Community of metazoan parasites of the cockfish Callorhinchus callorynchus (Linnaeus, 1758) (Chimaeriformes: Callorhinchidae) from artisanal fishing in Pisco, Ica, Peru

: There is an extensive lack of information on the parasitic fauna accompanying cartilaginous fish in Peru. The objective of this work was to identify the community of parasite metazoans of the cockfish Callorhinchus callorynchus (Linnaeus, 1758) (Chimaeriformes: Callorhinchidae) from artisanal fishing in San Andrés, Pisco, Ica, Peru. Thirty-one specimens of C. callorynchus were collected between June 2018 and October 2019. Following nec-ropsy, six species of parasites were obtained: Gyrocotyle rugosa Diesing, 1850 (Cestoda), Callorhynchocotyle callo- rhynchi (Manter, 1955) (Monogenea), Callorhynchicola branchialis Brinkmann, 1952 (Monogenea), Branchellion lobata Moore, 1952 (Hirudinea), Prokroyeria meridionalis (Ramírez, 1975) (Copepoda) and an unidentified spe- cies of the Anisakidae family (Nematoda). The G. rugosa tapeworm was the most prevalent parasite (77.42%), followed by the monogenean Cle. callorhynchi (54.84%) and finally the copepod P. meridionalis (16.13%). The total body length and sex of Ca. callorynchus showed no association with respect to the parasitological indices of prevalence, intensity, and mean abundance. Cle. callorhynchi and Cla. branchialis were registered for the second time in the same host, but for the first time in the department of Ica. However, the present study constitutes the first geographic record of G. rugosa , B. lobata , and P. meridionalis , and a new host for an unidentified species of the Anisakidae family in cockfish in Peru.


INTRODUCTION
The number of studies on parasitic metazoan communities and estimation of the relative biodiversity of parasites to the search relationships parasite-host in marine bony fishes in Peru have increased in recent years (Céspedes-Chombo et al., 2017;Chero et al., 2019;Ferré-Alcántara et al., 2019;Londoñe-Bailón et al., 2020;Minaya et al., 2020a,b). Currently, there is a remarkable relevance in studies on the biodiversity of the parasitic fauna because they are important for maintaining the balance in the ecosystem by regulating the density and abundance of their hosts, structuring food chains (Poulin et al., 2016). Unfortunately, the same cannot be said for parasitological studies in elasmobranch fish, being one of the least studied lines of research within ichthyoparasitology in Peru, despite being a country with a wide diversity of cartilaginous fish (Cornejo et al., 2015).
Among the 1,188 species of cartilaginous fish worldwide, holocephali or chimera fish constitute 49 species (Carrier et al., 2004;Weigmann, 2016). In the last five years, multiple aspects of parasitology in holocephalan fishes have been studied worldwide, including studies on the structure of the parasite community as a tool to predict the population structure of its host, the discovery of new parasite species by molecular and morphological analysis, parasite-host phylogenetic relationships and the lack of geographic patterns in the distribution of their species, and their utility as bioindicators of heavy metal contamination, among others (Morris et al., 2016;Derouiche et al., 2019;Morris et al., 2019;Bray et al., 2020;Barčák et al., 2021).
The cockfish Callorhinchus callorynchus (Linnaeus, 1758) (Chimaeriformes: Callorhinchidae) belongs to the group of cartilaginous fish and is representative of the holocephalans associated with the marine coasts. It is distributed only in the southern hemisphere, being found on the Southeast Pacific side, from northern Peru including the coast of Ecuador, to the Strait of Magellan. On the Atlantic side, it is distributed along the coast of Brazil, Uruguay, and Argentina, up to the Beagle Channel. Ca. callorynchus is a demersal marine fish, with a depth range of 10 to 116 m. It is usually found in sandy and muddy substrates; it is oviparous and according to the International Union for Conservation of Nature its conservation status is vulnerable. It is caught as bycatch and is consumed by the local population on the marine coasts of South America (Chirichigno & Cornejo, 2001;Cousseau & Perrotta, 2000;López et al., 2000;Swing & Beárez, 2006;Bernasconi et al., 2015;Weigmann, 2016;Finucci & Cuevas, 2020;Froese & Pauly, 2021).
This species is economically important in the artisanal fishery of the town of San Andrés, Pisco, Ica, because its commercialization and human consumption is almost constant, despite being caught as bycatch. It is consumed in the form of ceviche, fish stew, and fried. Considering the commercialization of this species, there is a need to investigate the parasitology of cockfish from an ecological aspect, seeing the diversity of species of parasites described in this host in studies carried out in Peru compared to other countries such as Chile and Argentina (Luque & Iannacone, 1991;Di Giácomo & Perier, 1994;Tantaleán & Huiza, 1994;Di Giácomo & Perier, 1996;Aedo et al., 2010;Luque et al., 2016;Martínez et al., 2016;Carvalho-Azevedo et al., 2021).
Therefore, the present study aimed to determine the characteristics of the community of parasite metazoans of the cockfish Ca. callorynchus from the artisanal fishing landing of San Andrés, Pisco, Ica, Peru, and evaluate the ecological aspects of the host-parasite and observe possible interactions among these characteristics.

MATERIALS AND METHODS
Collection and execution place. Thirty-one specimens of Ca. callorynchus were obtained from Lagunillas (Paracas Bay) and Laguna Grande (Independencia Bay) through the artisanal fishing landing of San Andrés, Province of Pisco, Department of Ica (13°44'01" S; 76°13'30" W), Peru from June 2018 to October 2019. The fish were frozen and transported to the laboratory in Lima, Peru.

Data processing and statistical analysis.
The ecological parasitological indices of prevalence (P), mean abundance (MA), and mean intensity (MI) were estimated (Bush et al., 1997;Bautista-Hernández et al., 2015). The specific importance index (I) calculated as the importance of each parasitic species in the ecological assembly was used. I = Prevalence + (mean abundance [numerical or volumetric] x 100) in order to obtain an integrated infection index for both ecological descriptors (Iannacone et al., 2012). The type of strategy of each parasitic species was evaluated according to the percentage of P%. Species with a prevalence higher than 45% were classified as "core" species, while those with a prevalence between 10% -45% were defined as "secondary" species, and species with a prevalence lower than 10% were classified as "satellite" species (Bush & Holmes, 1986).
The following indices were used: dispersion index (dI), Poulin discrepancy index (PDI) and K of the negative binomial equation with its respective Chi square value (X 2 ) to determine the type of distribution and degree of aggregation (Bego & Von Zuben, 2010). The Pearson correlation coefficient (r) was used to determine the relationship of the total body length (TBL) of the host fish with the MA and MI of each parasitic species. The Spearman correlation coefficient (r s ) was applied to evaluate the association between TBL versus the prevalence of infestation, previously transforming the P% values to square root of arcsine. In all cases, the normality of the data was verified using the Kolmogorov-Smirnov test with the modification of Lillierfors and the homoscedasticity of variances based on the Levene test (Zar, 2014). The Student's t test was used to compare the MA and MI of each parasite and the sex of the host. The analysis of parasites in relation to TBL and host sex was performed only for species with a prevalence greater than 10% (Esch et al., 1990). 2 x 2 contingency tables were used to calculate the degree of association between the sex of the host and P% of each parasite by means of X 2 . Yates' correction was used to correct for possible X 2 errors with respect to the P% of parasites. For the determination of the descriptive and inferential statistics, a p value of 0.05 was considered significant.
The following alpha dI were calculated: richness: species richness and Margalef, and the richness estimator: Chao-1; equity: Shannon-Wiener and Equitability; and dominance: Berger-Parker (Bego & Von Zuben, 2010) for the total parasitic community component, and for males and females. A dendrogram with the Ward method and Euclidean index was constructed to compare the similarity between the parasites of the 31 specimens of Ca. callorynchus studied. For the determination of the dI, the statistical package PAST-Palaeontological Statistics ver.4.09 was used, and for the descriptive and inferential statistics the statistical package Quantitative Parasitology 3.0 was used (Reiczigel et al., 2019).
Finally, the representative specimens of the metazoan parasite species collected were deposited in the collection of parasitic helminths and related invertebrates belonging to the Zoological Collection of the Natural History Museum of the Universidad Nacional Federico Villarreal (UNFV) under the codes: MUFV: ZOO-HPIA: 177 -182.

RESULTS
The Callorhinchus callorynchus population consisted of 31 specimens distributed in 13 females (TBL = 64.12 ± 12.85 cm) and 18 males (TBL = 55.70 ± 9.30 cm). The female population had a larger TBL than the males (t = 2.02, p <0.05). At least one parasite specimen was found in 90.32% (n = 28) of the fish. A total of 263 specimens belonging to 6 parasites species were found and identified (Table 1).
The three parasites with the highest prevalence of infestation and which had a P%> 10% were Gyrocotyle rugosa Diesing, 1850 (Cestoda), followed by Callorhynchocotyle callorhynchi (Manter, 1955) (Monogenea) and Prokroyeria meridionalis (Ramírez, 1975) (Copepoda). Similarly, the parasites with the highest MA and MI of in-festation were Cle. callorhynchi, followed by G. rugosa and P. meridionalis. Regarding the type of strategy, only G. rugosa and Cle. callorhynchi were considered as core species in the cockfish parasite community (Table 1). The PDI for Cle. callorhynchi and P. meridionalis showed values close to 1, and thus, the type of distribution was considered aggregate or contagious (Table 1).
The correlation coefficients of the most prevalent parasites (G. rugosa, Cle. callorhynchi, and P. meridionalis) showed no degree of association between the ecological variables of the parasites and the morphological aspects of the host (TBL and sex). There was also no significant association between the sex of Ca. callorynchus and the ecological variables. The Student's t test showed no differences between males and females of Ca. callorynchus (Table 2). Likewise, for Cle. callorhynchi the homogeneity of variances was not fulfilled, and therefore, the Mann Whitney U test was used.
According to the alpha dIs, higher values were observed in richness, number of individuals, and dominance (Berger-Parker) in the population of females compared to males. On the other hand, equitability and diversity (Shannon-Wiener) was biased in favor of the male population of Ca. callorynchus. The sampling effort was optimal, which was corroborated with the Chao-1 richness estimator (Table 3).
A dendrogram was constructed based on Ward method and the Euclidean index to determine the similarity of the Ca. callorynchus parasites.
The most similar were Anisakidae gen. et sp. indet, Callorhynchicola branchialis Brinkmann, 1952 and Branchellion lobata Moore, 1952 based on the shortest distance presented. The monogeneous Cle. callorhynchi, the most abundant of all the parasites collected, presented a greater distance in the dendrogram in relation to the rest of the parasitic metazoans, followed by the cestode G. rugosa and the copepod P. meridionalis (Fig. 1). Finally, in the bibliographic search for reports of parasites in Ca. callorynchus, it was found that 13 species of metazoan parasites and three indeterminate species had been described in this host from 1927 to the present study. All reports are restricted to the southeast of the Pacific (Peru and Chile) and southwest of the Atlantic of the South American continent (Brazil, Uruguay, and Argentina). The finding of the parasites G. rugosa, B. lobata, and P. meridionalis constitutes new geographical records in Peru. Likewise, the unidentified nematode appears as a new record in the host of the cockfish (Table 4).

DISCUSSION
In the present study, the community of metazoan parasites of Callorhinchus callorynchus was determined, thereby increasing the geo-  graphic distribution of some parasites and range of hosts for others. It is known that the vast majority of these parasites are primitive organisms in their lineages, they infest almost exclusively chimeras and have established a co-evolutionary dependence with their hosts (Caira et al., 2012). Previous records have shown that chimeras are usually parasitized by cestodes of the Gyrocotylidae family in the spiral valve, a site that is specific for this helminth (Alves et al., 2017). The genus Gyrocotyle Diesing, 1850 is indicated as a parasitic cestodaria restricted only to holocephalan fishes of the genus Callorhinchus (Bray et al., 2020;Barčák et al., 2021). Gyrocotyle is a group reduced to 10 valid species (Barčák et al., 2021;WoRMS, 2021). In Peru, only Gyrocotyle maxima MacDonagh, 1927 was registered parasitizing Ca. callorynchus from Lima and La Libertad (Luque et al., 2016). The present study constitutes the first record of G. rugosa from Peru. This species has been previously recorded on the marine coasts of Argentina, Chile, South Africa, and New Zealand (Reed, 2015;Barčák et al., 2021).
According to parasitological parameters, the parasite with the highest prevalence was Gyrocotyle rugosa (P% = 77.4%), making it an important and core species in the communi-  (Morris et al., 2019). In the present study, no more than one Gyrocotyle species was found in the studied host. It has been argued that most of the cockfish studied are parasitized by one or two specimens per fish and by a single (rarely two) Gyrocotyle species, which is why they are considered oioxenic parasites (Barčák et al., 2021). It is suggested that Ca. callorynchus is infected with the G. rugosa cestode because of its benthic diet mainly based on bivalves, brachyurans, gastropods, isopods, and anomurans (Roman et al., 2020), which are invertebrates that have previously been reported as intermediate hosts of several species of helminths. Gyrocotyle seems to remain within the digestive tract of its hosts for a long time and possibly throughout the life of the holocephalans, which could explain why the Gyrocotyle species were the most prevalent in south american Ca. callorynchus as well as those studied in South Africa (Halvorsen & Williams, 1968;Williams et al., 1987).
Although G. rugosa is a species with a high P%, the degree of aggregation was the lowest among the most prevalent parasites, probably due to the low number of parasitic individuals per fish. Nonetheless, the distribution of this parasite encompassed the majority of holocephalans evaluated.
On the other hand, the monogenean Callorhynchocotyle callorhynchi presented the highest value of MA and MI in fish from Peru (MA = 6.68; MI = 12.18) which was also simi-lar in C. capensis from South Africa (MA = 1.55; MI = 4.79) (Morris et al., 2019). This is due to the greater number of specimens per host, which varied from 1 to 61 individuals of Cle. callorhynchi for each cockfish. It is known that the MI index is higher when a population of parasites is high in a small group of hosts, which is why it is argued that the species Cle. callorhynchi is characterized by presenting this high value in the host populations.
Many studies on cockfish carried out in Peru have registered the monogeneans Callorhynchocotyle marplatensis Suriano & Incorvaia, 1982(Luque & Iannacone, 1991Tantaleán & Huiza, 1994), Cla. branchialis (Martínez et al., 2016), and Cle. callorhynchi (Carvalho-Azevedo et al., 2021) in the gills of cockfish, all from the town of Chorrillos, Lima. In this study, Cle. callorhynchi and Cla. branchialis were registered for the second time in the same host but were registered for the first time in the department of Ica. In the mentioned surveys, both species of monogenean were evaluated qualitatively and descriptively, and therefore, this is the first determination of the quantitative nature of this population of parasites in Peru.
The third species considered important in this community of parasites was the population of Prokroyeria meridionalis, which had a secondary category due to the prevalence presented (P% = 16.13%). This host-parasite association has been previously described in Argentina and Chile and now for the first time in Peru (Ramírez, 1975;Castro & Baeza, 1984;Fernández et al., 1986;Deets, 1987;Castro-Romero et al., 2016). The   Rego et al. (1974), Alves et al. (2017) Fernández et al. (1986, Alves et al. (2017) Tantaleán (1991, Tantaleán & Huiza (1994), Alves et al. (2017) Szidat (1968 ASPIDOGASTREA Multicalyx elegans ----Chile Fernández et al. (1986) Rugogaster callorhinchi Rectal gland Brasil Amato & Pereira (1995) Rugogaster hydrolagi Rectal gland Brasil Amato & Pereira (1995) COPEPODA MI of infestation of this species was the second highest (MI = 2.4 ± 0.46) recorded in this study, similar to that observed in G. rugosa. Likewise, it was the species with the highest degree of aggregation (DOA = 0.85). Branchellion Savigny, 1822 is a genus that includes blood-sucking leeches of strictly marine hosts, with a preference for batoid fish to which they adhere to the surface and / or body orifices (Rohde, 2005;Caira et al., 2012). In the Eastern Pacific, the species B. lobata and B. callorhynchus Szidat, 1972, both in Chile, are qualitatively reported (Szidat, 1972;Ringuelet, 1985;Fernández et al., 1986). In the present study, only one individual of B. lobata was found in the anal orifice of a cockfish, making it difficult to determine the behavior of this species in Ca. callorynchus.
In our study, no correlation was observed between the TBL of the fish and the P%, MA, and MI of the parasites Gyrocotyle rugosa, Cle. callorhynchi, and Prokroyeria meridionalis. In contrast, Morris et al. (2019) found a correlation between the abundance of Cle. callorhynchi and the TBL and weight of C. capensis. Additionally, these same authors found a significant positive relationship between the weight of C. capensis and the abundance of G. rugosa. The findings of Morris et al. (2019) suggest that smaller fish contain a lower abundance of parasites compared to larger fish. This idea is reinforced by Poulin (2011) who indicated that the largest hosts can provide a greater supply of nutrients to the parasites and, consequently, they would be the most susceptible to a greater parasite diversity and load. In our case, the lack of association between the parameters evaluated indicates that other factors may influence the population dynamics of the parasites, such as host infection at an early age. Halvorsen & Williams (1968) speculated that Gyrocotyle infections begin when young hosts begin to feed, which was also observed in this study as fish with a shorter total length (33.1 cm) presented a moderately elevated parasitic infection indicating that the infection begins in the early stages of life.
According to the alpha dIs, the Berger Parker dominance index showed low values of parasites found in male fish and high values in females (0.46 and 0.88, respectively) and consequently, a greater diversity in the population of males. Iannacone et al. (2012) argue that the selection of parasites by either sex of the host fish could be attributed to differences in the ecological relationships (habitat, behavior, and feeding) of males and females (Minaya et al., 2020c).
Regarding the Chao-1 richness estimator, the number of parasitic species present in the sampled Ca. callorhynchus, in both males and females and the total population (males + females) showed a richness similar to that estimated by this index. Therefore, the sampling effort was optimal for the 31 specimens collected.
Addressing the historical aspects in the parasitological studies in Ca. callorynchus, Fernández et al. (1986) found the largest number of species in a single population of cockfish from central-southern Chile to date, describing nine species of parasites (Table 4). Of these, six corresponded to phylogenetic type parasites conditioned by historical and zoogeographic factors: Cla. branchialis, Cle. callorhynchi, Multicalyx elegans (Olsson, 1869), G. rugosa, G. maxima, and P. meridionalis. The other three species corresponded to ecologically-acquired parasites: Branchellion callorhynchus, Caligus teres Wilson, 1905 and Ceratothoa sp.

CONCLUSION
The three parasites with the highest prevalence of infestation were Gyrocotyle rugosa (Cestoda), followed by Callorhynchocotyle callorhynchi (Monogenea) and Prokroyeria meridionalis (Copepoda). Similarly, the parasites with the highest MA and MI of infestation were Cle. callorhynchi, followed by G. rugosa and P. meridionalis. Regarding the type of strategy, only G. rugosa and Cle. callorhynchi were considered as core species in the cockfish parasite community. The sampling effort was optimal, which was corroborated with the Chao-1 richness estimator. This work provides the first extensive quantitative analysis of the parasitic community in the cockfish Ca. callorynchus in Peru, as well as three new geographic records for G. rugosa, P. meridionalis, and B. lobata in Peru and a new record for a host of a nematode infesting Ca. callorynchus.

ACKNOWLEDGMENTS
We thank the DPA retailers José Olaya de San Andrés and fishermen Wilkins Poves, Germán Avalos, and María del Pilar Saldaña. We are also grateful to Oscar Méndez for his contributions about monogeneans p that affect elasmobranchs, Luis Ñacari, for providing scientific articles and to Obla Paliza for providing relevant scientific information.