Proceedings of The Physiological Society

Experimental Models (Exeter, UK) (2018) Proc Physiol Soc 40, PC08

Poster Communications

Novel electromechanical method for measuring toxic response in aquatic invertebrates

A. M. Whiteoak1, P. Penson1

1. Pharmacology and Biomedical Science, Liverpool John Moores University, York, United Kingdom.

  • Legend: Heart (circles) and leg (triangles) beats (Hz) of individual Daphnia filmed in darkfield are compared to EMM readouts of the same individuals (on the x-axis.) Error bars show standard deviation. The grey diagonal indicates a Line of Identity through which we would expect data to pass should the EMM readout, and either the heart, or leg, movement data be one and the same. Leg movement data is close to the line of identity while the heart data is not, suggesting that the legs, not the heart, are the source of Whole Daphnia EMM based readouts. P values are given in the main text.

  • Legend: Leg movement rate is normalised to 100% for individual starting values so that rate after exposure is given in comparison to the start point. Records were taken at 5 minute intervals. Circles show mean, error bars show standard error for population data. Here we see that leg rate declines and then plateaus at around 60.8% of the original mean rate when 64uM imidacloprid is applied.

Using principles from ECG, a novel method of recording electromechanical movement (EMM) from whole Daphnia was created. Comparative investigation using simultaneous high speed film revealed EMM signals derived from feeding limbs rather than the heart (fig1). Dependent two-tailed t-tests were performed for paired samples. Null hypotheses assumed no difference in the randomness of the data, therefore paired data would be the same. Comparing heart vs EMM data p=1.39x10-9, there is 99% confidence the EMM does not record the heart. Leg vs EMM data p=0.083, giving 95% confidence that EMM records leg movement. Leg vs heart data were also paired, p=4.52Ex10-10, showing 99% confidence that the leg movement is not a proxy for heart activity. Observation of limb activity has been used as a toxicity measure in previous studies (Ren et al 2015, Lari et al 2017). Legs beat a continuous repeating rhythm to push waterborne food particles towards the digestive opening. EMM signals from entire swarms have been performed (Freund et al 2002) however this has never been done with individual Daphnia; dedicated equipment was built for the task. The EMM method presents an efficient real time method of recording limb beat patterns in Daphnia. It is here applied to the investigation of toxic chemicals in the freshwater environment. The neonicotinoids' effect on pollinators are a hot topic both politically and scientifically. These nicotine receptor agonists show biphasic hormesis giving low dose stimulation and high dose inhibition. Imidacloprid is popular and well studied of these compounds, as a partial agonist, it specifically binds and activates post synaptic nicotinergic acetylcholine receptors (nACHR) in the central nervous system. Binding is nearly irreversible, holding the channel open to cause continuous nervous system stimulation, which eventually leads to the death of the neuron. This affinity is equivalent across the neonicotinoids. Permanent effects accumulate with time. Neonicotinoids are primarily used in corn seed coating. There they form a prophylactic which is taken up by growing plant tissues to provide long-lasting toxicity against insect pests. However, these are also highly leachable compounds prone to water source contamination. The presence of neonicotinoids is frequent and long term due to their long 1000+ day half life in soil and freshwater streams. This work compares results obtained via the EMM method to current peer-reviewed data on imidacloprid exposure in Daphnia (Qi et al 2018, Zein et al 2014). Using equivalent doses we show that this method produces directly comparable results. The effects of various concentrations of imidacloprid over time are shown, an example is shown in figure 2. Whole Daphnia EMM measurement removes the manual/ observation step from current practice, which may improve accuracy as well as efficiency in toxicological studies.

Where applicable, experiments conform with Society ethical requirements