﻿Two new species of Acanthobothrium Blanchard, 1848 (Cestoda, Onchoproteocephalidea) from Urobatisjamaicensis (Cuvier, 1816) (Elasmobranchii, Batoidea, Myliobatiformes) of the Mexican Caribbean

﻿Abstract Eight specimens of Urobatisjamaicensis were collected from four localities of the Yucatan Peninsula, Mexico, of which four specimens were infected with cestodes of two new species of Acanthobothrium. Acanthobothriumgarciaprietoisp. nov. differs from congeners by a combination of characters including the size of the scolex and bothridia, length of the cephalic peduncle, length of the axial and abaxial prongs and total length of the abaxial prongs of the hooks, size of the cirrus sac and testes in mature proglottids, and the total number of proglottids. The most prominent characteristic distinguishing A.pulidofloresaesp. nov. from other species of the Atlantic Ocean/Caribbean Sea/Gulf of Mexico is the form of the scolex, which has the “clover leaf” configuration. In addition, it can be distinguished by the total length of the worm, total number of proglottids, small accessory suckers, the form of the bothridia, length of the axial and abaxial prongs and total abaxial prong length of hooks, and the number of testes. According to the current category scheme, A.garciaprietoisp. nov. and A.pulidofloresaesp. nov., belong to categories 1 and 5, respectively.


Introduction
The Family Urotrygonidae (round rays) is represented by approximately 20 species in two genera, Urobatis Garman, 1913 andUrotrygon Gill, 1863.Family members are confined to tropical warm water continental shelves of the Western Atlantic and the Eastern Pacific coasts of the Americas, mainly in demersal inshore habitats (Del Moral-Flores et al. 2015;Last et al. 2016a, b).The yellow stingray, Urobatis jamaicensis (Cuvier, 1816) is the only member of the genus found in the Western Atlantic and Caribbean waters, with a latitudinal range from Florida to Venezuela (Lesniak et al. 2015).However, it also has been reported in North Carolina, south to Brazil, and in the Gulf of Mexico (Bigelow and Schroeder 1953;Robins et al. 1986;Hoese and Moore 1998;McEachran and Fechhelm 1998;McEachran and de Carvalho 2002;Spieler et al. 2013).
The genus Acanthobothrium Blanchard, 1848 currently is comprised of 211 species, and is the most diverse genus of Onchoproteocephalidea.Its members principally parasitize batoids and sharks (Caira and Jensen 2017).Recent descriptions include 23 species, eleven from the greater Atlantic Ocean (Franzese and Ivanov 2018;Rodríguez-Ibarra et al. 2018;Franzese and Ivanov 2020;Van Der Spuy et al. 2020, 2022), six from the Persian Gulf and Gulf of Oman in the northwest Indian Ocean (Maleki et al. 2018(Maleki et al. , 2019) ) and six from the Pacific Ocean (Zaragoza-Tapia et al. 2019, 2020b;Gallagher and Caira 2020).Of the 211 species, 17 species have been described from Mexican waters and three species known from other localities have been reported off México.
As part of a continuing study of the helminths of marine fish, eight specimens of U. jamaicensis were collected in the Yucatan Peninsula, Mexico; four of these were found to be infected with two species of Acanthobothrium.These two and ten other recently described species of Acanthobothrium were added to the checklist by Zaragoza-Tapia et al. (2020a).The current paper is a continuation of the process of documentation of the DAMA protocol (see Brooks et al. 2014).

Materials and methods
Eight specimens of Urobatis jamaicensis were collected by local fishermen in the Yucatan Peninsula, Mexico, who donated the spiral intestines to the authors.The locations and number of rays from each location are as follows: two from Xcalak, Quintana Roo (18°15'58.37"N,87°50'9.00"W)(collected in October 1998), one from Río Lagartos, Yucatán (21°35'58.05"N,88°9'32.44"W)(February 1999), four from Isla Contoy, Quintana Roo (21°31'44.45"N,86°48'11.53"W)(February 1999), and one from Isla Cozumel, El Paso de los Cedros, Quintana Roo (20°21'57.74"N,87°1'32.48"W)(February 1999).Cestodes were removed from the spiral valve of the host, killed with hot water, transferred immediately to AFA (alcohol-formalin-acetic acid) for 24-48 hours, and then stored in 70% ethanol.Specimens were stained either with Mayer's carmalum or Ehrlich's hematoxylin following Pritchard and Kruse (1982), dehydrated in a graduated series of ethanol, cleared in methyl salicylate, and mounted in Canada balsam for viewing.Worms that were too large to be mounted on a single slide were cut into sections and mounted sequentially on separate slides.The prevalence of infection was calculated according to Bush et al. (1997) and Bautista-Hernández et al. (2015).Images were acquired using a Zeiss Axio Zoom V16 stereo microscope equipped with a Zeiss Axiocam M8c5 (5 megapixels) camera and controlled with the Zeiss Zen 2012 (blue edition) software.
Because the specimens had been mounted on slides in 1997-1998 (but not studied at that time), one specimen was dismounted from its single slide, placed in 100% Xylol for 24 hours, after which the excess Canada balsam was removed as much as possible with a fine brush in order to process it for scanning electron microscopy (SEM).This specimen was dehydrated through a graded series of ethanol, subjected to critical point drying with CO 2 Table 1.Species of Acanthobothrium different categories that were compared with A. garciaprietoi sp.nov.and A. pulidofloresae sp.nov.Note: * = Species with characters used are mostly assigned to category 5; ** = species with characters used are mostly assigned to another category, but some of them allow them to be in category 5; † = species considerate as species inquirenda by Caira et al. (2012).Etymology.The species is named in honor of Dra.Griselda Pulido-Flores (Universidad Autónoma del Estado de Hidalgo, Centro de Investigaciones Biológicas, Hidalgo, Mexico) for her contributions to the academic training of the first author, and to knowledge of helminths, as well as for her friendship.
Finally The following species of Acanthobothrium are described as having a clover-leaf scolex configuration: Category 3, A. robertsoni; Category 4, A. adlardi, A. cestraciontis, A. dasybati, A. grandiceps, A. karachiense, A. ijimai, A. macrocephalum and A. pingtanensis; A. mujibi has not been assigned to a category because of the lack of data in the description.Acanthobothrium cribbi is illustrated as having a scolex with bothridia that extend almost to have a petaloid scolex; however, the species is not described as having a clover-leaf scolex configuration (Campbell and Beveridge 2002) (Table 1).
Site of infection.Spiral valve.Prevalence of infection.14.28% Etymology.The species is named in honor of M. C. Luis García-Prieto (Universidad Nacional Autónoma de México, Instituto de Biología, México) for his friendship and for his contributions to the knowledge of helminths.
Acanthobothrium cartagenensis and A. himanturi have been reported from the Caribbean Sea.Acanthobothrium cartagenensis differs from and A. garciaprietoi sp.nov.by having a greater number of proglottids (13 vs. 7-9, respectively); its scolex is square (300-300), while that of A. garciaprietoi sp.nov. is wider than long (225-320 long 275-323 wide); the loculi of A. cartagenensis are shorter (1:0.3:0.3 vs. 1:0.4:0.4).The apical pads of A. cartagenensis are 67 in diameter, and they are relatively elliptical with no invaginations in the hook region; in contrast, those of A. garciaprietoi sp.nov.are larger (76-83 long by 95-110 wide), with an invagination in the region between the hooks.The cephalic peduncle of A. caragenensis is shorter than that of A. garciaprietoi sp.nov.(180 long by 120 wide vs. 175-288 long by 58-90 wide).The testes of A. cartagenensis are smaller in diameter (30-45 vs. 50-75 long by 30-55 wide of A. garciaprietoi sp.nov.), but the cirrus sac of A. garciaprietoi sp.nov. is larger.The lobes of the ovary of A. cartagenensis are longer than those of A. garciaprietoi sp.nov.(210-390 long by 60-112 wide vs. 133-375 long).

Discussion
It is good practice to describe a species based on as large number of specimens as possible and, if the material permits, include molecular information on the species.However, sometimes the material is limited and, in name of "the progress of knowledge and science" (Thomson et al. 2018), this sub-representation is accepted because the alternative is to fail to recognize new aspects of biodiversity.Recent examples are the descriptions of species of Acanthobothrium by Caira and Burge (2001), Campbell and Beveridge (2002), Fyler and Caira (2010), Maleki, Malek, and Palm (2015), and Gallagher and Caira (2020).The entire coast of Quintana Roo now is an Área Natural Protegida (SEMARNAT 2018), making it difficult to collect new material that would augment the samples collected more than 23 years ago.Of course, if future circumstances permit, it is advisable to supplement these specimens with freshly-collected specimens.
As can be seen from the cited works, the majority of the species from elasmobranchs that have been described to date from Mexican waters are cestodes.Many of them have been assigned to the genus Acanthobothrium, which currently is represented world-wide by 213 nominal species.Of these, 25 have been recently described: 13 from the Atlantic (Franzese andIvanov 2018, 2020;Rodríguez-Ibarra et al. 2018;Van Der Spuy et al. 2020;Van Der Spuy et al. 2022;this study), six from the Persian Gulf, Gulf of Oman, and Indic Ocean (Maleki et al. 2018(Maleki et al. , 2019)), and six from the Pacific Ocean (Zaragoza-Tapia et al. 2019, 2020b;Gallagher and Caira 2020).Table 2 includes the most recently-described species as a complement to the information presented by Zaragoza-Tapia et al. (2020a).These data are integrated into the original table from that work (Suppl.material 1).The geographical distribution of the known localities of cestodes of elasmobranchs continues to be very unequal, representing the geographical distribution of the taxonomists that described the species rather than the expected global distributions of members of the order (Zaragoza-Tapia et al. 2020a).

Species with a "clover leaf" configuration of the scolex
The majority of species of Acanthobothrium have scolex moderately to completely attached to the scolex proper by membranous tissue (velum) that holds the bothridia close to the scolex.In contrast, the species with a "clover-leaf" scolex (Yoshida 1917) have bothridia either completely unattached along almost the entire length or attached only by a long, tenuous velum membrane.This allows the bothridia to project laterally from the anterior-most part of the bothridia, thus forming a laterally-wide surface (Figs 1A, 2A) used to attach to the intestinal wall of the host.This configuration has been called "clover leaf" for a general resemblance to the leaf pattern of the clover plant (Trifolium Linnaeus), which commonly has 3-5 leaves attached around the terminal point of the stem.Some authors (e.g., Yoshida 1917;Fyler and Caira 2010) have considered this conformation of the scolex to be a valuable morphological character to differentiate a number of species.However, this type of conformation suggests three possibilities: an adaptive change to increase the area of bothridial attachment in a monophyletic group; a parallel evolution of a similar function in species not closely related; or secondary functional increase in bothridial attachment as a result of lengthening of the velum allowing better movement of the bothridia.Since the length of the velum is variable within Acanthobothrium spp.and no phylogenetic hypothesis for all putative species exists, either is possible or it might be the result of some other mechanism.
Within Acanthobothrium, 13 species from Australian, the Western Pacific region, and Indian Ocean were described as having a "clover-leaf" scolex: A. adlardi, A. cestraciontis, A. dasybati, A. grandiceps, A. karachiense, A. ijimai, A. macrocephalum, A. mujibi, A. pingtanensis, and A. robertsoni. Three other species (A. majumdari, A. tsingtaoensis, and A. zugeinensis) are not included in the list above because they are not considered valid taxa by Caira et al. (2012) and Caira and Jensen (2017).The latter authors categorized them as species inquirendae.These species were included in the comparisons with A. pulidofloresae sp.nov.using the data recorded in the original descriptions because they were described as having a "clover-leaf" scolex, described as reminding a "fourleaved clover" or drawn as having this conformation.All of the aforementioned species occur in the greater Pacific Basin.In contrast, A. pulidofloresae sp.nov.occurs in the Caribbean.How this interpretation of the distribution awaits a phylogenetic hypothesis for the members of the genus.

Figure 5 .
Figure 5. Acanthobothrium garciaprietoi sp.nov.SEM images.Paratype (not deposited) A scolex B bothridial muscular pad with pair of hooks C central area of scolex proper between the bothridia D gladiate spinitriches located on the proximal bothridial surface, see image C E papilliform filitriches located on the scolex proper, behind the bothridia, see image C F coniforme spinitriches located on the cephalic peduncle.Scale bars: 5 μm (D-F); 30 μm (C); 100 μm (A, B).