New species of Urocleidoides (Monogenoidea: Dactylogyridae) from the gills of two species of Anostomidae from the Brazilian Amazon

gills marcosidney2012@hotmail.com Abstract Three species (2 new) of Urocleidoides are described and/or reported from the gills of Schizodon fasciatus and Laemolyta proxima (Anostomidae) from the Jari River in the eastern Brazilian Amazon. Urocleidoides jariensis n. sp. presents a sclerotized, tubular, spiral male copulatory organ (MCO) with one counterclockwise coil, a circular sclerotized tandem brim associated with the base of the MCO; a heavily sclerotized, funnel-shaped vaginal vestibule; and a broadly V-shaped ventral bar with anteromedial constriction and enlarged ends. Urocleidoides ramentacuminatus n. sp. has a sclerotized, tubular, spiral MCO with one counterclockwise coil; an accessory piece with a hook-shaped distal portion; and a dorsal anchor with a short, straight shaft; anchor point with ornamentation as sclerotized shredded filaments. Urocleidoides paradoxus is reported for the first time parasitizing S. fasciatus


Introduction
Neotropical characiforms fish of the Anostomidae family have wide distribution in the watersheds of South and Central America (Nelson et al., 2016). This family currently comprises 14 genera and 155 species, many of which occur in the Amazon basin, with species of Schizodon Agassiz, 1829 and Laemolyta Cope, 1872 representing approximately 16% of the diversity (Nelson et al., 2016). Members of this family are important hosts for the life cycle of a broad diversity of parasites (Guidelli et al., 2006;Oliveira et al., 2017;Yamada et al., 2017), including platyhelminths belonging to the Class Monogenoidea (Cohen et al., 2013). Among anostomid species, only 7.7% has been investigated for monogenoidean parasites. Currently, 12 species of monogenoids are known to parasitize the gills, nostrils, body surface, urinary bladder, and ureter of members of 13 species of Anostomidae (see Table 1). During a field survey of parasites from the gills of Schizodon fasciatus Spix & Agassiz, 1829 and Laemolyta proxima Garman, 1890 (Anostomidae) from the Jari River, a tributary of the Amazon River basin (northern Brazil), two new species of Urocleidoides Mizelle & Price, 1964 were found and are described herein. We also recorded for the first time Urocleidoides paradoxus Kritsky, Thatcher & Boeger, 1986 parasitizing the gills of S. fasciatus from the eastern Amazon.

Host fish collection
In March 2018, 15 specimens of S. fasciatus and 3 specimens of L. proxima were collected with fishing nets in the lower Jari River, near Jarilândia district, in municipality of Vitória do Jari, State of Amapá, Brazil (1°9'4.01"S; 52°0'53.22" W). Host scientific names were validated in accordance with Fricke et al. (2019), and identification was carried out in accordance with specialized literature (Queiroz et al., 2013). This study was developed in accordance with the principles adopted by the Brazilian College of animal Experimentation (COBEA). Authorization from Ethic Committee in the Use of Animal of the Embrapa Amapá (Protocol N o 014/2018) was also carried out.

Parasitological procedures
Gill arches were removed and placed in vials containing hot water (65° C). Each vial was vigorously shaken, and formalin was added to obtain a 5% solution. In the laboratory, the contents of each vial were examined under a dissecting microscope (Leica S6D) and monogenoids were removed from the gills or sediment using small probes. Some specimens were stained with Gomori's trichrome (Humason, 1979;Boeger & Vianna, 2006) and mounted in Damar Gum or Canada balsam to determine the internal soft structures, while others were mounted in Hoyer's or Gray & Wess medium (Humason, 1979) for the study of the sclerotized structures. Measurements, all in micrometers, were obtained according to the procedures described by Mizelle & Klucka (1953). The measurements inner and outer for the anchors follow the scheme illustrated in Figures 1j. Dimensions of organs and structures represent the straight line distances between the extreme ends, the total lengths of the male copulatory organ were carried out using ImageJ (Rasband, 1997) on drawing tube images. The mean is followed by the range and the number (n) of specimens measured in parentheses. Illustrations were prepared with the aid of a drawing tube on a Leica DM 2500 microscope with differential interference contrast and phase contrast optics. Illustrations of the soft and hard structures were prepared using pen and ink. Plates were prepared using PhotoPaint software. Prevalence and mean intensity follow Bush et al. (1997). Type specimens and vouchers were deposited in the Invertebrate Collection of the Museu Paraense Emílio Goeldi (MPEG), Belém, Pará state, Brazil. Urocleidoides jariensis n. sp. (Figure 1a-k) Description: Based on nine specimens; three mounted in Gomori's trichrome, six mounted in Hoyer' medium. Body fusiform (Figure 1a) total length excluding haptor 255 (218-298; n = 9) long, 107 (59-154; n = 9) wide at level of gemarium. Tegument smooth. Cephalic margin tapered; moderately developed terminal lobes; three bilateral pairs of head organs with rod-shaped secretion; cephalic glands unicellular, posterolateral to pharynx (observed only in paratypes). Four eyes (two pairs); posterior pair larger than anterior pair; accessory chromatic granules present in cephalic area, elliptical ( Figure 1a). Mouth subterminal, midventral; pharynx 21 (17-25; n = 8) long, 23 (13-30; n = 8) wide, muscular, glandular; moderately elongated esophagus. Two intestinal caeca, posteriorly confluent to gonads, lacking diverticula. Genital pore opening midventral; genital atrium muscular. Testis, seminal vesicle, vas deferens, prostatic reservoir not observed. Copulatory complex comprising male copulatory organ (MCO) and accessory piece ( Figure 1b); MCO sclerotized, tubular, spiral, counterclockwise, with one coil, 39 (28-49; n = 6) long; circular sclerotized tandem brim associated with base of MCO; proximal portion of MCO slightly expanded, distal aperture slightly acute. Accessory piece sclerotized, non-articulated with MCO, comprising a variable distal sheath, surrounding the MCO (Figure 1b Ventral bar (Figure 1e) 48 (30-55; n = 8) long, slightly curved, broadly V-shaped with anteromedial constriction and enlarged ends. Dorsal bar (Figure 1f) 45 (33-55; n = 8) long, U-shaped, with slightly enlarged ends, slightly curved in posterior direction. Hook pair 1, 21 (19-23; n = 8) long ( Figure 1h) and hooks pairs 2-4, 6-7, 27 (21-32; n = 8) long (Figure 1i), similar in shape, composed of two subunits, shank with inflation comprising approximately 2/3 of total shank length; erected thumb, lightly curved long shaft, delicate point; filamentous hook (FH) with loop extending to union of shank subunits; pair 5 smaller than other hooks, 15 (13-16; n = 8) long, composed of single unit and inflated base (Figure 1g).  Mean intensity: 14.0 (11-21).

Etymology:
The specific epithet derives from the Latin (rament = shreds + acumin = point) and refers to the dorsal anchor morphology with ornamentation as sclerotized shredded filaments.
Comparative measurements: These are shown in Table 2.

Remarks
The specimens available are conspecific with U. paradoxus, mainly due to sharing the morphology of the anchors, bars, hooks, copulatory complex, vagina dextral and vaginal sclerite. The specimens studied here differ morphometrically in some measurements from specimens from the type locality ( Table 2).

Discussion
Species of monogenoids belonging to Urocleidoides Mizelle & Price, 1964, Jainus Kritsky & Crane, 1968Tereancistrum Kritsky, Thatcher & Kayton, 1980;Trinibaculum Kritsky, Thatcher & Kayton, 1980;Rhinoxenus Kritsky, Boeger & Thatcher, 1988;Scleroductus Jara &Cone, 1989 andKritskyia Kohn, 1990 have been reported parasitizing Anostomidae species (Table 1). However, none of these taxa seems to be restrict to anostomid fish. Except by species of Jainus, Tereancistrum, Trinibaculum and Rhinoxenus that are restrict to characiform hosts, the other genera are reported from at least two different Neotropical fish order. Species of Kritskya and Scleroductus are parasites of ureters and body surface, respectively, from characiform and siluriform hosts, whereas members of Urocleidoides can be found parasitizing the gills of species of 13 families from 4 different host fish orders (i.e., Characiformes, Cyprinodontiformes, Gymnotiformes and Siluriformes). In the present study, U. paradoxus is reported for the first time on the S. fasciatus gills, which has two new species of Urocleidoides described herein.
Urocleidoides paradoxus was described in Rhytiodus microlepis Kner, 1858 from the Solimões River, in Amazonas State, Brazil. This unique species of genus with a vagina that present dextral opening, and this characteristic  was added for diagnose of this genus (see Kritsky et al., 1986). Urocleidoides jariensis n. sp. and Urocleidoides ramentacuminatus n. sp. resemble U. paradoxus because both species share the presence of the vaginal dextral opening, a characteristic present only in the species of Urocleidoides reported to members of Anostomidae family. Studies suggest that most monogenoids species are restricted to their hosts at highest taxonomic levels (i.e., family or order), probably due to broad historical restrictions (e.g., immunological, morphological and/or evolution), which acts at a large scale (Boeger & Kritsky, 1997;Desdevises et al., 2002).
The present study increased the species number of Urocleidoides sensu stricto to 27, as well as that of monogenoids in Amazonian fish, thus contributing to knowledge of the diversity of these parasites in this important Neotropical region, which includes a great, yet underestimated diversity of monogenoids. However, Urocleidoides is one of the most specious and taxonomically problematic genera among Neotropical dactylogyrids. It is possible that, in the future, the taxonomic position of incertae sedis species are clarified with the aid of molecular studies together with morphological analyses of holotypes and paratypes.