A new species of Cosmocerca (Nematoda, Ascaridomorpha) from the marine toad Rhinella marina (Linnaeus) (Anura, Bufonidae) in Australia

Abstract The marine toad Rhinella marina (Linnaeus) (Anura, Bufonidae) is a notorious, exotic amphibian species in Australia. However, our present knowledge of the composition of the nematode fauna of R. marina is still not complete. In the present study, a new cosmocercid nematode, Cosmocerca multipapillatasp. nov., was described using both light and scanning electron microscopy, based on specimens collected from R. marina in Australia. Cosmocerca multipapillatasp. nov. can be easily distinguished from its congeners by the body size, the presence of lateral alae and well sclerotized gubernaculum, the number and arrangement of plectanes and rosettes and the length of spicules, oesophagus and tail.


Introduction
The marine toad Rhinella marina (Linnaeus) (Anura, Bufonidae) is a large, terrestrial toad, which is natively distributed in Central and South America (Zug and Zug 1979;Lever 2001). The species has been widely introduced to the United States, Fiji, Philippines, Papua New Guinea, Australia, Japan, the Caribbean and some Pacific islands for controlling agricultural pests (Alford et al. 1995;Frost 2016). The helminth fauna of R. marina was studied by many authors and over 30 species of nematode parasites have been recorded from this host (Brenes and Bravo-Hollis 1959;Speare 1990;Goldberg and Bursey 1992;Barton 1996;Bursey et al. 2000;Kuzmin et al. 2007;Espinoza-Jimenez et al. 2007;Bursey and Brooks 2010;Drake et al. 2014).
During a helminthological survey in Australian amphibians, some nematodes belonging to the Cosmocercoidea Travassos, 1925 were collected from R. marina. Their examination using light and scanning electron microscopy revealed that these specimens represented a new species of Cosmocerca Diesing, 1861.

Light and scanning electron microscopy
Nematodes were collected from the intestine of the marine toad R. marina (Linnaeus) (Anura, Bufonidae) in various locations from Queensland, Australia. Specimens were fixed and stored in 70% ethanol until study. For light microscopy studies, nematodes were cleared in lactophenol. Drawings were made with the use of a Nikon microscope drawing attachment. For scanning electron microscopy (SEM), specimens were refixed in a 4% formaldehyde solution, post-fixed in 1% OsO4, dehydrated via an ethanol series and acetone, and then critical point dried. Samples were coated with gold and examined using a Hitachi S-4800 scanning electron microscope at an accelerating voltage of 20 kV. Measurements (the range, followed by the mean in parentheses) are given in micrometers (μm) unless otherwise stated. Type specimens were deposited in College of Life Sciences, Hebei Normal University, Hebei Province, China.
Type locality. Bloomfield (approximately 180 km north of Cairns), northern Queensland, Australia. Etymology. The specific epithet is derived from a combination of the Latin words multi-(multiple) and papillata (bearing papillae), referring to the characteristic numerous pre-cloacal plectanes.  Baker and Green 1988;Bursey et al. 2015).

Species of Cosmocerca
Cosmocerca multipapillata sp. nov. can be easily distinguished from the three above-mentioned species by having males with many more plectanes (10-12 pairs in the new species vs only 4-5 pairs in the other three) and a distinctly longer tail in females (0.22-0.38 mm, representing 6.65-12.4% of body length in the new species vs 0.14-0.22 mm, representing 3.25-6.33% of body length in the other three species) (Johnson and Simpson 1942; Baker and Green 1988;Bursey et al. 2015). Johnson and Simpson (1942) described C. australiensis Johnson &Simpson, 1942 andC. propinqua Johnson &Simpson, 1942 both from L. dorsalis in Australia. Both of them should be treated as incertae sedis, because only female specimens were found. In fact, Inglis (1968) considered that C. australiensis and C. propinqua should be transferred to Parathelandros Baylis, 1930 (Oxyurida, Pharyngodonidae) based on the morphological characters of the female. Nevertheless, the new species differs from C. australiensis and C. propinqua by the distinctly smaller body size in the female (2.68-3.73 mm in C. multipapillata sp. nov. vs 5.0-9.0 mm in the other two species). In addition, the position of the vulva and the morphology of the female tail of C. multipapillata sp. nov. are also different from C. propinqua (vulva situated in front of oesophageal bulb in this species). Moreover, the new species has a relatively longer oesophagus than that of C. australiensis (oesophageal length representing 9.08-12.8% of body length in C. multipapillata vs representing 3.89-4.67% of body length in C. australiensis) (Baker and Green 1988).
Based on morphological characters of the new species (i.e., the body size, the number of plectanes and the presence of well developed spicules and gubernaculum), we speculate that C. multipapillata sp. nov. could have been introduced to Australia along with its host R. marina, because all the recorded Cosmocerca species in the Australasian Region, including C. archeyi, C. australis and C. limnodynastes, have small body size (body length not over 2.00 mm in males), few plectanes (not over 5 pairs) and rudimental spicules and gubernaculum (Johnson and Simpson 1942;Baker and Green 1988;Bursey et al. 2015). However, some species of Cosmocerca found in the Neotropical Region have some common characters with the new species, for example, the body length of C. brasiliensis, C. travasssosi Rodrigues &Fabio, 1970, C. cruzi Rodrigues &Fabio, 1970 andC. podicipinus is more or less 3.0 mm or over 3.0 mm in males; C. brasiliensis, C. uruguayensis Lent &Freitas, 1948 andC. vrcibradici Bursey &Goldberg, 2004 all with many plectanes (7-9 pairs); C. brasiliensis, C. rara Freitas & Vicente, 1966 and C. vrcibradici with well developed spicules and/or gubernaculum (Lent and Freitas 1948;Freitas and Vicente 1966;Rodrigues and Fabio 1970;Bursey and Goldberg 2004). However, we need some more direct evidence to elucidate the origin of C. multipapillata sp. nov. in the Australasian Region or the Neotropical Region (i.e. if this new species is distributed in the Neotropical Region). Moreover, further studies on the composition of the Cosmocerca nematode fauna of native Australasian amphibians and rigorous phylogenetic studies to determine the interspecific relationships of Cosmocerca using genetic data including broad representatives worldwide (especially species from the Australasian and Neotropical Regions) are required to solve the evolutionary problem.