Aporocotyle mariachristinae n. sp., and A. ymakara Villalba & Fernández, 1986 (Digenea: Aporocotylidae) of the pink cusk-eel, Genypterus blacodes (Ophidiiformes: Ophidiidae) from Patagonia, Argentina

Aporocotyle mariachristinae n. sp. and A. ymakara Villalba & Fernández, 1986 were collected from the bulbus arteriosus and ventral aorta of pink cusk-eels, Genypterus blacodes (Forster, 1801) from Patagonia, Argentina. A. mariachristinae n. sp. can be distinguished from all the species of Aporocotyle by the asymmetrical extension of posterior caeca (right posterior caecum longer, terminating at the area between mid-level of ovary and posterior body end; left posterior caecum shorter, terminating at the area between mid-level of cirrus sac and posterior to reproductive organs), the distribution of spines along the ventro-lateral body margins and the number of testes. The new species clearly differs from A. ymakara, from the same host species, in the esophagus / body length ratio, the absence of distal loops at caeca, the anterior caeca / posterior caeca length ratio, and the number of testes. Additionally, in A. ymakara the left posterior caecum may be longer than right posterior caecum, while in the new species left posterior caecum is always shorter. The specimen of A. ymakara collected from Argentina is also described. We also provide observations of the distribution of spines in different species of Aporocotyle, including new specimens of A. argentinensis Smith, 1969 from Merluccius hubbsi Marini, 1933. Molecular sequence data obtained from partial 18S and 28S rDNA regions were compared between the new species and other two species of Aporocotyle (A. argentinensis and A. spinosicanalis Williams, 1958). This is a new locality record for A. ymakara, extending the known geographical distribution for this species from Chile to Argentina, and the first report of two species of Aporocotyle in the same host species and locality.

During a parasitological survey of teleosts collected from the Argentine Patagonian shelf, some specimens of Aporocotyle spp. were found in the bulbus arteriosus and ventral aorta of pink cusk-eels G. blacodes. One single specimen was identified as A. ymakara, previously found in the same host from the coast of Chile (Villalba & Fernández, 1986b). The rest of the specimens clearly differed from all the known species of Aporocotyle. The aim of present study is to describe the new species of Aporocotyle and the specimen of A. ymakara from Argentina.

MORPHOLOGICAL DESCRIPTION
15 blood flukes from 13 fish, fixed in 70 % ethanol (11 parasites from fresh fish and 4 from thawed fish), were used for morphological study, stained with iron acetocarmine (n = 13) or alum carmine (n = 2), dehydrated in ethanol, cleared in dimethyl phthalate, and mounted in Canada balsam. Specimens were examined using a compound microscope equipped with bright field and differential interference contrast optics. Morphometric measurements were taken from drawings made with the aid of a drawing tube. Measurements are reported in micrometers and presented as the mean followed in parentheses by the range and the number of structures measured. The same procedure was followed for the specimens collected from the Argentinean hakes (4 worms stained with iron acetocarmine). Blood flukes were identified as A. argentinensis according to Smith (1969), and were used for morphological comparison with the specimens collected from G. blacodes. For morphological comparison with the species studied type specimens of the following Aporocotyle spp., deposited in the Zoology Museum of the University of Concepción (Concepción, Chile (ZMUC)) and the US National Parasite Collection (Beltsville, Maryland, USA (USNPC)), were examined and measured: A. keli -one paratype (ZMCU No. 7811); A. kuri -one paratype (ZMCU No. 7808); A. wilhelmi Villalba & Fernández, 1986 -three paratypes (USNPC No. 79462: MT24-19K, MT24-19L, and MT24-19N); and A. ymakara -five paratypes (ZMCU No. 7759 and USNPC No. 79463: MT24-20A, MT24-20B, MT24-20G, and MT24-20H). Additionally, 3 vouchers of A. spinosicanalis Williams, 1958, collected from the heart of Merluccius merluccius L. from the Western Mediterranean from the private collection of Dr. F. E. Montero, were studied. Measurements from previously described species were obtained from specimens or original descriptions (manuscripts or drawings), depending on the availability (see Table I). Ecological terms follow Bush et al. (1997) and Rósza et al. (2000). The prevalence, mean abundance and mean intensity are presented as the number followed in parentheses by the 95 % confidence intervals (CI). The 95 % CI for prevalence was set with Sterne's exact method (Reiczigel, 2003), while the 95 % CIs for the mean abundance and mean intensity were estimated with 20,000 bootstrap replications with the statistical software Quantitative Parasitology v. 3.0 (Reiczigel & Rósza, 2005 Smith, 1969Fernández & Durán, 1985Tantaleán & Martínez, 1990Kamegai et al., 1990Villalba & Fernández, 1986bVillalba & Fernández, 1986bHolmes, 1971Smith, 1967Parukhin, 1985Yamaguti, 1934Yamaguti, 1970Thulin, 1980Parukhin & Tkachuk, 1980Williams, 1958Ichihara, 1970 Villalba & Fernández,
• Comparison Among the 16 known species of Aporocotyle, A. mariachristinae is distinguished from the other species by its unique asymmetric arrangement of posterior caeca. This is the only species known of the genus with the right posterior caecum ending at the area between mid-level of ovary and posterior body end, and the left posterior caecum, always shorter, ending at the area between mid-level of cirrus sac and posterior to reproductive organs, never ending near posterior body end. Posterior caeca reach near to the posterior body end in most of the species of Aporocotyle, except for those in fishes of the Ophidiiformes (Genypterus spp. and H. armata, see Table I) in which posterior caeca never reach to ovary (Parukhin & Tkachuk, 1980;Villalba & Fernández, 1986b;Tantaleán & Martínez, 1990;Kamegai et al., 2002). A. mariachristinae would be the first species in an ophidiiform fish in which at least one posterior caecum extends beyond the anterior ovary end. The new species clearly differs from A. ymakara, the other species described from G. blacodes, by the higher number of testes (37-39 instead of 18-21), the absence of distal curves at caeca (see  Scale bars: Fig. 7: 500 μm; Fig. 8: 10 μm; Fig. 9 Fig. 1 in Villalba & Fernández, 1986b), the different esophagus/body length ratio (1:2.7-3.9 instead of 1:2.1-2.6), and the different anterior caeca/posterior caeca length ratio (1:2.6-4.7 instead of 1: 1.1-1.3). Additionally, in A. ymakara, the left posterior caecum can be equal or longer than the right posterior caecum (see Villalba & Fernández, 1986b), while in the new species is always shorter. The most similar species is A. keli, described from G. chilensis from the Southeast Pacific, with similar size and number of testes. This species differs from A. mariachristinae by the presence of a "muscular fold at posterior body region at level of genital atrium" (Villalba & Fernández, 1986b, see discussion for comments on this trait) together with the different host and the different posterior caeca arrangement.
Aporocotyle mariachristinae also differ from A. argentinensis, A. australis, A. margolisi, A. nototheniae, A. orientalis, A. pacifica, A. smithi, A. spinosicanalis, A. theragrae, and A. wilhelmi, in which the clusters of spines can join ventrally at sagittal axis (Fig. 10). Other specific differences with the other Aporocotyle species are reported in Table I Host size: total length, 51.2 cm.

• Remarks
The morphological traits of the specimen collected from Argentina coincide with those described by Villalba & Fernández (1986b) for A. ymakara from Chile: body size, esophagus/body length ratio, number of testes, and distribution of spines along the body (see Table I). The extension of the caeca showed a slight variability in specimens of both localities. Right posterior caecum ends before cirrus sac (three paratypes), or at cirrus sac level (two paratypes and the specimen from Argentina). Left posterior caecum ends at last testis level in all specimens (Chilean and Argentinean). Villalba & Fernández (1986b) reported that the distal end of caeca of some specimens was curved, however this curvature was not observed in the paratypes or in the Argentine specimen analysed in present study. The ratio between anterior caeca length/posterior caeca length of A. ymakara was 1:2.8-3.5 according to Villalba & Fernández (1986b), however revision of paratypes from Chile revealed that the ratio was 1:1.1-1.3.

DISCUSSION
T he most used morphological traits to differentiate species of the genus Aporocotyle are body shape, distribution of spines along the body, esophagus/body length ratio, anterior caeca/posterior caeca length ratio, number of testes, shape of cirrus sac, shape of ovary, presence of a "muscular fold" at level of genital atrium, and extension of posterior caeca (Smith, 1967(Smith, , 1969Holmes, 1971;Parukhin & Tkachuk, 1980;Villalba & Fernández 1986b). The distribution of spines along the body has been commonly used to separate species of Aporocotyle (see Smith, 1967Smith, , 1969Ichihara, 1970;Holmes, 1971). From the species examined in this study, two different patterns of distribution of spines have been observed: (i) in A. argentinensis, A. spinosicanalis, and A. wilhelmi, the cluster of spines are arranged along the ventromarginal areas of the body joining at sagittal axis from posterior margin of mouth to caeca bifurcation (Fig.  10); and (ii) in specimens of A. keli, A. mariachristinae, and A. ymakara, spines are arranged along the ventro-marginal area of the body, never joining at sagittal axis (Figs 11, 12). The spine arrangement of the paratype of A. kuri was in poor conditions and was unable to be described. The first type of spine arrangement is similar to those of A. argentinensis, A. australis, A. margolisi, A. simplex, A. spinosicanalis, A. theragrae, and A. wilhelmi (Williams, 1958;Smith, 1967Smith, , 1969Ichihara, 1970;Thulin, 1980;Fernández & Durán, 1985;Villalba & Fernández, 1986a), while the second type of spine arrangement is similar to those described for A. garciai, A. keli, A. kuri, A. macfarlani, and A. ymakara (Holmes, 1971;Villalba & Fernández, 1986b;Tantaleán & Martínez, 1990). A third pattern of distribution of spines has been described in A. orientalis, A. pacifica, A. smithi, and A. nototheniae where the full body surface is covered with spines (Yamaguti, 1934(Yamaguti, , 1970Parukhin & Tkachuk, 1980;Parukhin, 1985). However, this trait should be revised as the description of the distribution of spines in these species is not sufficiently detailed. The presence of a "muscular fold" was proposed by Villalba & Fernández (1986b) as a morphological character to differentiate A. kuri and A. keli from other species. These authors described this structure as "a muscular fold at posterior body region at level of genital atrium" ("un repliegue muscular en la región posterior a nivel del atrio genital"). Villalba & Fernández (1986b) also reported that in these species, due to this fold, the region posterior to genital atrium was sinistrally bent, oblique to perpendicular to longitudinal body axis. The authors did not provide more information or drawings on what kind of musculature existed within this fold. In Figures 2 and 3 of Villalba & Fernández (1986b) (A. kuri and A. keli, respectively) a marked lateral body depression at level of genital atrium can only be noticed. Thereafter, Tantaleán & Martínez (1990) again referred to the absence of this trait to describe A. garciai. Moreover, Kamegai et al. (2002) mistranslated the trait "repliegue muscular" as "muscular sphincter at female pore" in their description of new specimens of A. garciai from Japan. We did not observed a body depression or a muscular fold at level of genital atrium, or a curvature of the posterior region in the paratypes of A. kuri and A. keli examined in this study. Therefore this character does not appear to be recommendable for these species' diagnosis. Species of Aporocotyle have been classified in two groups according to the extension of posterior caeca (Villalba & Fernández, 1986b): (i) species of Aporoco-Original contribution Parasite, 2012, 19, 319-330 tyle described from fishes of the orders Gadiformes, Perciformes, Pleuronectiformes and Scorpaeniformes which have posterior caeca ending near the posterior body end (Fig. 10) (Yamaguti, 1934(Yamaguti, , 1970Williams, 1958;Smith, 1967Smith, , 1969Ichihara, 1970;Holmes, 1971;Thulin, 1980;Fernández & Durán, 1985;Parukhin, 1985;Villalba & Fernández, 1986a); and (ii) species of Aporocotyle described from ophidiid fishes, with posterior caeca never ending near the posterior body end and never reaching to the level of the ovary (Fig.  12) (Parukhin & Tkachuk, 1980;Villalba & Fernández, 1986b;Tantaleán & Martínez, 1990). According to this classification, A. mariachristinae could be considered to belong to a third intermediate group with at least one posterior caecum reaching to the area from mid-level of ovary to posterior body end (Fig. 11). Unfortunately, the existence of different phylogenetic groups of Aporocotyle species could not be confirmed in present study with molecular analyses as only three sequences exist (two of them provided in this study). We encourage authors to provide more molecular data on aporocotylid genera to explore phylogenetic relationships. Currently, there are six species of Aporocotyle described from four species of fishes from the genus Genypterus (see Parukhin & Tkachuk, 1980;Villalba & Fernández, 1986b;Tantaleán & Martínez, 1990;present study). To our knowledge, this is the only fish genus in which two different species of Aporocotyle infect the same host species. Moreover, A. mariachristinae and A. ymakara could not be the only two Aporocotyle species infecting the same host species: although the specific identity of the definitive host for A. garciai is unknown (reported as Genypterus sp. in Tantaleán & Martínez, 1990), G. chilensis or G. maculatus must be the type host as they are the only species of Genypterus distributed along the Peruvian coast (Nielsen et al., 1999), and both fish species harbour other Aporocotyle species (A. keli and A. kuri respectively, see Table I). More than one blood fluke species of the same genus in the same host have been previously reported: i.e., Cardicola Short, 1953(see Nolan & Cribb, 2006) and Paradeontacylix McIntosh, 1934(see Ogawa & Egusa, 1986Repullés-Albelda et al., 2008).