Christian Tudorache

This study presents nanofractionation analytics coupled with in vivo profiling of zebrafish embryo paralysis and lethality in response to toxins in cone snail venoms. The focus of this study is on the development of this approach using venoms of Conus marmoreus, Conus ebraeus, and Conus bandanus. In brief, cone snail venoms were separated using reversed-phase chromatography following high-resolution nanofractionation on microplates with parallel mass spectrometry, enabled via a post-column flow split. All collected fractions were dried overnight, followed by assays on zebrafish embryos. For the paralysis assessment, we monitored swimming behavior and swimming distance and found that exposure to cone snail toxins led to paralysis and decreased movement and swim distance. To correlate the masses of eluted toxins with their paralyzing effects and potency, we compared the fractionation retention time versus normalized swimming distance. This allowed identification of the masses of toxins with paralyzing bioactivity, which were predominantly conopeptides. To assess lethality, zebrafish embryos were exposed to fractionated toxins for 24 h, after which they were inspected. The lethal doses and correlated toxins were identified by comparing retention times of fractionation versus the lethal dose values calculated for each fraction. We found that the most lethal venom was from C. bandanus, displaying the largest number of lethal peptides, followed by C. marmoreus and C. ebraeus. On the other hand, the most paralytic venom was from C. ebraeus, presenting a higher number of peptides with non-lethal paralytic effects, followed by C. bandanus and C. marmoreus. This study provides a pipeline to rapidly identify paralytic and lethal cone snail venom toxins using the zebrafish embryo model.

Coping styles are individually coherent sets of behavioural and physiological responses to stress. Coping styles are thought to remain consistent across context and time, and display a certain level of heritability. Here, we examined whether risk taking is a predictor for consistency and heritability of stress coping styles in both larval and adult zebrafish (Danio rerio). A group emergence test where fish emerge from a familiar housing compartment into a potentially dangerous novel environment, established the level of risk taking of F0 generation adult zebrafish. The degree of risk taking appeared to be consistent over time and context. Then, the F0 risk taking degree was further correlated with various behavioural parameters related to stress coping of the F1 and F2 generations. In larval fish, these parameters were measured during a light dark challenge which elicits an anxiety like response. In adults, they were measured during a single emergence test and a combined open field and mirror biting test, estimating the degree of risk taking and the level of explorativeness and aggressiveness. The results show that (i) parental risk taking behaviour is a good predictor for a large number of larval and adult behavioural parameters, within and between generations; (ii) a number of these parameters are consistent over ontogenetic (larval and adult) stages within the same generation, and (iii) four of these parameters representing risk taking, aggressiveness, and swimming behaviour, were correlated over multiple generations, establishing heritability of coping styles. We conclude that risk taking behaviour is a strong predictor of coping style within and between generations and behavioural parameters associated with risk taking are consistent over time and heritable over generations.

Whilst previous studies have described the impact of various environmental conditions on behaviour and abundance of great white sharks (GWS), existing knowledge gaps must be addressed in order to turn the tide on their population declines. This study used data collected by a diving tour operator, to investigate how environmental and anthropogenic variables affected the rate of GWS sightings. Observation data were collected by trained crew and volunteers alongside tourists, and combined with externally sourced environmental data. Hurdle modelling identified that the probability of sighting at least one GWS fluctuated seasonally (peaking during winter), but was also correlated with minimum running air temperature, water visibility and the length of time the boat stayed at anchor. The rate GWSs were sighted also rose in winter, and was associated with maximum running air temperature, the arrival time, seal activity, and the wind direction and speed. These findings indicate that environmental conditions directly impacted upon the sighting frequency, but also influenced habitat selection on a fine spatial scale. This study emphasises that collaboration with ecotourism companies could represent a valuable, inexpensive alternative for scientific data collection, as long as powerful statistical methods are used, the influence of human activity is considered and results are interpreted with consideration of the data collection methodology.

Research on stress coping style, i.e., the response of an organism to adverse conditions, which is constant over time and context, gained momentum in recent years, to better understand behavioural patterns in animal welfare. However, knowledge about the ontogeny of stress coping style is still limited. Here, we performed a detailed analysis of the light dark challenge behavioural assay in zebrafish larvae, where after acclimation in ambient light sudden alternating dark and light phases elicit an anxiety-like response. A principal component analysis on parameters related to locomotion (distance moved, swimming velocity, acceleration, mobility) and directionality (angular velocity, meandering of swimming path) revealed independence between the parameters determined in the light and the dark phases of the assay, indicating unrelated generalised behaviours per phase. However, high collinearity was observed between behavioural parameters within the same phase, indicating a robust response to the stimulus within behavioural phenotypes. Subsequently, this assay was used to determine the correlation between individual hatching time and the behavioural phenotype. The results show that fish that had hatched during daytime have a stronger behavioural response to the dark phase at 5 days post-fertilisation in locomotion related parameters and a weaker response in directionality related parameters, than fish that had hatched during nighttime. These results show that behavioural responses to the light dark challenge assay are robust and can be generalised for the light and the dark phase, and that diel hatching time may determine the behavioural phenotype of an individual.

Standardization and reduction of variation is key to behavioural screening of animal models in toxicological and pharmacological studies. However, individual variation in behavioural and physiological phenotypes remains in each laboratory population and can undermine the understanding of toxicological and pharmaceutical effects and their underlying mechanisms. Here, we used zebrafish (ABTL-strain) larvae to explore individual consistency in activity level and emergence time, across subsequent days of early development (6–8 dpf). We also explored the correlation between these two behavioural parameters. We found inter-individual consistency over time in activity level and emergence time, but we did not find a consistent correlation between these parameters. Subsequently, we investigated the impact of variation in activity level on the effect of a 1% ethanol treatment, suitable for our proof-of-concept case study about whether impact from pharmacological treatments might be affected by inter-individual variation in basal locomotion. The inter-individual consistency over time in activity level did not persist in this test. This was due to the velocity change from before to after exposure, which turned out to be a dynamic individual trait related to basal activity level: low-activity individuals raised their swimming velocity, while high-activity individuals slowed down, yielding diametrically opposite response patterns to ethanol exposure. We therefore argue that inter-individual consistency in basal activity level, already from 6 dpf, is an important factor to take into account and provides a practical measure to improve the power of statistical analyses and the scope for data interpretation from behavioural screening studies.