Helminths of 13 species of microhylid frogs (Anura: Microhylidae) from Papua New Guinea

ABSTRACT In an attempt to better document the invertebrate biodiversity of the threatened fauna of Papua New Guinea (PNG), 208 microhylid frogs representing 13 species collected in 2009 and 2010 in PNG were examined for endoparasitic helminths. This study found mature individuals of one species of Digenea (Opisthioglyphe cophixali), adults of two species of Cestoda (Nematotaenia hylae, Cylindrotaenia sp.) and cysticerci of an unidentified cestode species; adults of nine species of Nematoda (Aplectana krausi, Bakeria bakeri, Cosmocerca novaeguineae, Cosmocercella phrynomantisi, Falcaustra papuensis, Icosiella papuensis, Ochtoterenella papuensis, Parathelandros allisoni, Parathelandros andersoni), and one species of Acanthocephala (cystacanths in the family Centrorhynchidae). There was a high degree of endemism among the helminth species infecting the microhylids, with 83% of the species known only from PNG. Yet the helminth fauna infecting Papuan microhylids are generalists with direct life cycles (no intermediate host) that also infect other anuran species. We thus conclude infection is more dependent upon habitat conditions than diet. Twenty-nine new host records are reported.


Introduction
The landscape of Papua New Guinea (PNG) is threatened with habitat loss due to logging and resultant forest loss, conversion of land for agriculture, mining, removal of oil and natural gas, and habitat modification and competition from invasive exotic species (Marshall and Beehler 2007a). In view of these threats to the environment, it is crucial to document both invertebrate and vertebrate diversity in Papuan fauna while it is possible. Frogs are particularly sensitive to environmental degradation and many species have suffered serious population declines (Lips and Donnelly 2005). With the above in mind, an examination of frogs for helminths will add to our knowledge of invertebrate biodiversity from a threatened area.
Knowledge of tropical biodiversity is still very imperfect, and this is especially true for the invertebrates, which encompass the vast majority of species diversity. Helminths form a major portion of this diversity, and they can be important in in PNG where the frogs were collected are indicated in Figure 1. Geographic ranges for the above frog species can be found by searching under the species name at http://www.iucnredlist.org.
Immediately after capture, frogs were euthanised and fixed in neutral buffered 10% formalin, and were later stored in 70% ethanol in the herpetology collection of the Bishop Museum (BPBM), Honolulu, Hawaii (Appendix 1). Subsequently, the body cavity was opened by a longitudinal abdominal incision, and the gastrointestinal tract was removed by cutting across the esophagus and the rectum and then shipped to Whittier College, Whittier, CA, where a detailed dissection of each digestive organ was carried out using a dissecting microscope. Endoparasites from individual hosts were removed to vials of 70% ethanol, and later placed on a microscope slide in a drop of lactophenol under a coverslip and allowed to clear. Nematodes and acanthocephalans were identified from these temporary preparations. Digeneans and cestodes were washed in water, regressively stained in hematoxylin, and mounted in balsam for identification under a compound microscope. Parasite terminology used herein is in accordance with Bush  (1997). The number of parasites, prevalence, mean intensity, range of infection and new host records are given in Table 1. Hosts for helminths reported in Paupan frogs are summarised in Table 2. Selected helminth specimens were deposited in the Harold W. Manter Parasitology Laboratory (HWML), University of Nebraska, Lincoln, USA (Appendix 2).

Comparative endoparasite distributions
The digenean Opisthioglyphe cophixali was originally described from two specimens taken from Cophixalus parkeri collected at Kotuni, Mt. Otto, PNG by Moravec and Sey (1989). Other reports of Opisthioglyphe cophixali are in Table 2. Our records here expand the range of this helminth to several other frog species: Choerophryne darlingtoni, Cophixalus balbus, Oreophryne notata and Sphenophryne cornuta (Table 1); all are new host records.
The cestode Nematotaenia hylae was originally described from specimens taken from two frog species, the pelodryad Litoria ewingii (as Hyla ewingii) and the myobatrachid Crinia signifera collected in Burnie, Tasmania, by Hickman (1960). Reports of Nematotaenia hylae in other anurans are in Table 2. Nematotaenia hylae in Cophixalus variabilis and Oreophryne notata (Table 1) are new host records.
The nematode Aplectana krausi was described from Cornufer boulengeri (as Platymantis boulengeri) by . It has been reported from other microhylids, one pelodryad and ceratobatrachids (Table 2). Sphenophryne cornuta represents a new host record for Aplectana krausi.
The nematode Falcaustra papuensis was described from the scincid lizard Sphenomorphus simus from PNG by , and was also reported from the skinks Emoia atrocostata (Goldberg et al. 2010) and Prasinohaema flavipes (Goldberg et al. forthcoming) from PNG. It was previously found in Papuan ceratobranchids, pelodryads and microhylids (Table 2). Cophixalus desticans and Oreophryne geislerorum represent new host records for Falcaustra papuensis.
The nematode Parathelandros allisoni was described from the hylid Nyctimystes trachydermis from PNG . It was also reported from pelodryads and a microhylid (Table 2). Oreophryne biroi represents a new host record for Parathelandros allisoni.
Centrorhynchid acanthocephalan cystacanths are larval forms that utilise paratenic (= transport) hosts (never an obligatory host) in which development to the adult does not occur (Kennedy 2006). Cystacanths have been reported in other Papuan frog species, namely ceratobatrachids, pelodryads, microhylids and a ranid (Table 2). Cophixalus variabilis represents a new host record for centrorhynchid cystacanths.

Conclusions
There was a high degree of endemism among the adult helminth species infecting our sample of microhylids, with 83% of the species known only from PNG. Of the 12 helminth species represented by mature individuals reported in this study, nine species (75%) were originally described from Papuan anurans, whereas one of the 12 species, Falcaustra papuensis, was described from the skink Sphenomorphus simus ) from PNG. Of the nine nematode species capable of reaching maturity in microhylids, Aplectana krausi, Bakeria bakeri, Cosmocerca novaeguineae, Cosmocercella phrynomantisi, Parathelandros allisoni and Parathelandros andersoni are considered monoxenous and infect either orally or by skin penetration (Anderson 2000). Two others (Icosiella papuensis and Ochoterenella papuensis Onchocercidae) are transmitted by haematophagous insect vectors (Anderson 2000). Falcaustra nematodes are generally considered to require an ingested intermediate host. Among the other kinds of helminths infecting the Papuan microhylids discussed herein, the life cycle of the digenean Opisthioglyphe cophixali is not known but its congener Opisthiogylphe locellus utilises Lymnaea snails as intermediate hosts (Macey and Moore 1958). Both cestodes Nematotaenia hylae and Cylindrotaenia sp. belong to the Nematotaeniidae (Jones 1987) for which life cycles are not conclusively known. The data presented here suggest that Papuan microhylid frogs are largely infected by helminth generalists that have direct life cycles; thus, infection is more dependent upon habitat conditions than diet.