Parasite detection in the ornamental fish trade using environmental DNA

Abstract

Effective border control relies on stringent biosecurity protocols to detect and prevent introductions of exotic pests and diseases. Detection of pathogens and parasites in the live ornamental fish trade using environmental DNA (eDNA) techniques has the potential to improve current biosecurity practices. We examined water samples from 11 target consignments (cyprinids susceptible to Dactylogyrus spp. infections) and seven non-target fish consignments (non-cyprinids, not susceptible to Dactylogyrus spp. infections) imported from Southeast Asia to Australia for the presence of eDNA from five Dactylogyrus species (Monogenea: Dactylogyridae). A four-step predictive framework was used to predict putative positive and putative negative detections from quantitative PCR assays. Both target and non-target consignments were positive for Dactylogyrus spp. eDNA as confirmed by Sanger sequencing. Positive detections for Dactylogyrus spp. eDNA in non-target fish consignments demonstrates the possibility of source water contamination, limiting the applicability of eDNA screening methods at border control. This study suggests that screening for parasite eDNA within ornamental fish consignments should be tested during pre-export quarantine periods to avoid false positive detections at border control. Lastly, the proposed predictive framework has a broad utility for minimizing false positive and false negative eDNA detections of aquatic organisms.

Pooled samples and eDNA-based detection can facilitate the “clean trade” of aquatic animals

“…The formulae presented below can account for false positives (i.e., less than perfect specificity, Sp). It will be important to consider the sources and likelihood of false positives (e.g., from carry over of target DNA from the water rather than infected hosts 34,73 vs. from contamination in a laboratory) as well as the consequences (e.g., slowing shipments, economic costs, loss of trust 32,34 ). In the case of Bsal detection, I would hope that any positive test would be investigated further with additional, independent diagnostic tests.…”

“…In the case of Bsal detection, I would hope that any positive test would be investigated further with additional, independent diagnostic tests. Moreover, setting aside the issue of carry over contamination 34 , which might be avoided by transferring animals to clean water prior to sampling, there is no evidence that false positive rates are higher in pools of samples or eDNA than typical individual samples. In fact, to the extent that fewer tests are conducted with pooling or eDNA one would expect fewer false positives at a given level of surveillance sensitivity.…”

“…(Semi-aquatic species or life stages are often shipped in moist sphagnum moss or paper, but can then be placed in water to collect eDNA during a quarantine period 26 .) Environmental DNA sampling is being used to detect pathogens in a growing number of settings, from natural environments [27][28][29][30] to ballast water 31 and even in trade [32][33][34] . However, while the statistical framework for making proper inference from eDNA results is well-developed in certain contexts (e.g., the presence or absence of a target species in ponds using occupancy models 29,35 ), these approaches do not translate well to sampling small captive populations or consignments.…”

In-situnon-lethal rapid test to accurately detect the presence of the nematode parasite,Anguillicoloides crassus, in European eel,Anguilla anguilla

“…The mitochondrial gene COI, has been widely used to detect the presence of nematode parasites in commercially important fish species (Godínez-González et al, 2017;Herrero, Vieites, & Espiñeira, 2011;Paoletti et al, 2018;Santos et al, 2006). Many studies rely on qPCR or Real Time PCR for fish pathogens detection, but such approaches are challenging and expensive to deploy in the field (Chlo Suzanne Hutchins et al 2018;Trujillo-González et al 2019).…”

Detection of the parasitic nematode, Pseudocapillaria tomentosa, in zebrafish tissues and environmental DNA in research aquaria

“…Water tests have been developed for a variety of parasites of fishes, including Ceratonova shasta (Hallett & Bartholomew, 2006), Nanophyetus salmonicola, Gyrodactylus salaris (Rusch et al, 2018), Dactylogyrus spp. (Trujillo-González, Edmunds, Becker, & Hutson, 2019), Neobenedenia girellae (Agawa, Tani, Yamamoto, Hirano, & Shirakashi, 2016) and Chilodonella hexasticha (Bastos Gomes et al, 2017).…”