Hemiurid and lecithasterid digenean trematodes and camallanid and cucullanid nematodes parasitizing flounders collected off the coast of Rio de Janeiro State, Brazil

Abstract A total of 132 flounder specimens (60 Paralichthys isosceles, 36 Paralichthys patagonicus and 36 Xystreurys rasile) were collected off the coast of the state of Rio de Janeiro, Brazil. The fish were measured, necropsied, and had their organs investigated for hemiurid and lecithasterid digenean trematodes and camallanid and cucullanid nematodes. Taxonomic identification of the parasites was based on morphological and morphometric characters and was conducted using bright-field and scanning electron microscopies. The trematodes Lecithochirium monticellii and Aponurus laguncula were found parasitizing P. isosceles, P. patagonicus and X. rasile while the nematodes Procamallanus (Spirocamallanus) halitrophus and Cucullanus bonaerensis were found parasitizing P. isosceles and X. rasile and P. isosceles, P. patagonicus and X. rasile, respectively. Parasite indices of prevalence, mean intensity, mean abundance, and range of infection, as well as infection site, were evaluated for each parasite species. This study allowed to evidence the first occurrence of P. patagonicus by L. monticellii; X. rasile by A. laguncula and P. (S.) halitrophus; and P. isosceles and P. patagonicus by C. bonaerensis in the Western South Atlantic Ocean.


Introduction
The flounder species Paralichthys isosceles Jordan, 1890, P. patagonicus Jordan, 1889 and Xystreurys rasile (Jordan, 1891) (Paralichthyidae) represent important primary fishery resources in the coastal waters of Brazil (Figueiredo & Menezes, 2000). According to Cerqueira et al. (1997), Massa et al. (2005) and Bernardes et al. (2005), the flounder fishery is referred to as "fine fishing" due to high commercial interest, meat quality and market price and because of broad sale in domestic and foreign markets.
Digenean trematodes present a certain degree of specificity in relation to their site of infection and their definitive host, although some species may infect different sites, such as adult individuals of species of the families Hemiuridae and Lecithasteridae when parasitizing the digestive system of fish (Eiras et al., 2006).
Parasitic nematodes are important pathogens associated with human and non-human animal health (Acha & Szyfres, 2003). Nematodes inhabit hosts of fresh, brackish and marine waters around the world, and some are known to be agents of serious fish diseases that cause considerable losses to the fishing industry (Klimpel & Palm, 2011). Adult nematodes of the families Camallanidae and Cucullanidae are commonly found parasitizing the intestine of various fish, their definitive hosts (Williams & Jones, 1994;Lanfranchi et al., 2004;Cárdenas & Lanfredi, 2005).
Studies, including morphological and ecological surveys, have reported some groups of helminths parasitizing the flounder species P. isosceles, P. patagonicus and X. rasile (Paralichthyidae) in Brazil (Felizardo et al., 2009a(Felizardo et al., , b, 2010(Felizardo et al., , 2011(Felizardo et al., , 2018Fonseca et al., 2012Fonseca et al., , 2016Fonseca et al., , 2019Knoff et al., 2012;Alarcos et al., 2016;Eiras et al., 2016;Justo et al., 2017) including species of trematodes and nematodes. The present paper continues these studies on flounders occurring off of the Brazilian coast. Thus, aiming to continue the study of these helminths was investigated the presence of hemiurid and lecithastherid digenean trematodes and camallanid and cucullanid nematodes parasitizing paralichthyid flounders from off the coast of the state of Rio de Janeiro, Brazil. These helminths were identified morphologically and morphometrically by bright-field and scanning electron microscopies, calculated their parasitic indices and determined their sites and ranges of infection.

Morphological identification and parasitic indices
Hemiurid species were identified using Looss (1908), lecithasterid species using Skrjabin & Guschanskaja (1955), camallanid species using Fusco & Overstreet (1978) and Cárdenas & Lanfredi (2005) and cucullanid species using Lanfranchi et al. (2004). The taxonomic classification of digenean trematodes was according to Gibson et al. (2002) while that of nematodes followed De Ley & Blaxter (2004). Samples were analyzed using an Olympus BX-41 brightfield microscope and images captured using a Canon Power Shot A640 digital camera coupled to a Zeiss Axiophot microscope using a Nomarski's differential interference contrast (DIC) apparatus. Illustrations were made using a drawing tube connected to the microscope. Measurements are provided in millimeters (mm) unless otherwise indicated, with averages in parentheses. Scanning electron microscopy (SEM) was used to elucidate topographic details of specimens of Cucullanus, with processing according to Lopes Torres et al. (2013). Samples for SEM were fixed in Karnovsky solution, dehydrated in an ethanol series (70-100%), CO 2 critical-point dried, coated in gold and examined and photographed using JEOL SM -25 SII scanning electron microscope under an acceleration voltage of 15 kV. The parasitic indices of prevalence (P), mean intensity (MI) and mean abundance (MA) were calculated according to Bush et al. (1997). Range of infection (RI) and sites of infection (SI) are also presented. Representative specimens were deposited in the Helminthological Collection of the Oswaldo Cruz Institute (CHIOC), FIOCRUZ, Rio de Janeiro, RJ, Brazil.

Results
A total of 1,127 parasite specimens were collected from the sampled flounders, with 783 digenean trematodes of two species of two families (398 Hemiuridae, 385 Lecithasteridae) and 344 nematodes of two species of two families (46 Camallanidae, 298 Cucullanidae). The total number of helminths per flounder species were: 401 from P. isosceles, 584 from P. patagonicus and 142 from X. rasile. The species taxonomic identifications follow.
Lecithasteridae Odhner, 1905Lecithasterinae Odhner, 1905 Aponurus Looss, 1907 Aponurus laguncula Looss, 1907 ( Figures 3 and 4)  Features observed in 31 specimens, 10 from P. isosceles, 13 from P. patagonicus and 8 from X. rasile: Body small, narrow, widest near posterior extremity. Tegument smooth. Pre-oral lobe small or not evident. Oral sucker subglobular, subterminal. Ventral sucker subglobular; immediately anterior to mid-body. Pharynx oval. Excretory system generally obscured by eggs, pore terminal, arms unite dorsal to oral sucker. Testes oval, oblique, in anterior hindbody. Seminal vesicle subglobular, in posterior forebody, near anterior margin of ventral sucker. Pars prostatica narrow, curved, with gland cells surrounding external ejaculatory duct. Sinus-sac oval, thin-walled. Genital pore medial. Ovary oval to sub-triangular, posterolateral to posterior testis and posterior to anterior testis. Seminal receptacle inconspicuous. Uterus in mature worms fills most of hindbody, also in median region dorsal to ventral sucker region and up to about middle of forebody, metraterm entering sinus sac ventrally joining male duct immediately at the beginning of sinus sac at the internal ejaculatory duct. Vitellaria paired, seven irregular follicles, one trilobed and one quadrilobed with follicles somewhat longer than wide, overlapping posterior portion of ovary. Eggs numerous, thin-shelled and operculate. Table 2.
Female: Body longer than that of males. Muscular vagina opens in vulva, a transversal ventral opening at midbody. Anus at posterior end with transversal opening. Tail rounded. Pair of phasmids located laterally near tip of tail and anus. Table 3.

Morphometrics shown in
Hosts: P. isosceles and X. rasile.
Infection sites: intestine (P. isosceles and X. rasile). Features observed in 40 specimens, 10 from P. isosceles, 20 from P. patagonicus and 10 from X. rasile: Body slender. Cuticle finely striated throughout. Lateral alae absent. Anterior end rounded, dorsoventrally expanded.     Cephalic extremity with usual features of Cucullanus, with two pairs of prominent outer papillae, a pair of amphids, and three pairs of small labial inner papillae. Mouth slit-like dorsoventrally, surrounded by collarette armed with numerous triangular denticles. Pseudobuccal cavity well developed with internal cuticular lining, esophagus long and narrow, expanded at both extremities, opening into intestine through small valve; anterior end wider than posterior end. Nerve ring surrounding esophagus at its second third. Deirids and excretory pore situated between the second half and the distal end of esophagus. Right deirid preequatorial, left postequatorial. Tail conical.
This study allowed to evidence the first occurrence of parasitism of P. patagonicus by L. monticellii; X. rasile by A. laguncula and P. (S.) halitrophus; and P. isosceles and P. patagonicus by C. bonaerensis in the Western South Atlantic Ocean.

Discussion
The morphology and morphometry of the specimens of L. monticellii collected in the present study were in accordance with the original description (Linton, 1940) and redescriptions (Skrjabin & Guschanskaja, 1955;Nasir & Díaz, 1971;Fernandes et al., 1985;França et al., 2020) of the species. Morphometrically, the present specimens were smaller than those collected from Trichiurus lepturus L. from off the coast of Cumaná, State of Sucre, Venezuela (Nasir & Díaz 1971), and from Trachurus lathami Nichols, 1920 and T. lepturus from off the coast of the state of Rio de Janeiro, Brazil (Fernandes et al., 1985;França et al., 2020), which can be considered intraspecific variation.
The morphology and morphometry of the specimens of A. laguncula collected in the present study were in accordance with the redescriptions of Manter (1947), Szidat (1961), Nahhas & Short (1965) and Fernandes et al. (1985). Although Bray & MacKenzie (1990) demonstrated the entrance of the metraterm at the base of the sinus sac in an illustration in their redescription of this species, they did not describe this in the text, which is provided by the present study as "metraterm entering sinus sac ventrally joining male duct immediately to the beginning of sinus sac to the internal ejaculatory duct".
The species A. laguncula was found parasitizing P. patagonicus in Argentinean waters by Szidat (1961) and in waters off Necochea, Argentina, by Alarcos & Timi (2012), which differs from the present study, which also found it parasitizing P. isosceles and X. rasile. The species A. laguncula was found in all three of the flounder species studied, which differs from Alarcos et al. (2016), who reported it only in P. isosceles from off the state of Rio de Janeiro, Brazil.  (Kohn et al., 2007;Fernandes et al., 2009;Cárdenas et al., 2012a, b;Eiras et al., 2016). Therefore, the list of hosts for A. laguncula is expanded to include the flounders studied in the present study. This is the first report of A. laguncula parasitizing P. isosceles and X. rasile.
The morphology and morphometry of the specimens of P. (S.) halitrophus collected in the present study were in accordance with redescription of the species using specimens collected from Syacium papillosum L. and Citharichthys macrops Dresel, 1889, from off the coast of the state of Rio de Janeiro, Brazil (Cárdenas & Lanfredi, 2005).
The morphology and morphometry of the specimens of C. bonaerensis collected in the present study were in accordance with the description of the species using specimens collected from Urophycis brasiliensis (Kaup, 1858) from off the coast of Mar del Plata, Argentina by Lanfranchi et al. (2004). (2012) found Cucullanus sp. and C. bonaerensis parasitizing X. rasile off the Argentine Coast, while the present study found only C. bonaerensis in the three paralichthyid hosts studied. Alarcos et al. (2016) reported Cucullanus sp. in P. isosceles from off the coast of the state of Rio de Janeiro, Brazil, which differs from the present study, which found only C. bonaerensis. Therefore, the present study expands the occurrence of this species in South America. This is the first report of C. bonaerensis parasitizing P. isosceles and P. patagonicus. Alarcos & Timi (2012) found gravid females of C. bonaerensis in X. rasile from off the Argentine Coast and suggested this species as their definitive host. In the present study, gravid females of this nematode were only found in P. patagonicus. Lanfranchi et al. (2004) did not find any gravid females of C. bonaerensis, which they attributed to seasonality in the life cycle of the parasites and/or host suitability.

Alarcos & Timi
The sites of infection found here were the same as reported by Alarcos & Timi (2012), stomach for hemiurid and lecithasterid digenean trematodes and intestine for camallanid and cucullanid nematodes.
Comparisons of the parasitic indices of the present study with those for the hemiurid and lecithasterid digeneans and camalanid and cucullanid nematodes collected from flounders in Argentine waters by Alarcos & Timi (2012) found that the latter had a lower prevalence (10.42%) and mean abundance (0.17) for L. microstomum in X. rasile; a lower prevalence (23.53%) and mean abundance (0.94) for A. laguncula in P. patagonicus; and a higher prevalence (39.58%) and lower mean abundance (0.73) for C. bonaerensis in X. rasile.
Comparisons with the parasitic indices of helminths parasitizing P. isosceles off of the state of Rio de Janeiro, Brazil, reported by Alarcos et al. (2016), revealed that these authors found lower prevalences (32.80%, 5.30%,11.10%) and mean abundances (0.95, 0.10, 0.17) for L. microstomum, A. laguncula and P. halitrophus (respectively). These differences in parasitic indices could be related to the greater number of collection locations of the present study since, beyond the municipalities of Cabo Frio and Niterói, the present study also collected in the municipalities of Rio de Janeiro and Angra dos Reis. The differences could also be due to various environmental and seasonal factors intrinsic to the collections themselves.
Differences observed in helminthofauna composition and parasitic indices for the same hosts collected in Necochea, Argentina, and Rio de Janeiro, Brazil, could be correlated with the different ecoregions of these areas. According to Spalding et al. (2007), marine ecoregions comprise relatively homogeneous sets of species that are clearly distinct from adjacent systems. The dominant biogeographic agents that define ecoregions vary from location to location but may include physico-chemical and biological factors. Thereby, features observed within ecoregions can influence their fish parasite communities and, thus, explain the parasite compositions found for flounder species from waters off Argentina by Alarcos & Timi (2012) and those found for the same flounder species from waters off of the state of Rio de Janeiro, Brazil, by the present study and for that found for P. isosceles by Alarcos et al. (2016).