A new flatworm species of Temnocephala (Rhabdocoela, Temnocephalidae) ectosymbiont on the freshwater crab Valdivia serrata (Decapoda, Trichodactylidae) from Amazonas, Colombia

Abstract A new species of temnocephalan is described from the branchial chambers of Valdivia serrata in Colombia as Temnocephala ivandarioisp. nov. The most distinctive characters of the new species are in the cirrus and the epidermal ‘excretory’ syncytial plates. In the present study, the terminology to describe the cirrus of species of Temnocephala is updated. Comparison between the shape of the cirrus of the temnocephalans associated with trichodactylid crabs is also provided.


Materials and methods
Eleven specimens of Valdivia serrata White, 1847 (crab) were manually collected from the Reserva Natural Tanimboca, Leticia, Amazonas (4°07'39.8"S, 69°57'13.0"W), Colombia. The specimens were transported alive to the Programa de Estudio y Control de Enfermedades Tropicales (PECET) laboratory and identified using a decapod key (Campos 2014). Specimens of Temnocephala were removed from the branchial chambers of host crabs under a stereomicroscope, rinsed in saline solution, and preserved in cold alcohol-formalin-acetic acid (AFA) and 70% ethanol for morphological identification by light microscopy; or fixed in 2.5% glutaraldehyde for observation by Scanning Electron Microscopy (SEM). The temnocephalans were identified by focusing on the morphology of the reproductive complex, the shape of the epidermal excretory syncytial plates (DLSPs) and the deposit areas of the eggs in the host. Terminology to describe the cirrus of the temnocephalans was updated from Sewell et al. (2007), Seixas et al. (2011), Garcés et al. (2013 and Ponce de León et al. (2015). Morphology of the male and female reproductive system was studied by light microscopy in specimens mounted as permanent slides in Canada balsam, stained in Meyer's paracarmine and Borax carmine. Samples were also observed by SEM to determine the shape and position of the egg filament, the fracture plane of the eggshell, the shape of the DLSPs and the relative position of the excretory pore. Measurements were in micrometres (µm) unless otherwise indicated; ranges were determined followed by the arithmetic mean, the standard deviation and the number of specimens measured for a given character (mean, standard deviation, n). Photomicrographs of the temnocephalans were taken with a Nikon Alphaphot YS-2 microscope. Drawings were made using a drawing tube Nikon 1.25X. Line drawings and photographic images were prepared using Inkscape 0.92. The SEM preparations were examined with a Hitachi S-4800 SEM at the Servicio de Microscopía, SCSIE, Universitat de València, Spain. Type specimens were deposited in the Colección Colombiana de Helmintos (CCH.116), Universidad de Antioquia, Medellín, Colombia.  Fig. 3E). DLSPs small, elliptical-shaped (Fig. 3A, B), 167 long by 141 wide (N = 2); excretory pore "subcentral" in the DSLP, displaced towards the internal limit (Fig. 3A); length ratio of DLSPs:total body length, without tentacles, 1.0:10.7.
Glands. Rhabditogenic glands forming bunches in the lateral fields of the body extending from the pharynx to the middle level of the adhesive disk. Haswell cells in front of the eyespots and the brain. Disk glands between the adhesive disk and the genital complex ( Fig. 2A).
Etymology. The new species is dedicated to Dr. Iván Darío Vélez Bernal for his outstanding contributions to the study of helminthology and the understanding of tropical diseases in Colombia.  Taxonomy of temnocephalans is based on morphology of adult specimens with emphasis on the reproductive system. The structure of the cirrus is the trait of greatest taxonomic value (Damborenea 1991, Damborenea and Cannon 2001, Sewell et al. 2007, Garcés et al. 2013. Other traits important for species differentiation include composition of the female reproductive complex, eggs deposit areas in the host, and the shape of the DLSPs (Damborenea and Brusa 2008, Amato et al. 2010, Volonterio 2010, Seixas et al. 2011. Nine species of Temnocephala are known for their association with crabs of the Trichodactylidae family. Of these, T. ivandarioi sp. nov., T. longivaginata Seixas, Amato & Amato, 2011, and T. lutzi Monticelli, 1913(Amato et al. 2005) present a similarsized cirri and have the Amazon River basin as a biogeographical connection. Temnocephala longivaginata and T. ivandarioi sp. nov. are most similar to each other in the length of the vagina and the presence of sclerites in the distal portion of the cirrus.
Temnocephala ivandarioi sp. nov. can be distinguished by the combination of the following features: cirrus with a circle of small sclerites (range 18-20) in the distal portion of the introvert, without spines or ridges in the inner wall of the introvert (Fig.  2C). The ovary lies ventral to vesicle resorbens followed by an elongated vagina with two vaginal sphincters similar in size, one symmetric and proximal, and one symmetric and distal; the vagina connects to the genital atrium dorsally. The seminal vesicle is located anterolateral to the prostatic bulb. The DLSPs are small and 'elliptical-shape', with a partially sinuous contour.
On an ecological-level T. ivandarioi sp. nov., T. longivaginata, and T. lutzi inhabit the branchial chambers of trichodactylid crabs from the middle basin and lower basin of the Amazon River (Leticia, Amazonas, Colombia; Peixe-Boi, Pará State; Rio Amapá, Amapá State, northern Brazil, respectively). Temnocephala ivandarioi sp. nov. is the third species described from Colombia, and therefore V. serrata is registered as a new trichodactylid host for neotropical temnocephalans. Valdivia serrata is widely distributed throughout the Orinoco and Amazon River basins in Venezuela, the islands of Trinidad and Tobago, the Guianas, Colombia, Brazil, Peru and Bolivia (Cumberlidge 2008). In Colombia this species is found in the eastern region of the country (Amazonas, Arauca, Caqueta, Meta, Putumayo, and Vichada Departments) in the Putumayo and Maqueta rivers that drain into the Amazon River, and the Guaviare, Meta, and Arauca rivers that drain into the Orinoco River (Campos 2005(Campos , 2014. It is likely that T. ivandarioi sp. nov., T. longivaginata, and T. lutzi are closely related due to their morphological similarities and geographical proximity. The implementation of molecular studies will reveal the phylogenetic relationships between the different species of Temnocephala in the Neotropics. In Colombia more than 132 species of decapod crustaceans have been recorded (Campos 2014), while only two associated species of temnocephalans have been reported to date: T. icononcensis (Arias-Pineda et al. 2015) and T. ivandarioi sp. nov. The great diversity of these potential hosts (Campos 2014) suggests that most temnocephalans remain undescribed.  Garcés et al. (2013), and Ponce de León et al. (2015); diagrams modified from Sewell et al. 2007: 205, fig. 2).
Comparative notes. The cirrus is the only rigid structure and therefore of constant general morphology in juveniles and adults (except for small intraspecific variations) for each species. The morphology of the cirrus constitutes one of the few characters used and is the most valuable taxonomic character for species identification  Monticelli, 1913(Amato et al. 2005) E Temnocephala microdactyla Monticelli, 1903FTemnocephala pignalberiae Dioni, 1967(Amato et al. 2010) G Temnocephala santafesina Dioni, 1967H Temnocephala trapeziformis Amato, Amato & Seixas, 2006I Temnocephala travassosfilhoi Pereira & Cuocolo, 1941 (Seixas et al. 2015b). In the present study, terminology describing the temnocephalan cirrus is updated for neotropical species (Fig. 5), according to Sewell et al. (2007), Seixas et al. (2011), Garcés et al. (2013) and Ponce de León et al. (2015). The cirrus of the species of Temnocephala described from trichodactylid crabs (Fig. 6) are compared based on this terminology.
The cirrus is defined as the entire sclerotised male copulatory organ comprised of a 'shaft' (rigid, tubular region tapering distally; Fig. 5A) and an 'introvert' (flexible distal eversible region armed with grooves, spines, sclerites or ridges, Fig. 5B) (modified from Sewell et al. 2007). Furthermore, the degree of shaft curvature is a reliable taxonomic characteristic of neotropical temnocephalans (Garcés et al. 2013).
The shape of the shaft may be described as a 'funnel', 'goblet', or 'cone'. Funnel-or goblet-shaped shafts have a wide proximal region which tapers rapidly into a narrow tubular distal region (Sewell et al. 2007). The cirrus may be more or less curved, and it may be described as 'curved up', 'straight', or 'curved down'. Similarly, the position of the cirrus with respect to the body may be described as 'towards the forebody', 'horizontal', or 'towards the hindbody' (modified from Garcés et al. 2013). The position of the cirrus can or may not depend on the cirrus curvature i.e. cirrus 'curved up' directed 'towards the forebody', but cirrus 'straight' are directed towards the 'forebody', 'horizontal' or 'towards the hindbody'. The cirrus position can be examined only from a complete diagram of the temnocephalan.
The introvert shape can be described as 'cylindrical', 'cone'; 'scoop', or 'goblet'. Scoop-or goblet-shaped introvert have a wide middle region, which tapers into a narrow distal region. In addition, the introvert may be 'unarmed', armed with 'grooves' in the proximal limit of the introvert, or armed with 'spines', 'sclerites', and 'ridges' in the inner wall of the introvert. The distal opening of the introvert may be at right angles with respect to the proximal limit of the introvert i.e. 'not oblique', 'oblique', or 'very oblique' (modified from Sewell et al. 2007). Additionally, the distal region of the introvert may be curved (with or without spines, sclerites, or ridges), and described as 'forward curved', 'straight' or 'backward curved' (described as with non-spined region or without non-spined region by Sewell et al. 2007).
The morphology of the cirrus is necessary for species identification and should be clearly described based on the terminology proposed in the present study. This new terminology can be applied to species of neotropical temnocephalans described to date. of an early warning system for the prevention and control of the main VTE in the Department of Amazonas, Colombia", General Royalty System (BPIN 2013000100240). All the specimens were collected under the "Permiso Marco", Universidad de Antioquia, to collect research specimens. This permission has been granted to us by the National Authority of Environmental Sciences -ANLA. Ministry of Environmental, Republic of Colombia. Resolution 0524, 27 May 2014.