 specimens and 6.8 parasites per fish, on average.
Nematoda was the most representative taxon, since it presented 94.4% parasitism rate (707 specimens) and 6.93 parasites per fish, on average. They presenting the largest number of parasite specimens in the two collection periods analyzed in separate, as well as in the whole collection period. Eucestoda and Digenea specimens were the least representative groups in the endoparasitic community found in P. piraya fish living in upper São Francisco river -their parasitism rate was lower than 10% (Table I).
The endoparasite community found in P. piraya revealed one central species (P. (S.) inopinatus), one secondary species (Hysterothylacium sp.) and eight satellite species, based on their importance values (Table I).
Simpson's dominance index (C) indicated that was dominance in the endoparasitic community of P. piraya (C= 0.27), with P. (S.) inopinatus presenting higher frequency dominance value (Table II).
Components of the parasite community found in P. piraya have shown typical aggregated or superdispersed distribution pattern, even in C. fischeri, whose aggregation was not statistically significant (Table III). The parasite community found in P. piraya presented H'= 0.63, Dbp= 0.38 and E= 3.21; it recorded mean richness and mean abundance of 1.89±1.21 and 6.82±8.32, respectively.

Parasite infracommunities
The total number of 737 endoparasite specimens were collected in P. piraya; mean number of parasites per infracommunity was 6.82±8.32 and its amplitude reached 1-38 parasites per host.
Female P. piraya were significantly more parasitized by Hysterothylacium sp. larvae than males. Parasitic indices recorded for other parasite species analyzed in the current study were not influenced by host's sex (Tables V and  VI).
Parasitic indices recorded for C. sentinosa (abundance) and Hysterothylacium sp. (intensity and abundance) were higher in larger fish (positive correlation). Spiroxys sp., despite not show significant values, they presented negative correlation, which indicated higher incidence in smaller fish (Table VII). Both the diversity and richness of infracommunities were greater in larger fish (r s = 0.35, p= 0.0002, r s = 0.38, p<0.0001, respectively).
The total number of 53 piranha fish were collected during the dry season, whereas 55 were collected during the rainy season -their parasite indices are shown in Table VIII. Fish collected during the dry season have shown mean parasite diversity 0.11±0.16 (0-0.66) and mean parasite richness 1.28±0.88 (0-5 species). Fish collected during the rainy season have shown mean parasite diversity 0.28±0.19 (0-0.66) and mean parasite richness 2.49±1.20 (0-6 species). Both diversity and parasitic richness were significantly higher in the rainy season (U= 688.0, p<0.0001; U= 598.5, p<0.0001).
Procamallanus (S.) inopinatus intensity and abundance, as well as C. sentinosa and Hysterotyhlacium sp. prevalence and abundance, were positively correlated to the rainy season (Table IX). During this period, P. (S.) inopinatus abundance was significantly higher in female fish. In the dry period, P. (S.)      inopinatus prevalence and abundance, as well as Hysterothylacium sp. prevalence, were higher in female fish (Table X).

Moravec
The elevated position of P. piraya fish in the trophic web features this species as the final host of adult endoparasites found in the current study, such as P. (S.) inopinatus, C. fischeri and C. sentinosa. Specimens belonging to forage fish species Tetragonopterus chalceus Spix & Agassiz 1829 and Triportheus guentheri (Garman, 1890) living in Três Marias reservoir were considered intermediate hosts of C. fischeri by presents these larvae in their endoparasitic community (Albuquerque et al. 2016). The prey-predator relationship established between these forage fish and P. piraya, which is predatory and top carnivore, helps maintaining the life cycle of these nematodes in Três Marias reservoir.
Although fish species P. piraya is considered a top web predator, the five larval nematodes observed in the endoparasitic community found in this species have indicated that piranha fish can be intermediate or even paratenic hosts. According to Eiras (1994), nematodes use oligochaetes, crustaceans, insect larvae, mainly ephemeropterans, and fish as intermediate hosts.
Trindade & Jucá-Chagas (2008) have stated that although P. piraya fish are primarily piscivore, terrestrial insects can be found in their stomach content. This finding explains the presence of larval and adult nematodes in the endoparasitic community found in P. piraya. Likely final hosts of some of these nematodes, such as alligators, were seen feeding on P. nattereri in the Brazilian Pantanal region (Sazima & Machado 1990).
Female P. piraya fish were significantly more parasitized by Hysterothylacium sp. larvae than male individuals, likely when they were preparing for the reproduction season. According to Pavanelli et al. (1997), the stress experienced by fish during the reproduction season can make them susceptible to infections.
Descriptors such as parasitic richness and diversity are often associated with fish size and collection period (Guégan & Hugueny 1994, Tavares-Dias et al. 2014. Endoparasite diversity, richness and mean total abundance rates in the current study have significantly increased as the fish grew. The abundance of C. sentinosa was higher in larger hosts, as well as the intensity and abundance of Hysterothylacium sp. larvae. This outcome be explained by the cumulative effect of these parasites on their hosts. According to Neves et al. (2016), larvae who are not eliminated by hosts' immune system can remain viable for a long period-of-time until they reach their final hosts. Larger fish can eat greater amounts and diversity of food items; by doing so, they acquire intermediate and/or paratenic hosts of several parasite species and increase the likelihood of developing infections (Poulin 1995).
The transmission of the most prevalent endoparasite species was favored in the rainy season, since their ecological parasitic descriptors were higher at this time. According to Moravec (1998), the most important biotic factors in Nematoda's life cycle lie on the presence and density of intermediate hosts, as well as on the trophic relationship between them and their definitive hosts. According to López & Sampaio (2003), rainfall regime and temperature are the factors affecting the composition and abundance of zooplankton communities. In addition, the abundance of Copepoda, among other aquatic invertebrates, is often higher in the rainy season. A mix of these factors seems to influence the greater incidence of these endoparasites during the rainy season.
Proteocephalidae gen. sp., Contracaecum sp. and Hysterothylacium sp. -larval parasites found in P. piraya -are generalist because they parasitize several taxonomically unrelated fish species. Besides P. piraya, several fish species living in upper São Francisco river were infected by these larvae (Brasil-Sato 2003, São-Sabas & Brasil-Sato 2014, Santos-Clapp & Brasil-Sato 2014, Albuquerque et al. 2016, Vieira-Menezes et al. 2017, Almeida-Berto et al. 2018; thus, they acted as intermediate and/or paratenic hosts for these cestodes and nematodes, who would become adults when these infected fish were ingested by piscivorous birds or other final hosts.
Significant positive associations observed in pairs of adult endoparasites species C. fischeri -C. sentinosa, and in pairs of larval endoparasite species Hysterothylacium sp. -Spiroxys sp., may indicate that these associated species have the same ecological requirements and that there are probably frequent and common intermediate hosts for the associated species, as the carnivore host P. piraya, revealed in this study.
Some studies have reported some of these helminths parasitizing P. piraya living in upper São Francisco river. Brasil-Sato (2003)