Ligophorus uruguayense sp. nov. (Monogenea, Ancyrocephalidae), a gill parasite from Mugil platanus (Mugiliformes, Mugilidae) in Uruguay

The present study describes a new species, Ligophorus uruguayense, parasitizing the gills of Mugil platanus Günther, 1880 from the coast of Uruguay. It differs from all other species of the genus mainly in the shape of the ventral bar, the thick process at the distal end of the inner root of ventral anchors, the J-shaped penis accessory piece and the vaginal tube showing transverse annulations at its distal end, the host species, and geographical distribution. This is the first description of a species of Ligophorus from a mullet in the South Atlantic Ocean.


Introduction
The fish species of the family Mugilidae have a wide geographic distribution and are mainly restricted to coastal estuarine waters of tropical and subtropical regions. The taxonomic status of Mugil platanus Günther, 1880 is still controversial, with some authors considering it as a distinct population of Mugil cephalus Linnaeus, 1758 (see Fraga et al. 2007), and others as a different species (Castro et al. 2008). The parasites of mullets have been thoroughly studied because of their importance for human consumption (Paperna and Overstreet 1981, Juárez-Arroyo and Salgado-Maldonado 1989, Valles-Ríos et al. 2000. The species of the monogenean genus Ligophorus Euzet et Suriano, 1977 have received considerable attention in the past few years. The genus Ligophorus was established for species recovered from Mediterranean mugilids. In the Northern Hemisphere, new species of Ligophorus were found parasitizing mugilid species from the Black Sea, Azov Sea, Mediterranean Sea, North Atlantic Ocean, and Pacific Ocean (Dmitrieva and Gerasev 1996;Sarabeev and Balbuena 2004;Sarabeev et al. 2005;Rubtsova et al. 2006Rubtsova et al. , 2007Dmitrieva et al. 2007). In the Southern Hemisphere the only species so far described is L. huitrempe Fernández Bargiela, 1987 from a mugilid in the Chilean coast, Pacific Ocean.
The present study describes a new species of Ligophorus, and is the first report of this genus parasitizing Mugil platanus from the South Atlantic Ocean.

Materials and methods
Mugil platanus specimens were collected from the coastal lagoon Laguna de Rocha (34°33´-34°41´S and 54°02´-54°22´W), Uruguay, in 1993 and 1994. Fish were dissected in the field, and their gills and other organs were immediately removed and fixed in 4% formalin. In the laboratory, gills were examined under a stereoscopic microscope. Ancyrocephalid monogeneans were cleared in lactophenol (Amman) and mounted in glycerine jelly or stained with chlorhydric carmine (Langeron 1942), dehydrated in an ethanol series and mounted in Canada balsam. To study live monogeneans additional mugilids of the same species were captured in 2007 from Las Flores (34°50´S-55°25´W), Department of Maldonado, Uruguay.
A total of twenty-five characters of sclerotized structures were measured, of which twenty-two were defined by Mariniello et al. (2004), Sarabeev and Balbuena (2004), and Rubtsova et al. (2007), and the remaining three are defined here, namely distance between upper and lower lobe of the penis ac-cessory piece; the vaginal plate (corresponding to the saucershaped opening); and the vaginal ring (corresponding to transverse annulations at the distal end). All these structures were measured on fixed and live specimens, except the vaginal plate and the vaginal ring, which were measured on live specimens only. The abbreviations of the characters used throughout the text, and the measurements in micrometers, with the mean followed by the standard deviation (SD) within parentheses, range and number of specimens measured (n), are given in Table II. A diagram showing measurements of the sclerotized elements is presented in Figure 1. Drawings were made with the aid of a camera lucida attached to an Olympus BX-50 microscope. Micrographs were taken with a Sony Cyber-shot 6.0 DSC-S600 digital camera. Representative specimens were mounted in Canada balsam and deposited in the Helminthological Collection of the Laboratorio de Zoología de Invertebrados, Facultad de Ciencias, Montevideo, Uruguay, and in the Helminthological Collection of the Museo Argentino de Ciencias Naturales "Bernardino Rivadavia", Buenos Aires, Argentina. Data were analyzed with χ 2 test and Mann-Whitney U-test.

Results
Thirty-one out of 123 fish collected in 1993 and 1994 were found infected with a new species of Ligophorus. Specimens were recovered from both the right and left gill arches, with a higher number of parasites on the first and second gill arches  Table II for abbreviations of metric variables)  Table II. Comparative morphometrical data of specimens of Ligophorus uruguayense sp. nov., with L. mugilinus (Hargis, 1955), according to Sarabeev et al. (2005), and L. huitrempe Fernández Bargiela, 1987 (Table I). Prevalence of infection was 25.2% and mean intensity 13.7 (24.0) worms per fish.
Ligophorus uruguayense sp. nov. (Figs 2-4) Description: Worms with characters of the genus as defined by Euzet and Suriano (1977) and supplemented by Sarabeev and Balbuena (2004). Table II shows the morphometric measurements of fixed and live specimens of the studied species and those of L. huitrempe (Southern Pacific Ocean) and L. mugilinus (Hargis 1955) (North Atlantic Ocean) for comparison.
Body fusiform, with two pairs of pigmented eye-spots, posterior one with lens. Posterior haptor armed with 14 uncinuli (Fig. 2). One pair of ventral anchors connected by ventral transverse bar (Figs 3A, 4A), one pair of dorsal anchors connected by dorsal transverse bar (Figs 3B, 4A). Ventral anchors with elongate thin blade and recurved point, forming obtuse angle (about 100°). Base of anchor markedly thicker than blade, separated by a notch. Inner root (guard) somewhat longer than outer root (shaft), VAD/VAC = 1.2 ± 0.2 (1.0-1.5; n = 22). Inner root shows thick process at distal end where ventral connecting bar articulates with anchor. Angle between roots about 50°. Filaments present. Ventral connecting bar massive, slightly curved, with two sclerotized thickenings at base of two membranous anterior medial processes; heavily sclerotized median process between open V-shaped membranous processes (not always seen in stained specimens) (Figs 3A, 4A). Dorsal anchors similar in shape to ventral anchors (except for thick process at distal end of inner root (Fig. 4B). Inner root much longer than outer root, DAD/DAC = 1.5 ± 0.2 (1.0-2.0; n = 25). Base of dorsal anchors somewhat thinner than that of ventral anchors. Dorsal transversal bar slightly V-shaped, with rounded ends (Figs 3B, 4A). All 14 uncinuli similar in shape and size, with slightly curved handle and sickle (Fig. 3C). Male copulatory complex consisting of tubular penis about 1.0 μm in diameter and with J-shaped accessory piece, showing short lower lobe joined to longer, simple and tubular upper lobe at proximal end, separated by short distance (Figs 3E, 4C, 4D). Accessory piece supports distal end of penis. Vaginal armament constituted by convoluted, sclerotized tube; two proximal thirds thin, distal third thick; transverse annulations at distal end; with wide saucer-shaped opening (Figs 3D, 4E) placed submidventrally.
Etymology: The specific name refers to the geographic location where the worm was found.
Remarks: There were significant differences in the measurements of characters between live and glycerine-or balsammounted specimens (Table II) . Thus, the characters that were not affected by the mounting methods used are the ventral bar (VBL), the ventral (VAB) and dorsal (DAB) anchor main part lengths and the accessory piece (PAPL) of the male copulatory complex. The ventral bar is a heavily sclerotized piece rather than a slightly curved piece and its morphology remained unchanged. On the contrary, the dorsal bar is less sclerotized, and its shape was modified by the pressure of the coverslip. No significant differences were found in the distance between shaft and guard of both ventral and dorsal anchors in live specimens (VDIOR, DDIOR: U = 0.46; p = 0.64; n = 22). The distance between the membranous processes of the ventral bar is not a reliable measurement because it differed between mounted and live specimens or the membranous processes could not be seen clearly. In contrast to Rubtsova et al. (2006), no difficulty was found in obtaining an accurate measurement of the whole length of the penis either for live or mounted specimens.

Discussion
This study reports for the first time the occurrence of a Ligophorus species in the host Mugil platanus and of this genus in the South Atlantic Ocean.
The similarity among species has led to erroneous identifications. In many cases, a single species was split into different species based on detailed morphological and morphometric analyses of the sclerotized structures of the haptor (dorsal and ventral bars, dorsal and ventral anchors) and of the genital organs (terminal genitalia of male copulatory complex, vagina), and on the occurrence of Ligophorus specimens in mullets from different geographic areas. This is illustrated in the following examples: (1) the separation of L. gussevi Miroshnichenko et Maltsev, 2004(synonymous of L. pilengas Sarabeev et Balbuena, 2004, according to Balbuena et al. 2006 from L. chabaudi Euzet et Suriano, 1977; (2) the separation of