Use of iDNA in detecting elusive mammals: a case study on the Indonesian island of Seram

More than a fifth of all mammalian species on the planet are categorised as threatened. A significant proportion of these live in remote tropical forest and are difficult to survey hence their actual numbers cannot be determined. Distribution and abundance data for most tropical mammal species is data-deficient and therefore the development of cheap and reliable surveying methods is crucial to successfully assess these species. The use of carrion-feeding flies as vectors of mammalian DNA (referred to here as iDNA) and their application as a cost-effective tool for the assessment of mammalian biodiversity was first shown in 2013; however iDNA technology remains constrained by the persistence period of amplifiable iDNA in fly guts and the limitations of DNA preservation in remote locations. Fieldwork was carried out on the island of Seram, Indonesia. Archive data and interviews with indigenous forest users were used to optimise locations for the detection of Rhynchomeles prattorum, an endemic marsupial recorded from Seram in 1920. Carrion feeding flies were collected in montane forest, initially in the vicinity of a butchering site of Cuscus (Phalangeridae) (n = 99), and subsequently at remote sites in pristine forest (n = 50). At all locations camera trapping was conducted to provide independent evidence of mammal species present. Flies were individually dissected in the field and gut contents smeared onto FTA cards to preserve iDNA, or, if too small to facilitate dissection (n = 57), were placed individually into 95% ethanol at ambient temperature. Following extraction, a PCR using pan-mammalian 12S mitochondrial DNA (mtDNA) primers yielded bands of the expected size. These bands were purified and sequenced, and a BLAST search confirmed the presence of Cuscus DNA, as well as other mammal species from the island. A lower recovery of amplifiable iDNA from flies collected in remote locations correlated with a longer processing time for flies post capture, potentially indicating a limitation of current iDNA methodologies. In order to extend the time interval required to remove flies from traps for successful iDNA amplification, a new fly trap incorporating Propylene glycol as a fixative was designed and its capture efficacy evaluated. Through a laboratory feeding experiment using newly pupated blow flies Calliphora vomitoria the persistence period of amplifiable iDNA in the new trap was evaluated in comparison to a conventional dry fly trap design. After exposure to beef liver, flies were subsequently trapped in conventional dry traps and in the new fly trap. At 24, 48, 72, 96, 120 and 144 hours post-trapping flies were removed and their guts dissected onto FTA cards. Following extraction, a PCR using Beef Specific Primer (BSP) yielded bands of the expected size. An average higher intensity of band derived from flies caught in the new trap indicates its partial success in extending the persistence period of amplifiable iDNA.