Description of Pudica wandiquei n. sp. (Heligmonellidae: Pudicinae), a nematode found infecting Proechimys simonsi (Rodentia: Echimyidae) in the Brazilian Amazon

Abstract A new species of nematode parasite of the subfamily Pudicinae (Heligmosomoidea: Heligmonellidae) is described from the small intestine of Proechimys simonsi (Rodentia: Echimyidae) from the locality of Nova Cintra in the municpality of Rodrigues Alves, Acre state, Brazil. The genus Pudica includes 15 species parasites of Neotropical rodents of the families Caviidae, Ctenomyidae, Dasyproctidae, Echimyidae, Erethizontidae, and Myocastoridae. Four species of this nematode were found parasitizing three different species rodents of the genus Proechimys in the Amazon biome. Pudica wandiquei n. sp. can be differentiated from all other Pudica species by the distance between the ends of rays 6 and 8 and the 1-3-1 pattern of the caudal bursa in both lobes.

The echimyid rodents of the genus Proechimys Allen, 1899 have been reported as reservoirs of parasitic zoonoses and a potential threat to public health (Cordeiro et al., 2015a). A total of 22 Proechimys species have been described from localities in Central and South America (Patton and Leite, 2015), and four Pudica species have been found parasitizing three of these species -Proechimys (2009), Vicente et al. (1997) and Durette-Desset et al. (2017). The holotype, allotype, and paratypes were deposited in the helminthological collection of the Oswaldo Cruz Institute (CHIOC) in Rio de Janeiro.

Description
General: Very small nematode, body coiled irregularly with two coils in both sexes. Round deirids close to excretory pore, both located halfway between nerve ring and esophagus-intestine junction. Cephalic vesicle present ( Figure 1A). Oral opening triangular, surrounded by small, thick ring. Four cephalic papillae, four external labial papillae, and two amphids in apical view ( Figure 1B).
Synlophe: Cuticle with longitudinal and uninterrupted ridges in both sexes. Ridges posterior to cephalic vesicle, extending close to copulatory bursa in male and anus in female. Females with eight ridges (careen, three dorsal, two ventral and one comarete), careen more developed The Amazon biome is the world's largest tropical forest, a region of high temperatures and abundant rainfall, with an extremely diverse biota, which provide ideal conditions for the evolution of an equally diverse parasitic fauna. However, ongoing anthropogenic impacts in the Amazon has provoked the widespread loss of habitats, and many parasite species, even before they have been discovered (Penna et al., 2009;Olival et al., 2017;Fernandes et al., 2018).
More and more studies on conservation biology are becoming necessary, and investigations of helminth fauna can help us in monitoring parasites, including in hosts that are threatened with extinction (Gomes et al., 2019;Souza et al., 2021).
The present study describes a new species of the genus Pudica found infecting the small intestine of Proechimys simonsi Thomas, 1900 at the locality of Nova Cintra, in the municipality of Rodrigues Alves, Acre state, Brazil.

Study site
Ten rodent specimens were collected during a five-day survey in south western Amazonia in May 2016. The specimens were collected in the municipality of Rodrigues Alves, Acre state, Brazil (7.8149S, 72.6799W), using a Tomahawk® Live Trap (USA) baited with a mixture of peanut butter, banana, oats, and bacon. All necessary biosafety procedures were respected during the handling of the specimens and the collection of the biological samples (Lemos and D'Andrea, 2014). The rodents were anesthetized and euthanized for the extraction of helminths and other biological samples following Costa- Neto et al. (2016). Voucher specimens (rodents) were deposited in the scientific collection of the UFRJ National Museum (Museu Nacional, Universidade Federal do Rio de Janeiro). All capture and handling procedures followed the guidelines of the Ethics Committee for the Experimental Use of Animals (CEUA) of the Oswaldo Cruz Institute -IOC (Instituto Oswaldo Cruz), under permit L-39/14. The collection of animal specimens was authorized by the Chico Mendes Institute for Biodiversity Conservation -ICMBio (Instituto Chico Mendes de Conservação da Biodiversidade) of the Brazilian federal government, through license number 13373.

Analysis of the helminths
The worms were washed in a 0.85% saline (NaCl) solution and stored in 70% ethanol. For light microscopy, the nematodes were clarified in lactophenol (40% glycerin, 20% lactic acid, and 20% phenol in q.s. 100 mL of water). The specimens were drawn using a camera lucida attached to a Nikon Eclipse E200MVR light microscope (Nikon Corporation, Tokyo, Japan). The morphological structures were measured using a Zeiss Axio Cam HRC digital imaging system (Zeiss, Germany), with the Axio Vision Rel accessory software, version 4.7. All measurements are given in micrometers. The measurements of holotypes and allotypes are followed by the ranger between brackets, and mean and standard deviation between parentheses of the paratypes. The nematodes were identified following Anderson et al. and comarete larger than other ridges in mid-body. Careen poorly developed at infundibulum, with ridges of equal size. Axis of orientation is from right to left in both sexes (Figure 2A, B, C). In male with eight ridges (careen, three dorsal, two ventral and one comarete), varying in size along body with careen more developed in the first third and comarete slightly larger than other ridges. In the median portion, more developed comarete; in posterior third, ridges decrease progressively in size ( Figure 2D, E, F).
Etymology: The species epithet is a tribute to José Wandique Fraga da Costa, a valuable and long-serving contributor to the Laboratory of the Biology and Parasitology of Wild Mammal Reservoirs (LABPMR) at the Oswaldo Cruz Institute in Rio de Janeiro, Brazil.

Discussion
Caviomorph rodents were the first to occupy the South American continent. Throughout its evolutionary history, it has undergone successive dispersion processes forming several taxa, with the Echimyidae family being considered the most diverse family of the infraorder Hystricognathi, occupying several ecological niches, distributed in different Neotropical regions with several species acting as a helminth host and reservoir of zoonosis (Patterson and Wood, 1982;Huchon and Douzery, 2001;Fabre et al., 2016;Eler et al., 2020).
Up to now, 15 Pudica species have been described, of which, seven are parasites of rodents of the family Echimyidae, Four of these species (P. minima, P. evandroi, P. tenua, and P. ginsburgi) were found parasitizing three different echimyid rodents of the genus Proechimys (P. roberti, P. semispinosus and P. longicaudatus) in the Amazon biome (Serrano et al., 2019). However, it is not yet known what the evolutionary relationship between the helminths parasites and these rodents.
According to Durette-Desset et al. (2017) the genus Pudica is characterized by a synlophe with a careen, made up of two continuous ridges, three to five continuous dorsal and ventral ridges, and at least two ventral comaretes; and a bursa of the 2-2-1 or 1-3-1 type, with the dorsal ray divided in its anterior half. In the present study, the presence of careen, three dorsal ridges, two ventral ridges, one ventral comarete, and a caudal bursa of 1-3-1 type, jointly with the absence of a gubernaculum, supported the inclusion of the helminth specimens collected in the municipality of Rodrigues Alves in the genus Pudica.
Compared with its congeners, Pudica wandiquei n. sp. has the same number of ridges found in Pudica gonosoma Cassone and Durette-Desset, 1991, that is, eight continuous ridges in both sexes (exclusive to these two species) therefore distinguishing itself from all other species of the genus. However, P. gonosoma can be differentiated from Pudica wandiquei n. sp. by the configuration of the caudal bursa. While Pudica wandiquei n. sp. presents a 1-3-1 pattern in both lobes, P. gonosoma has a 1-4 pattern, as described by Durette-Desset and Digiani (2012) when conducting a study on the caudal bursa in the Heligmonellidae. In addition the presence of a bursa membrane connecting the base of the rays 6 in P. gonosoma is not observed in Pudica wandiquei n. sp. Furthermore, female P. gonosoma specimens present a cuticular post vulvar expansion, structure not observed in Pudica wandiquei n. sp.
The pattern of the bursa observed in the new species resembles that found in Pudica cattani Digiani et al. 2017, P. evandroi (Travassos, 1937, and Pudica cercomysi (Durette-Desset and Tchéprakoff, 1969), but in general according to the classification key of Serrano et al. (2019) Pudica wandiquei n. sp. can be distinguished from these three species by the distance between the ends of rays 6 and 8, which are close in P. cattani, P. evandroi, and P. cercomysi and separate in Pudica wandiquei n. sp.
Furthermore, these three species differ from the new species by the following features: P. cattani has lateral and dorsal rays robust but not hypertrophied, rays 4 curved anteriorly, rays 6 curved posteriorly, and the dorsal lobe moderately developed; P. evandroi is characterized by the strong thickening of the lateral trunks of rays 3-5 and the trunk of the dorsal ray, and P. cercomysi has a dorsal ray dividing at proximal quarter of length, rays 6 and 8 parallel and similar in size. All these characters contrast markedly with Pudica wandiquei n. sp. which has the rays 2 to 6 reaching the edge of the caudal bursa, trunk of the dorsal ray not hypertrophied and rays 8 relatively short.
In conclusion, these diagnostic characters support the classification of the nematodes found infecting Proechimys simonsi in southwestern Amazonia as a new species, Pudica wandiquei n. sp., the sixteenth species of the genus described to date. Moreover, the study of biodiversity in the tropics, especially in the Amazon, is important and urgent, given the accelerated degree of loss of this biome by anthropic action. Knowing biodiversity and the relationship between species generates subsidies for conservation actions.