qPCR-based environmental monitoring of Myxobolus cerebralis and phylogenetic analysis of its tubificid hosts in Alberta, Canada.

Myxobolus cerebralis is the causative agent of whirling disease in salmonid fishes. In 2016, this invasive parasite was detected in Alberta, Canada, for the first time, initiating a comprehensive 3 yr monitoring program to assess where the parasite had spread within the province. As part of this program, a qPCR-based test was developed to facilitate detection of the environmental stages of M. cerebralis and from the oligochaete host, Tubifex tubifex. During this program, ~1500 environmental samples were collected and tested over 3 yr. Fish were collected from the same watersheds over 2 yr and tested as part of the official provincial monitoring effort. Substrate testing identified sites positive for M. cerebralis in 3 of 6 watersheds that had been confirmed positive by fish-based testing and 3 novel detections where the parasite had not been detected previously. Testing of individually isolated Tubifex from each sample site was used to further confirm the presence of M. cerebralis. DNA barcoding of the cytochrome oxidase I (cox1) gene of 567 oligochaete specimens collected from 6 different watersheds yielded 158 unique sequences belonging to 21 genera and 37 putative species. Phylogenetic analyses of sequences assigned to the genus Tubifex predicted 5 species of Tubifex arising from this assessment. Based on our results, we propose that environmental and worm samples can be a valuable complement to the gold-standard fish testing and will be especially useful for monitoring in areas where fish collection is challenging or prohibitive because of site accessibility or vulnerability of the fish populations.

Shoaling as an antiparasite defence in minnows (Pimephales promelas) exposed to trematode cercariae.

1. Individuals that live in groups benefit from increased foraging success and decreased predation. Protection from some types of parasites may provide an additional benefit of group-living. For fish, the extent to which shoaling can reduce an individual's risk of exposure to the infective stages of parasites is unknown. 2. We tested for antiparasite benefits of shoaling in fathead minnows exposed to larvae (cercariae) of two of their most common species of trematode, Ornithodiplostomum ptychocheilus and Posthodiplostomum minimum. As developing stages (metacercariae) of these trematodes cause reductions in minnow activity, growth and survival, natural selection should favour the evolution of cercariae-avoidance behaviours. 3. We evaluated shoal dimensions in groups of minnows exposed to O. ptychocheilus and to other chemical/physical stimuli within aquaria. To compare risk of exposure in shoaling vs. non-shoaling fish, we confined groups of minnows into mesh cages in outdoor mesocosms, exposed them to cercariae, then compared mean worm numbers in grouped vs. solitary fish. Lastly, we tested whether fish located within the centre of an artificial shoal reduced their risk of cercariae exposure compared with those along peripheral edges. 4. Minnows distinguished infective cercariae from other potential aquatic threats and responded with activity that reduced the 2-dimensional area of their shoals 15-fold compared with water-only controls. Fish confined within artificial shoals had 3-fold fewer worms than single fish and minnows located within the centre of artificial shoals had significantly fewer worms than those without peripheral minnows. 5. These results show that shoaling reduces a minnows' risk of exposure to cercariae, either directly via detection of cercariae in the water column followed by behavioural avoidance or indirectly via behaviour-mediated differences in exposure between shoaling vs. non-shoaling fish.

Epidermal 'alarm substance' cells of fishes maintained by non-alarm functions: possible defence against pathogens, parasites and UVB radiation.

Many fishes possess specialized epidermal cells that are ruptured by the teeth of predators, thus reliably indicating the presence of an actively foraging predator. Understanding the evolution of these cells has intrigued evolutionary ecologists because the release of these alarm chemicals is not voluntary. Here, we show that predation pressure does not influence alarm cell production in fishes. Alarm cell production is stimulated by exposure to skin-penetrating pathogens (water moulds: Saprolegnia ferax and Saprolegnia parasitica), skin-penetrating parasites (larval trematodes: Teleorchis sp. and Uvulifer sp.) and correlated with exposure to UV radiation. Suppression of the immune system with environmentally relevant levels of Cd inhibits alarm cell production of fishes challenged with Saprolegnia. These data are the first evidence that alarm substance cells have an immune function against ubiquitous environmental challenges to epidermal integrity. Our results indicate that these specialized cells arose and are maintained by natural selection owing to selfish benefits unrelated to predator-prey interactions. Cell contents released when these cells are damaged in predator attacks have secondarily acquired an ecological role as alarm cues because selection favours receivers to detect and respond adaptively to public information about predation.