Occurrence of Dipetalonema gracile in a wild population of woolly monkey Lagothrix poeppiigii in the northeastern Peruvian Amazon

Dipetalonema gracile (Rudolphi, 1809) (Filarioidea: Onchocercidae) is one of six species of cavities filarial parasites of Neotropical non-human primates. The present study recorded the occurrence of D. gracile, provides morphological and morphometric data and extends the geographical distribution. Adult filariae were obtained from the thoracic and abdominal cavities of 38 specimens of woolly monkey, which were used for local human consumption, in the northeastern Peruvian Amazon. Male and female filarids were processed and analysed using light and scanning electron microscopy. Details of the cephalic papillae, post-cloacal bands and papillae, vulva, phasmid position and lateral appendages are showed by scanning electron microscopy and is recorded the occurrencce of Lagothrix poeppigii monkey as a new host of this filaria in the Yavari-Mirin river basin, Peruvian Amazon.

However, the geographical distribution of Dipetalonema throughout the Neotropics is imprecise because most records are from captive primates or only one host, such as D. freitasi in a necropsy of Cebus capucinus at the London Zoo (BAIN et al., 1987), D. robini in Saimiri sciureus in Guyana (PETIT et al., 1985), D. graciliformis in Saguinus midas in French Guiana (BAIN et al., 1986), and D. yatesi in Ateles chamek in the Beni region of Bolivia (NOTARNICOLA et al., 2007).In the case of D. caudispina and D. gracile, most records in the last century have been concentrated in Brazil and Guiana (FREITAS, 1943;NOTARNICOLA et al., 2008) and cover a larger number of non-human primate hosts (WEBBER, 1955;NOTARNICOLA et al., 2008).
According to Dunn & Lambrecht (1963), D. gracile has been reported in the Neotropics in Ecuador, Peru and Brazil as a parasite of S. sciureus, Ateles paniscus and Saguinus nigricollis, but like the other species of the genus, most reports are not geographically explicit.Furthermore, only the D. gracile specimens recorded in S. sciureus have complete descriptions of their morphometric data made by Bain et al. (1986) for specimens from French Guiana (49 ED, Collection of the National Museum of Natural History) and by Notarnicola et al. (2008) for specimens from the Ecuadorian Amazon (CHMLP Nro5556 and Nro5557, Collection of the Museo de La Plata).
The present study describes, for the first time, parasitism by D. gracile in the silvery woolly monkey, Lagothrix poeppigii (Schinz, 1844), which is one of the largest non-human primates found in the western Amazon and is commonly hunted for food by human subsistence hunters.In addition to extending the list of host species, this study performed an extensive morphological analysis of D. gracile with use of scanning electron microscopy.

Materials and Methods
Between 2009 and 2013, 38 silvery woolly monkeys (18 males and 20 females) were killed for subsistence hunting in the Yavari river basin, which is between the Yavari and Yavari-Mirin rivers (04°19'53" S, 71°57'33" W; UT-5:00) in the northeastern Peruvian Amazon in the Loreto region.According to Bodmer et al. (2003), hunting is a common activity in the area, so local hunters and residents have been incorporated into a natural resource management programme and taught to remove and identify the abdominal and thoracic organs of primate prey, preserve them in a 10% formaldehyde fixative solution and deliver them to researchers.
The protocol authorizing this study and the sample collection was approved by the Peruvian Forest and Wildlife Service (Servicio Forestal y de Fauna Silvestre; Committee for Ethical Wildlife Research, protocol no.0127-2010, 0229-2011 and 0350-2012-DGFFS-DGEFFS).The helminths were retrieved from the external surfaces of the thoracic and abdominal organs of the silvery woolly monkeys, washed in saline solution and preserved in 70% ethanol.The samples were then shipped to the Laboratory of Cell Biology and Helminthology of the Institute of Biological Sciences at the Federal University of Pará, Brazil, under import permit no.02309-MINAGRI-SERFOR.
Following Gardner's (1996) protocol, the helminths were dehydrated in ethanol and cleared in 50% Amann's lactophenol to study their morphological and morphometric characteristics, and cross-sections of the nematodes were placed on slides with coverslips and analysed.Some of the samples were analysed using an Olympus BX41 microscope and an Olympus SZX12 stereomicroscope (Melville, New York, USA), both of which were equipped with a light chamber.
Samples were prepared for scanning electron microscopy following the protocol of Furtado et al. (2010).Male and female helminths were sectioned at 2 cm from their anterior and posterior ends, and the parts were analysed using a TESCAN Vega 3 LMU scanning electron microscope (Kohoutovice -Czech Republic).The morphometric analysis was based on 20 adult male and 20 adult female specimens.Measurements of the total length of the nematodes and the caudal area rugosa of males were taken in millimetres, and measurements of the other anatomical structures were taken in micrometres.The obtained values are presented below in the following format: mean and standard deviation followed by the range between the lowest and highest values in parentheses.

Onchocercidae (CHABAUD & ANDERSON, 1959)
Dipetalonema gracile (RUDOLPHI, 1809) Description (Figures 1-4, Table 1) Long pale body with fine transverse striations and thickening on the sides along the length of the body beginning at the nerve ring and ending near the tip of the tail in both sexes (Figure 3A and E).The ratio of the size of females to males is 1:1.7.The cephalic structure has a smooth quadrangular shape with four cephalic papillae and four labial papillae located around a simple, small mouth composed of four dorsal and four ventral papillae; the amphids are arranged between the cephalic papillae (Figures 1B,2B,3A and 4A).The oesophagus is divided into the muscular region (anterior and short) and the glandular region (posterior and long), and the nerve ring is located in the muscular region of the oesophagus.Posteriorly to the nerve ring there is a small, discrete excretory pore (Figures 1A and 2A).In both males and females, the posterior extremity ends in a conic tail accompanied by caudal appendages on either side with the phasmid at the base (Figure 3F).

Discussion
Detailed analysis of the structures of male and female specimens found in 38 L. poeppigii hosts revealed their compatibility with D. gracile, initially denominated Filaria gracilis by Rudolphi (1809).Characteristics such as the difference in the size of the spicules and the divided oesophagus placed the specimens within the genus Dipetalonema, which was originated described by Diesing (1861).There are currently six filariae species of the genus Dipetalonema that are exclusive to the peritoneal and thoracic cavities of Neotropical primates: D. caudispina, D. graciliformis, D. yatesi, D. gracile, D. freitasi and D. robini.Specific denomination is possible due to differences in the morphology of the reproductive organs of these six species.
Among the female specimens of the six Dipetalonema species, the shape of the vagina is distinctive; D. caudispina, D. yatesi and D. freitasi have a simple, straight vaginal canal that is structurally less complex than those found in the other three species, D. robini, D. graciliformis and D. gracile; the latter two have vaginas with similar sinuous shapes.The vulvar opening of the D. gracile from L. poeppigii has well-developed labias as well as caudal appendages, of which those at the end of the tail have phasmids at the base.These characteristics are shown in detail for the first time by scanning electron microscopy, which also shows the lateral thickening along the body in both sexes of the nematode.The uterine microfilariae

Host
The structure of the left spicule of D. gracile described from L. poeppigii is similar to that described by Bain et al. (1986), with the division into three structures: the handle, membranous alae and flagellum; the latter two comprise what is termed the lamina, which has a ratio of 2.8 relative to the handle.Additionally, the membranous alae is larger, on average, than that in the only existing data reported by Bain et al. (1986).The right spicule is a simple structure with a mean length ratio of five relative to the left spicule, which was similar to the findings of Yeh ( 1956) and Notarnicola et al. (2008) and should be considered an important taxonomic trait in the species description.
In the male specimen, the cloacal papillae present marked differences, being 12 of circular format with difference in size and a papilla in format of central bar.These structures are more noticeable in scanning electron microscopy and were not highlighted in the studies of Bain et al. (1986) and Notarnicola et al. (2008).Another important male characteristic are the spiral twists in the area rugosa, which were found to be six to eight in number; these structures were larger in the present study than those found by Bain et al. (1986) and Notarnicola et al. (2008).This trait may vary given the difficulty of measuring that region of the body due to the twists in the tail.
A discrete excretory pore was observed in both sexes of the D. gracile parasite of L. poeppigii in this study, which was not previously reported.However, the location of the excretory pore relative to the anterior extremity coincides with the value described by Notarnicola et al. (2008) in the female.Compared to the data presented by Bain et al. (1986) and Notarnicola et al. (2008), additional morphometric differences were also observed in the present study, including the larger size of the ovijector and smaller sizes of the buccal capsule, oesophagus, gubernaculum, caudal appendages, distance of the vulva and nerve ring from the anterior extremity, and a significantly smaller tail in the female.However, there was no significant difference in the total body size found by those authors and in this study.These variations can be attributed to the inclusion of a much larger number of specimens in this study, which resulted in more complete morphometric data.Dunn & Lambrecht (1963) reported finding specimens of D. gracile in some primate hosts in the Peruvian Amazon without providing the correct taxonomic identification.These authors reported A. paniscus, T. nigricollis (S. nigricollis), S. sciureus and S. boliviensis (DUNN & LAMBRECHT, 1963) as hosts without specifying exact geographical locations.However, it's already known that the distribution of D. gracile or other species of filariae may be much wider, given that there are more than 40 species of primates in Peru and large geographical areas that have not yet been studied.Other initial reports for D. gracile or synonyms in the Americas (BOULENGER, 1920;WEBBER, 1955) are based on zoo animals or individual cases for which the precise geographic origin of the host, and hence the parasite, is unknown, hampering efforts to assemble an ideal and accurate record.The 38 infected hosts in our study represent a wide geographic distribution of filariasis in non-degraded natural areas and are not limited to representing hosts in captivity.
Due the great diversity of hosts and the difficulty of reaching large geographic areas for research, gaps in our knowledge of natural infections of filariae in Neotropical primates remain.Therefore, in situ studies are needed to gain an accurate view of the distribution of filariae in natural areas.We have presented the occurrence of D. gracile as a parasite in the thoracic and peritoneal cavities of L. poeppigii under natural infection conditions with additional details about its morphometry and morphology as well as images of its ultrastructure.This is the first record of its presence in this host and in the Amazonian region of northeastern Peru.

Figure 2 .
Figure 2. Morphology of D. gracile Female: (A) Cephalic end, lateral view with details of the nerve ring, excretory pore, glandular and muscular oesophagus.Bar = 100 µm; (B) Cephalic end, apical view.Bar = 50 µm; (C) Cross section of the body at the level of the intestine and ovijector.Bar = 100 µm; (D) Uterine microfilaria.Bar = 10 µm; (E) Posterior end with details of the lappets and anal aperture.Bar = 100 µm; (F) Lateral view with details of the vulvar aperture and vagina.Bar = 100 µm.
gracile from L. poeppigii have morphometric values similar to those found by