Intergenerational effects of manipulating DNA methylation in the early life of an iconic invader

In response to novel environments, invasive populations often evolve rapidly. Standing genetic variation is an important predictor of evolutionary response but epigenetic variation may also play a role. Here, we use an iconic invader, the cane toad (Rhinella marina), to investigate how manipulating epigenetic status affects phenotypic traits. We collected wild toads from across Australia, bred them, and experimentally manipulated DNA methylation of the subsequent two generations (G1, G2) through exposure to the DNA methylation inhibitor zebularine and/or conspecific tadpole alarm cues. Direct exposure to alarm cues (an indicator of predation risk) increased the potency of G2 tadpole chemical cues, but this was accompanied by reductions in survival. Exposure to alarm cues during G1 also increased the potency of G2 tadpole cues, indicating intergenerational plasticity in this inducible defence. In addition, the negative effects of alarm cues on tadpole viability (i.e. the costs of producing the inducible defence) were minimized in the second generation. Exposure to zebularine during G1 induced similar intergenerational effects, suggesting a role for alteration in DNA methylation. Accordingly, we identified intergenerational shifts in DNA methylation at some loci in response to alarm cue exposure. Substantial demethylation occurred within thesodium channel epithelial 1 subunit gammagene (SCNN1G) in alarm cue exposed individuals and their offspring. This gene is a key to the regulation of sodium in epithelial cells and may help to maintain the protective epidermal barrier. These data suggest that early life experiences of tadpoles induce intergenerational effects through epigenetic mechanisms, which enhance larval fitness.This article is part of the theme issue ‘How does epigenetics influence the course of evolution?’

Effect of Potential Sea Lamprey (Petromyzon marinus) Repellents on the Distribution of Juvenile Rainbow Trout (Oncorhynchus mykiss) in a Laboratory Environment

Recent studies have begun to consider the use of chemosensory alarm cues as potential repellents of invasive Sea Lamprey (Petromyzon marinus Linnaeus, 1758) in the Great Lakes Basin. An important factor in determining the efficacy of potential P. marinus repellents is whether they are species-specific. To that end, using laboratory stream channels, this study investigated whether a non-target species, the Rainbow Trout (Oncorhynchus mykiss Walbaum, 1792), would change their distribution in response to damage-released P. marinus alarm cues, potential mammalian predator cues, and damage-released conspecific cues. In groups of 10 individuals per replicate, with 10 replicates per stimulus type, subjects were exposed to one of the following treatment types: deionized water (control), P. marinus extract (heterospecific alarm cue), O. mykiss extract (conspecific alarm cue), 2-phenylethylamine hydrochloride (potential predator cue), and human saliva (potential predator cue). None of the stimuli induced a significant avoidance response during the stimulus observation period as compared to the control. These findings provide preliminary support for the species-specific nature of these proposed alternative P. marinus control measures.

Evidence of the peptide identity of the epidermal alarm cue in tadpoles of the toad Rhinella arenarum

Chemical cues associated with predation attempts allow prey to trigger defensive behaviours. Accordingly, tadpoles of several species of anurans display strong behavioural responses to chemical cues of injured conspecifics. As part of the antipredator response, tadpoles show rapid and sustained inhibition of activity when exposed to chemical cues of predation. Although the ability to respond to cues of conspecifics has been confirmed in a wide variety of anuran species, studies about the tissue source and the chemical aspects of the molecules involved are scarce and contradictory. In the present work, we analysed the chemical characteristics, tissue source and release mechanism of the chemical alarm cue in Rhinella arenarum tadpoles. Our results support the hypothesis that a peptide of epidermal origin in mediates amphibian tadpole communication.

Do Epigenetic Changes Drive Corticosterone Responses to Alarm Cues in Larvae of an Invasive Amphibian?

Synopsis The developmental environment can exert powerful effects on animal phenotype. Recently, epigenetic modifications have emerged as one mechanism that can modulate developmentally plastic responses to environmental variability. For example, the DNA methylation profile at promoters of hormone receptor genes can affect their expression and patterns of hormone release. Across taxonomic groups, epigenetic alterations have been linked to changes in glucocorticoid (GC) physiology. GCs are metabolic hormones that influence growth, development, transitions between life-history stages, and thus fitness. To date, relatively few studies have examined epigenetic effects on phenotypic traits in wild animals, especially in amphibians. Here, we examined the effects of exposure to predation threat (alarm cues) and experimentally manipulated DNA methylation on corticosterone (CORT) levels in tadpoles and metamorphs of the invasive cane toad (Rhinella marina). We included offspring of toads sampled from populations across the species’ Australian range. In these animals, exposure to chemical cues from injured conspecifics induces shifts in developmental trajectories, putatively as an adaptive response that lessens vulnerability to predation. We exposed tadpoles to these alarm cues, and measured changes in DNA methylation and CORT levels, both of which are mechanisms that have been implicated in the control of phenotypically plastic responses in tadpoles. To test the idea that DNA methylation drives shifts in GC physiology, we also experimentally manipulated methylation levels with the drug zebularine. We found differentially methylated regions (DMRs) between control tadpoles and their full-siblings exposed to alarm cues, zebularine, or both treatments. However, the effects of these manipulations on methylation patterns were weaker than clutch (e.g., genetic, maternal, etc.) effects. CORT levels were higher in larval cane toads exposed to alarm cues and zebularine. We found little evidence of changes in DNA methylation across the GC receptor gene (NR3C1) promoter region in response to alarm cue or zebularine exposure. In both alarm cue and zebularine-exposed individuals, we found differentially methylated DNA in the suppressor of cytokine signaling 3 gene (SOCS3), which may be involved in predator avoidance behavior. In total, our data reveal that alarm cues have significant impacts on tadpole physiology, but show only weak links between DNA methylation and CORT levels. We also identify genes containing DMRs in tadpoles exposed to alarm cues and zebularine, particularly in range-edge populations, that warrant further investigation.

Predation risk induces age- and sex-specific morphological plastic responses in the fathead minnow Pimephales promelas

Although comprehending the significance of phenotypic plasticity for evolution is of major interest in biology, the pre-requirement for that, the understanding of variance in plasticity, is still in its infancy. Most researchers assess plastic traits at single developmental stages and pool results between sexes. Here, we study variation among sexes and developmental stages in inducible morphological defences, a well-known instance of plasticity. We raised fathead minnows, Pimephales promelas, under different levels of background predation risk (conspecific alarm cues or distilled water) in a split-clutch design and studied morphology in both juveniles and adults. In accordance with the theory that plasticity varies across ontogeny and sexes, geometric morphometry analyses revealed significant shape differences between treatments that varied across developmental stages and sexes. Alarm cue-exposed juveniles and adult males developed deeper heads, deeper bodies, longer dorsal fin bases, shorter caudal peduncles and shorter caudal fins. Adult alarm cue-exposed males additionally developed a larger relative eye size. These responses represent putative adaptive plasticity as they are linked to reduced predation risk. Perhaps most surprisingly, we found no evidence for inducible morphological defences in females. Understanding whether similar variation occurs in other taxa and their environments is crucial for modelling evolution.

Fear contagion in zebrafish: a behaviour affected by familiarity

Emotional contagion has recently been described in fish but whether it is affected by familiarity is not known. We tested whether the sight of a distressed conspecific elicited fear in zebrafish, and whether this was modulated by familiarity. Groups of six zebrafish were housed together in the same tanks for 7 days to create familiar conditions. The behaviour of individual fish was then recorded in paired tanks within sight of either a familiar or an unfamiliar individual, before and after distilled water or an alarm substance was added to the demonstrator, but not to the observer. As expected, addition of distilled water did not elicit any behavioural change in either the demonstrator or the observer. However, addition of an alarm cue triggered anti-predatory behaviours in the demonstrator which caused the expression of anti-predatory behaviours in the observer, suggesting the existence of fear contagion. Furthermore, the extent of fear contagion was affected by familiarity, and observers were more active, swam closer to the bottom and further away from the demonstrator when they watched a distressed familiar neighbour than when they watched an unfamiliar fish. Our results have implications for fish welfare because they show that fish can become stressed by simply watching others become stressed. They also have implications for experimental design because fish housed in separate tanks cannot be assumed to be statistically independent if they can eavesdrop on their neighbours.

Push and pull of downstream moving juvenile sea lamprey (Petromyzon marinus) exposed to chemosensory and light cues

Visual and olfactory stimuli induce behavioural responses in fishes when applied independently, but little is known about how simultaneous exposure influences behaviour, especially in downstream migrating fishes. Here, downstream moving juvenile sea lamprey (Petromyzon marinus) were exposed to light and a conspecific chemosensory alarm cue in a flume and movement were monitored with overhead cameras and nets. When exposed to light, sea lamprey were more likely to be captured in a net closest to the light array. When exposed to the alarm cue, sea lamprey transit rate through the flume increased, but sea lamprey did not avoid the alarm cue plume by moving perpendicular to flow. When the alarm cue and light were applied simultaneously in a push and pull configuration, the alarm cue still triggered enhanced downstream movement (push downstream) and more sea lamprey was still captured in the net nearest the light (pull to the side), resulting in twice as many sea lamprey being captured in the lighted net relative to controls. To our knowledge, this is the first study using multiple sensory cues in a push-pull configuration to modulate fish outmigration. Push and pull of juvenile sea lamprey with sensory cues could be useful to reduce turbine entrainment where native and enhance trap catch where invasive.