Morphologic and genetic characterization of Pterygodermatites (Mesopectines) valladaresi n. sp. (Nematoda, Rictulariidae), a parasite of the mouse Mus musculus (Rodentia, Muridae) from the Canary Islands (Spain)

A new rictulariid nematode Pterygodermatites (Mesopectines) valladaresi n. sp., parasite of the house mouse Mus musculus (Rodentia: Muridae) in the Canary Islands (Spain) is described by means of light and scanning electron microscopy. The new species belongs to the subgenus Mesopectines characterized by a more or less dorsal orientation of the buccal capsule, the presence of three oesophageal teeth, the morphology of the oral denticles and the Spirurida type of arrangement of caudal papillae in males. The most discriminant characteristics between the new species and the existing species in the subgenus Mesopectines are (a) the number of cuticular projection pairs (62–64), (b) the size of right and left spicules (respectively, 62–90 µm and 123–139 µm), (c) the number of midventral fans in males (3–4), (d) the number of prevulvar/total cuticular projection pairs (38–42/63–71), (e) the posterior differentiation of combs into spines in relation to the position of the vulva and (f) the anterior position of the vulva in relation to the oesophagus-intestine junction in females. Parasitized hosts and geographical distribution are also useful criteria to distinguish P. (Me.) valladaresi n. sp. from the remaining species of the subgenus. In addition, the cox1 sequence of the new species is provided and compared with available data of related species.


Introduction
The genus Pterygodermatites Wedl, 1861 includes 68 rictulariid nematode species parasitizing a wide range of mammalian vertebrates [41]. This genus has five subgenera, namely P. (Mesopectines) , P. (Multipectines) , P. (Neopaucipectines) , P. (Paucipectines)  and P. (Pterygodermatites)   [6,31]. Species of these subgenera are mainly characterized by the orientation of the buccal capsule, the morphology of oral denticles and other peribuccal structures, the morphology of oesophageal teeth, the number of cuticular projection pairs (combs and spines), the size of spicules, and the type and arrangement of cloacal papillae. The geographical distribution of Pterygodermatites spp. is wide and it is also a feature that can be used to differentiate some of the species [6,31].
Recently, Simões et al. [41] compiled all the available literature providing the checklist of species of the genus Pterygodermatites. According to these authors, the subgenera P. (Paucipectines) and P. (Mesopectines) are the most diverse of the five subgenera with 27 and 25 described species, respectively. There are 16 additional species belonging to the remaining subgenera: six species of P. (Multipectines), four of P. (Neopaucipectines) and six of P. (Pterygodermatites). The subgenus P. (Mesopectines) includes species widely distributed in Africa and Asia parasitizing species of the orders Rodentia, Carnivora and Primates [31,41].
In the present study, we describe a new species, P. (Me.) valladaresi n. sp., parasitizing the house mouse (Mus musculus) in the Canary Islands (Spain). Additionally, the sequence of the mitochondrial cytochrome c oxidase subunit I gene (MT-CO1) is provided and compared with available data of related species.  [36]. The studied rodents were captured by means of Sherman and Firobin traps, sacrificed by cervical dislocation and then scanned for intestinal helminths. The new species described here P. (Me.) valladaresi n. sp. was found in mice from five islands: Fuerteventura, Gran Canaria, La Gomera, La Palma and El Hierro (Fig. 1) [36].

Light microscopy study
Specimens were mounted in Amann lactophenol on slides and then observed under the light microscope. Drawings were made with the aid of a drawing tube and later modified using Adobe Illustrator software (Adobe, San José, CA, USA). All measurements are given in micrometres (except where indicated).

Scanning electron microscopy study
Some worms (1 male and 4 females) were preserved for scanning electron microscopy (SEM) examination. Initially they were fixed in 70% ethanol in the field and later, in the laboratory, they were dehydrated in an ethanol series and critical point dried with carbon dioxide in an Emitech K850X (Quorum Technologies Ltd., Laughton, East Sussex, UK). Finally, specimens were mounted on stubs with conductive adhesive tape and colloidal silver, coated with carbon in an Emitech K950X (Quorum Technologies Ltd.) evaporator, and examined using a Field Emission SEM JSM-7001F (Jeol Ltd., Tokyo, Japan) at 10 kV in the "Centres Científics i Tecnològics" of the University of Barcelona (CCiTUB).

Molecular analysis and phylogenetic tree
Genomic DNA samples were isolated from the mid-section fragment of P. (Me.) valladaresi n. sp. following López et al. [23]. The DNA extraction procedure was checked using DeNovix DS-11+ Spectrophotometer (DeNovix Inc., Wilmington, DE, USA).
DNA amplification by PCR was conducted using the primer cocktail as described by Prosser et al. [30], for the barcode region of the mitochondrial cytochrome c oxidase subunit I gene (MT-CO1). The PCR amplification contained 1Â Buffer (Bioline Ltd., London, UK), 0.2 mM of each dNTP (Bioline Ltd.), 0.5 lL of each primer cocktail (10 lM of a threeforward-primers mix, and 10 lM of a three-reverse-primers mix), 1U of Taq DNA polymerase (Bioline Ltd.), 1.5 mM MgCl 2 (Bioline Ltd.), and 20-30 ng of total genomic DNA in a total volume of 50 lL. Amplification was conducted with XP Cycler (Hangzhou Bioer Technology Co. Ltd., Hangzhou, China) using the following parameters: 94°C for 1 min; five cycles at 94°C for 40 s, 45°C for 40 s, 72°C for 1 min; followed by 35 cycles at 94°C for 40 s, 51°C for 40 s, 72°C for 1 min; and a final extension at 72°C for 5 min [30]. The resulting amplifications were visualized on 1.2% agarose gel at 100 V for 45 min.
The PCR product was sequenced by Macrogen Spain Inc. (Madrid, Spain) with primers NemF1_t1 and NemR1_t1 [30]. The analysis of the sequences was carried out with software MEGA X [22], using the multiple alignment program ClustalW included in MEGA X, and minor corrections were made by hand.
A phylogenetic analysis based on the MT-CO1 gene sequences of P. (Me.) valladaresi n. sp. and other Pterygodermatites species available in GenBank was performed using the Neighbour-Joining distance method with the p-distance model [35] (Supplementary material Figure S1) and Maximum-Likelihood method with Tamura-Nei model [43] (Fig. 2), both with at least 1000 bootstrap replications in MEGA X [22]. The sequence of Plectus aquatilis Andrássy, 1985 (KX017524) was used as the outgroup.

Molecular analyses
The phylogenetic trees carried out with the Neighbour-Joining (Supplementary material Figure S1) and Maximum-Likelihood (Fig. 2)

Discussion
The new species P. (Me.) valladaresi n. sp. was included in the subgenus P. (Mesopectines) after an accurate analysis of the morphological characteristics of the buccal capsule and associated structures, and also of the male caudal extremity. The buccal capsule is oriented dorsally, with a more developed ventral wall in relation to the dorsal one. The oral opening is surrounded by a crown of regular denticles. There are three oesophageal teeth of similar development (one dorsal and two lateroventral). The lateroventral teeth are denticulated. Finally, the number, morphology and distribution of caudal papillae in males are other important features. They are arranged as follows: two precloacal pairs, one unpaired precloacal papilla, one pericloacal pair and seven postcloacal pairs. Moreover, they are sessile and aligned, and follow the Spirurida arrangement type of male cloacal papillae [7,31]. Because of all these features, these rictulariids were attributed to the subgenus P. (Mesopectines) [6,31].
Considering the caudal extremity of males, the arrangement of cloacal papillae is another particularly important feature to discriminate species between subgenera. The presence of ten pairs of cloacal papillae, plus an unpaired precloacal papilla and a pair of phasmids near the tail tip is a characteristic of all rictulariids [31]. However, cloacal papillae are difficult to observe and in several studies all the papillae have not been described [41]. Thus, male representatives of this nematode family have three types of posterior extremities according to the morphology and arrangement of cloacal papillae: the type Ascaridida, with a dorsolateral disposition of the pairs of papillae 1, 4 and 8; the type Spirurida, with a linear or almost linear arrangement of papillae; and a third type with grouped papillae and with the presence of some pedunculated papillae [7,31]. The posterior extremity type Ascaridida is present in species of the genus Rictularia and in those of the subgenera P. (Paucipectines), P. (Neopaucipectines) and P. (Pterygodermatites); the posterior extremity type Spirurida is present in species of the subgenus P. (Mesopectines) and the third type of posterior extremity with grouped and pedunculated papillae is present in representatives of the subgenus P. (Multipectines) [5,6,27,31].
The position of the vulva in relation to the oesophagusintestine junction is another important female character, which is useful to differentiate P. (Me.) valladaresi n. sp. from the remaining species of the subgenus P. (Mesopectines). Although there may be some intraspecific variability, the position of the vulva in relation to the oesophagus-intestine junction is, in general, a constant character for species [30]. In the new species, the position of the vulva is anterior to the oesophagus-intestine junction. A similar position is found in nine species, namely P.  [3,4,7,9,10,12,14,20,30,43]. In this case, using the previously discussed characters, all these species were already differentiated from the new species, except for P. (Me.) caucasica. The available data of P. (Me.) caucasica are very scarce and concern only females [13,44]; however, the number of prevulvar cuticular projection pairs is slightly lower than in the new species. Additionally, the parasitized host (Meriones meridianus) and the geographical distribution (Mongolia and North Caucasus, Russia) are other criteria to distinguish P. (Me.) caucasica from the new species. The remaining species of the subgenus P. (Mesopectines) present a posterior or unknown position of the vulva in relation to the oesophagus-intestine junction (see Table 1).
The geographical distribution and host range are other aspects considered in the present study. Species of P. (Mesopectines) are parasites of Rodentia, Carnivora and All measurements are given in lm. Primates and have been found in Asia and Africa, except for some species parasites of Primates detected in zoos, e.g. P. (Me.) nycticebi in zoos of Chicago, Oklahoma, Washington or Wisconsin (USA) [14,28,47,48], Germany [24,40], Japan [19,38] or Switzerland [1]. Pterygodermatites (Me.) valladaresi n. sp. was found on five islands of the Canary Archipelago parasitizing Mus musculus (Fig. 1). In the case of P. (Mesopectines) species parasitizing rodents, numerous individuals have been collected from rodents of the families Muridae and Sciuridae (see Table 1). In Muridae, other than P. (Me.) valladaresi n. sp., only two species have been described parasitizing species of the genus Mus, namely P. (Me.) magna in Mus sp. from Angola and P. (Me.) witenbergi in Mus musculus from Israel [13,31,34]. In the Canary Archipelago, there are only three species of murids: M. musculus, R. norvegicus and R. rattus. During the present study, between the years 2008 and 2012, we found specimens of another Pterygodermatites species in three R. rattus trapped in the same biotopes of Fuerteventura Island (La Oliva) and El Hierro Island (Lagartario) [36]. Among other aspects, the Pterygodermatites individuals found in R. rattus were characterized by having 65-66 cuticular projection pairs, subequal spicules (right: 62-64 lm; left: 69-72 lm) and 2 or 3 midventral fans (unpublished data). Thus, P. (Me.) valladaresi n. sp. may be specific to M. musculus. Quentin [31] argued about the geographic origin and evolution of rictulariids. For the genus Pterygodermatites, it seems that its geographic origin was an area of the Palearctic region between Siberia and Canada, and these archaic species belong to the subgenus P. (Paucipectines). Thus, this subgenus includes the most primitive species within the genus Pterygodermatites, which are parasites of Cricetidae rodents in the Palearctic and Nearctic regions. In fact, Quentin [31] considered the apical oral opening, the reduced number of prevulvar combs and the type Ascaridida for the arrangement of cloacal papillae as the primitive characters present in the subgenus P. (Paucipectines). According to this author, the remaining subgenera, including P. (Mesopectines), would have evolved from this archaic subgenus: the buccal capsule became progressively dorsal, oral ornamentations became increasingly diverse and cloacal papillae became more or less aligned and grouped. For the subgenus P. (Mesopectines), the buccal capsule is more or less dorsal according to the species and the oral ornamentations are variable, with species showing a crown of regular denticles as occurs in P. (Me.) valladaresi n. sp., while other species show quadrangular denticles [e.g. P. (Me.) whartoni] or with two ventral apophysis [e.g. P. (Me.) leiperi].
In conclusion, the morphologic characteristics of the buccal capsule and associated structures, the particular characters of males (arrangement of cloacal papillae, number of cuticular projection pairs, size of spicules, and number of fans) and females (number of prevulvar and total cuticular projection pairs, body level of the transition from combs to spines and position of the vulva in relation to the oesophagus-intestine junction), the molecular data, and the geographical distribution and parasitized host identify the discovered nematode as a new species of the subgenus P. (Mesopectines).