First record of Porocephalus cf. clavatus (Pentastomida: Porocephalida) as a parasite on Bothrops asper (Squamata: Viperidae) in Costa Rica

Pentastomids are parasites that infect respiratory cavities of vertebrates, they are pretty common but poorly known in wildlife veterinary. A Bothrops asper snake (Garman, 1884) was captured in the Caribbean region of Costa Rica and had its lung infested with pentastomids, identified as ca Porocephalus clavatus (Wyman, 1845). This represents the first record of Porocephalus (Humboldt, 1812) on B. asper as well as P. cf. clavatus in Costa Rica. Further studies are needed to clarify their taxonomic position, images and scanning electron microscopy photographs (SEM) of the specimens are given.


Introduction
Pentastomids are parasites in the respiratory tract of vertebrates (Almeida et al., 2007(Almeida et al., , 2008a with 144 recent species, particularly common in reptiles (Christoffersen and Assis, 2013;Riley, 1986). The pentastomiasis is well known in animals that come from the captivity but little information is available on wild animals. This group is neglected both in the books and in the curriculum of veterinary medicine, those veterinarians who practice herpetological medicine have a higher probability to find these wormlike organisms. The pentastomes of reptiles have zoonotic potential, but among these parasites only Armillifer, Raillietiella, and Porocephalus have been associated with human infections (Riley, 1986;Qiu et al., 2005;Meyers and Neafie, 2011).
The genus Porocephalus comprises nine species that infect lungs of snakes in America and Africa (Christoffersen and Assis, 2013;Gomez-Puerta et al., 2011;Poore, 2012). This genus is recognized by females' head swollen but without a neck, annuli obvious in nymphs but not in adults, oval or keyshaped mouth in the line of the inner hooks and by internal hooks simple and external hooks double in both, nymphs and adults (Riley and Self, 1979). This spinous extension over the external hook (so called First record of Porocephalus as parasite on Bothrops asper in Costa Rica double hook) is the only consistent character for this genus (Riley and Walters, 1980).
Porocephalus has been reported from at least a dozen species and subspecies of snake hosts ranging from North to South America where they could be found occasionally in intermediate hosts such as dogs (Brookins et al., 2009), primates (Pereira et al., 2010), opossums, rodents and other mammals (Christoffersen and Assis, 2013;Poore, 2012). The knowledge on Central American species and diversity remains mostly unknown. In this paper we inform for the first time the presence of Porocephalus cf. clavatus, in the lungs of a Bothrops asper (wild female specimen). Images and descriptions of the affected tissues and the pentastomids recovered are given.

Material and Methods
A Bothrops asper female was collected in Siquirres, Limón (28/III/2011) in the Caribbean region of Costa Rica and translated to the Department of Pathology at the Escuela de Medicina Veterinaria, Universidad Nacional (UNA). The animal presented low weight, severe dehydration, slow movements and an alike nodular lesion on the left eye. Euthanasia was performed immediately by Randall Arguedas and Gilbert Alvarado (both wildlife veterinarians), euthanasia solution was administrated via cardiac puncture and animal was previously anesthetized.
Necropsy revealed the presence of pentastomids in the lung and the gastrointestinal cavities, specimens were isolated and stored in 70% ethanol.
Identification of the pentastomids was made according to Vargas (1970) and Riley and Self (1979). Specimens' features were examined under a Nikon SM2800 stereomicroscope at the UNA and photographed with a Pentax Optio WG1 digital camera. Scanning electron images were obtained with a Hitachi S3700N scanning electron microscope in variable pressure mode at the Microscopic Structures Research Center (CIEMIC), University of Costa Rica (UCR). Annuli of all specimens recovered (but two males in bad conditions) were estimated directly at the UNA, length measures were obtained from photographs with the program UTHSCSA Image Tool for Windows Version 3.00. Data on immatures are not presented.

Results
Six females and 13 males were extracted from the snake examined. The number of annuli in females and males ranged between 34-36 and 39-45 respectively ( Table 1), bodysize was estimated in 67.54±6.63mm for females (N=6) and 20.39±2.38mm for males (N=13). Most of them were found in the lung cavity (Figure 1), only one male was isolated from visceral cavities.

Taxonomic status
According to Riley and Self (1979), the specimens found belong to P. clavatus based on the number of annuli and previous reports, however, the sizes in Table 1 for females exceed the ranges established for this species. Pentastomids found by those authors in Bothrops had the same number of annuli and the same hooks to those of P. clavatus but as the case of the specimens reported herein, the females size was much larger.
We agree with Riley and Self that this is probably a new species but just size is not enough support for segregation (Riley and Self, 1979) since it could be influenced by hosts and age (Vargas, 1970). Hook parameters could be key information for species segregation (Riley and Self (1979) but in this case such data could not be determined because the low number of specimens collected (see Figure 2 for hooks photographs). This is the first report of Porocephalus ex Bothrops in the asper species and of this cf. P. clavatus for Costa Rica. Regrettably, information on collection sites and host species are not accurate in all sources and the so-called "SouthAmerica" could be referring to the lands at the south of the United States of America, maybe including Central and SouthAmerica sensu stricto. In the latter region, Brazilian snakes are common host for pentastomids (Almeida et al., 2006(Almeida et al., , 2007(Almeida et al., , 2008bÁvila et al., 2013;Brito et al., 2012) and since some of these snake's species are distributed in northern areas (Central America i.e.) future records of Pentastomids might be possible if sampling and searching increase. Further studies on this taxa ex Bothrops asper should be addressed with a greater number of specimens and hosts to clearly define its taxonomic position.

Conclusion
Even though pentastomes are not well known in wild life veterinary, they could be very common to find in snakes and other reptiles. Further surveys are necessary to clarify the species definition within the genus Porocephalus and increase the knowledge on the diversity of this group in tropics. The pentastomids found in this case are the first report ex Bothrops asper and could belong to a new species.