Mortality events associated with microsporidian, Spraguea sp., in White Trevally culture in Japan.

In 2021, White Trevally or Striped Jack cultured in the western part of Japan exhibited mild, but chronic mortalities from late September through early October. The cumulative mortality rate was approximately 0.02% per a net pen containing approximately 50,000 fish. Although the cumulative mortality rate was not high, most of the fish in net pens showed characteristic gross signs and an abnormal swimming behaviour. The body of diseased fish became pale and the yellow lines on the lateral sides of fish body became darken. In addition, silver lines along the dorsal fin became apparent. Loss of schooling behaviour was noted during the mortality event. In addition, affected fish became lethargic and failed to swim against current, or frequently stopped swimming and sank to the bottom of net pens after feeding. The goal of this study was to identify the cause of the mortality event. To achieve the goal, we used histopathology and metatranscriptome analysis. Histopathological examination revealed that xenoma of microsporidian were frequently observed in the nerve axon in the brain and spinal cord. Spores observed in the sections were stained with a fluorescent dye, Uvitex 2B, indicating those spores are microsporidian. The data from metatranscriptome analysis indicated that the microsporidian is Spraguea sp. The microsporidian was frequently detected from diseased fish with similar symptoms collected in the same region, suggesting that the microsporidian was highly associated with abnormal swimming behaviour of fish.

Effects of hydraulic cues in barrier environments on fish navigation downstream of dams.

Understanding how hydraulic cues in the barrier environment affect fish navigation is critical to fish migration in dammed rivers. However, most of the current research on the effects of hydraulic cues on fish navigation focuses on the effects of a single hydraulic parameter on fish migration and usually ignores fish sensory perception and swimming ability. This study presents an effective approach that combines a computational fluid dynamics model of a river with a model of fish behaviour to elucidate the effects of hydraulic cues in the barrier environment on fish migration paths and strategies by simulating the fish's perception of flow direction and their regulation of multiple hydraulic parameters. Four release scenarios for the dam were reviewed and it was determined that the modelled fish movements realistically reflected actual observations. In various scenarios, the target fish (Schizothorax chongi) managed to move upstream to the tailrace downstream of the dam, despite the hydraulic barrier created by the mainstem area of the river; they overcame this obstacle by exploiting low-velocity zones on both sides of the mainstem and in the river's boundary layer. During upstream movement, the target fish preferred areas with flow velocities between 0.7 and 1.0 m/s and a turbulent kinetic energy of less than 0.3 m2/s2 to maintain aerobic activity. Additionally, the effects of alternative turbine release strategies on the fine-motor movement of target fish were reviewed and an optimised strategy was provided that could increase the proportion of target fish entering the fish passage facility from 0% to 53.8% in the original scenario to 82.6%. This study provides a feasible method for the simulation of fish fine motion in complex flow environments as well as a scientific basis for the management of fish resources in dammed rivers.

The effects of plastic additives on swimming activity and startle response in marine amphipod Echinogammarus marinus.

Plastic additives are widely used in plastic production and are found in the environment owing to their widespread applications. Among these additives, N-butyl benzenesulfonamide (NBBS) and triphenyl phosphate (TPHP) are under international watchlist for evaluation, with limited studies on amphipods. Di-ethylhexyl phthalate (DEHP) and dibutyl phthalate (DBP) are banned in some countries and categorised as substances of very high concern. This study aimed to investigate the effects of NBBS, TPHP, DEHP and DBP on the swimming activity of a coastal intertidal marine amphipod, Echinogammarus marinus. Furthermore, this study is the first to quantify startle response in E. marinus in response to light stimuli. Amphipods were exposed to 0, 0.5, 5, 50 and 500 µg/l concentrations of all test compounds. Swimming activity and startle responses were assessed by video tracking and analysis using an 8-min alternating dark and light protocol after exposure on days 7 and 14. We observed an overall compound and light effect on the swimming activity of E. marinus. A significant decrease in swimming distance was found in 500 µg/l NBBS and TPHP. We observed that the startle response in E. marinus had a latency period of >2 s and animals were assessed at 1 s and the sum of the first 5 s. There was a clear startle response in E. marinus during dark to light transition, evident with increased swimming distance. NBBS exposure significantly increased startle response at environmental concentrations, while significant effects were only seen in 500 µg/l TPHP at 5 s. We found no significant effects of DEHP and DBP on swimming behaviour at the concentrations assessed. The findings of this study affirm the necessity for a continuous review of plastic additives to combat adverse behavioural effects that may be transferable to the population levels.

Behavioural, developmental and biochemical effects in zebrafish caused by ibuprofen, irgarol and terbuthylazine.

The increasing use of chemicals and their release into aquatic ecosystems are harming aquatic biota. Despite extensive ecotoxicological research, many environmental pollutants' ecological effects are still unknown. This study examined the spatial avoidance, behavioural and biochemical impacts of ibuprofen, irgarol, and terbuthylazine on the early life stages of zebrafish (Danio rerio) under a range of ecologically relevant concentrations (0-500 µg/L). Embryos were exposed following the OECD guideline "fish embryo toxicity test" complemented with biochemical assessment of AChE activity and behavioural analyses (swimming activity) using the video tracking system Zebrabox. Moreover, spatial avoidance was assessed by exposing 120 hpf-old larvae of D. rerio to a gradient of each chemical, by using the heterogeneous multi-habitat assay system (HeMHAS). The results obtained revealed that the 3 compounds delayed hatching at concentrations of 50 and 500 µg/L for both ibuprofen and irgarol and 500 µg/L for terbuthylazine. Moreover, all chemicals elicited a dose-dependent depression of movement (swimming distance) with LOEC values of 5, 500 and 50 µg/L for ibuprofen, irgarol and terbuthylazine, respectively. Zebrafish larvae avoided the three chemicals studied, with 4 h-AC50 values for ibuprofen, irgarol, and terbuthylazine of 64.32, 79.86, and 131.04 µg/L, respectively. The results of the HeMHAS assay suggest that larvae may early on avoid (just after 4 h of exposure) concentrations of the three chemicals that may later induce, apical and biochemical effects. Findings from this study make clear some advantages of using HeMHAS in ecotoxicology as it is: ecologically relevant (by simulating a chemically heterogeneous environmental scenario), sensitive (the perception of chemicals and the avoidance can occur at concentrations lower than those producing lethal or sublethal effects) and more humane and refined approach (organisms are not mandatorily exposed to concentrations that can produce individual toxicity).

Assessment of behavioural effects of three water-soluble polymers in zebrafish embryos.

The water-soluble polymers (WSPs) are widely used in many industrial applications and are present in several commonly used products due to their physical-chemical characteristics: as their name indicates, despite being synthetic polymers, they are able to solubilize in water. Because of this peculiar property, both the qualitative-quantitative evaluation in aquatic ecosystems and their potential (eco)toxicological effects have been neglected until now. The aim of this study was to evaluate the possible effects of three of the most widely used WSPs as polyacrylic acid (PAA), polyethylene glycol (PEG) and polyvinyl pyrrolidone (PVP) on the swimming behaviour of zebrafish (Danio rerio) embryos after the exposure to different concentrations (0.001, 0.5, 1 mg/L). The exposure lasted from the eggs' collection up to 120 h post fertilization (hpf) also using three different light intensity (300 lx, 2200 lx, 4400 lx) to better evaluate any effects related to different gradients of light/dark transitions. In order to analyze individual behavioural changes in embryos, their swimming movements were tracked and a number of parameters for locomotion and directionality were quantified. The main results showed that all three WSPs resulted in significant (p ≤ 0.05) variations in different movement parameters, suggesting a possible toxicity scale: PVP > PEG â‰« PAA.

Among-individual variation in the swimming behaviour of the amphipod Gammarus pulex under dark and light conditions.

In recent years, considerable computational advancements have been made allowing automated analysis of behavioural endpoints using video cameras. However, the results of such analyses are often confounded by a large variation among individuals, making it problematic to derive endpoints that allow distinguishing treatment effects in behavioural studies. In this study, we quantitatively analysed the effects of light conditions on the swimming behaviour of the freshwater amphipod Gammarus pulex by high-throughput tracking, and attempted to unravel among individual variation using size and sex. For this, we developed the R-package Kinematics, allowing for the rapid and reproducible analysis of the swimming behaviour (speed, acceleration, thigmotaxis, curvature and startle response) of G. pulex, as well as any other organism. Our results show a considerable amount of variation among individuals (standard deviation ranging between 5 and 115 % of the average swimming behaviour). The factors size and sex and the interaction between the two only explained a minor part of this found variation. Additionally, our study is the first to quantify the startle response in G. pulex after the light is switched on, and study the variability of this response between individuals. To analyse this startle response, we established two metrics: 1) startle response magnitude (the drop in swimming velocity directly after the light switches on), and 2) startle response duration (the time it takes to recover from the drop in swimming velocity to average swimming speed). Almost 80 % of the individuals showed a clear startle response and, therefore, these metrics demonstrate a great potential for usage in behavioural studies. The findings of this study are important for the development of appropriate experimental set-ups for behavioural experiments with G. pulex.

Rearing Environment during the Endogenous Feeding Stage of Acipenser baerii.

The aim of this study was to evaluate behaviour, growth, lipid composition, muscle development, and stress status of Siberian sturgeon larvae reared with two types of substrate: Bioballs1 (BB1) and Bioballs2 (BB2), when compared to no substrate (CTR). Sampling points were: hatching (T0), schooling (T1), and yolk-sac full absorption (T2). BB1 larvae were less active and showed no schooling behaviour. At T1 and at T2, BB1 larvae showed a significantly higher weight and total length than larvae reared in either CTR or BB2 (p < 0.05). The lipid content of larvae decreased over time, with little relevant differences between groups. At T2, total muscle area, slow muscle area and fast muscle area were significantly higher in larvae reared in BB1 (p < 0.05). No significant differences in muscle proliferation were found between groups. Real Time PCR was used for evaluating the relative expression of a pool of genes: myod, myog, mrf4, igf2, hsp70, hsp90a, hsp90b, and glut2. The expression of these genes did not seem to be much affected by the type of rearing substrate, except for myog and hsp70 at T1, which was greater in BB2 larvae. Our data suggest that the presence of a substrate during this developmental period seems to have positive effects but further studies would be necessary during the exogenous feeding stage.

Environmentally relevant concentrations of microplastics modulated the immune response and swimming activity, and impaired the development of marine medaka Oryzias melastigma larvae.

Microplastics (MPs), due to their impacts on the ecosystem and their integration into the food web either through trophic transfer or ingestion directly from the ambient environment, are an emerging class of environmental contaminants posing a great threat to marine organisms. Most reports on the toxic effects of MPs exclusively focus on bioaccumulation, oxidative stress, pathological damage, and metabolic disturbance in fish. However, the collected information on fish immunity in response to MPs is poorly defined. In particular, little is known regarding mucosal immunity and the role of mucins. In this study, marine medaka (Oryzias melastigma) larvae were exposed to 6.0 µm beads of polystyrene microplastics (PS-MPs) at three environmentally relevant concentrations (102 particles/L, 104 particles/L, and 106 particles/L) for 14 days. The experiment was carried out to explore the developmental and behavioural indices, the transcriptional profiles of mucins, pro-inflammatory, immune, metabolism and antioxidant responses related genes, as well as the accumulation of PS-MPs in larvae. The results revealed that PS-MPs were observed in the gastrointestinal tract, with a concentration- and exposure time-dependent manner. No significant difference in the larval mortality was found between the treatment groups and the control, whereas the body length of larvae demonstrated a significant reduction at 106 particles/L on 14 days post-hatching. The swimming behaviour of the larvae became hyperactive under exposure to 104 and 106 particles/L PS-MPs. In addition, PS-MP exposure significantly up-regulated the mucin gene transcriptional levels of muc7-like and muc13-like, however down-regulated the mucin gene expression levels of heg1, muc2, muc5AC-like and muc13. The immune- and inflammation and metabolism-relevant genes (jak, stat-3, il-6, il-1ß, tnf-а, ccl-11, nf-κb, and sod) were significantly induced by PS-MPs at 104 and 106 particles/L compared to the control. Taken together, this study suggests that PS-MPs induced inflammation response and might obstruct the immune functions and retarded the growth of the marine medaka larvae even at environmentally relevant concentrations.

Identifying three-dimensional swimming corridors for fish to match their swimming characteristics under different hydropower plant operations: Optimization of entrance location for fish-passing facilities.

Fish that require migration are often diverse in the dam-built river, and some of them are benthic. For fish to pass efficiently across the dams, it is necessary to identify the swimming corridors and possible cluster areas of the target fish. However, previous studies have only predicted the two-dimensional swimming corridors of a single species. In this study, the relationships between the swimming behaviours of different species of fish and the three-dimensional (3D) hydrodynamic field were determined, and a model of the 3D swimming corridors corresponding to the fish swimming characteristics was constructed. The model was verified by a fish acoustic telemetry experiment. By simulating the river 3D hydrodynamic field in different scenarios, the possible swimming corridors and clustering areas of the target fish were further evaluated. We showed that the swimming corridors of the target fish were different among different scenarios, and the swimming corridors were mainly concentrated on both sides of the mainstream. With increased flow due to power station operation, the proportion of potential swimming corridors of target fish throughout the basin gradually decreased, and the proportion of velocity barrier zones of target fish gradually increased. By analyzing the possible swimming corridors of the target fish, the slow-flowing areas near the suddenly narrowed swimming corridors were considered to be the areas of target fish clustering. The cluster areas of the target fish weres also the optimal choice of the fish passing facility entrances, so as to ensure the best fish-crossing effect. The reliable and quantitative 3D fish swimming corridors model proposed in this study can provide a scientific basis for the layout of fish-crossing facilities and facilitate the scientific operation and management of the reservoir in the fish spawning season.

Acute exposure of embryo, larvae and adults of Danio rerio to fipronil commercial formulation reveals effects on development and motor control.

The insecticide fipronil, one of the main pesticides used in Brazil, is often detected in natural aquatic environments, and causes neuronal hyperexcitation by inhibiting GABAergic neurotransmission, leading to putative alterations in behaviour and development. This work sought to analyse the toxicity of formulated Regent® 800WG (80% fipronil) on development (fish embryo toxicity test, FET), morphology, and swimming behaviour of larvae and adults of zebrafish (Danio rerio). FET was performed following OECD236 guidelines at concentrations ranging from 0.002 to 1600 µg.L-1 of formulated Regent® 800WG. Adults were exposed to 0.2, 2 and 20 µg.L-1 of the product for 24 and 96 h, and were submitted to the light-dark, novel tank and swimming endurance tests No lethal parameters were observed in larvae, but in concentrations above 400 µg.L-1, there was shortening of the body axis and decreased swimming behavior. In adults, exposure to the pesticide did not lead to changes in free swimming parameters. However, a marked decrease of swimming endurance was observed at all experimental treatments, although probably not in consequence of energetic depletion, since baseline blood glucose levels and condition factor were similar at all conditions. Furthermore, zebrafish adults did not show their natural preference for the dark environment. The pesticide likely has anxiolytic effects on zebrafish, as well as a compromising effect on locomotor control, illustrating that behavioural changes, which could affect activities on the natural environment, such as escape and predation, may occur even in environmentally relevant concentrations of this pollutant.

The Effect of Different Feeding Applications on the Swimming Behaviour of Siberian Sturgeon: A Method for Improving Restocking Programmes.

Restocking programmes of different fish species have been implemented worldwide. However, the survival of hatchery-reared fish after release to riverine ecosystems is at a very low level. One of the reasons for the high mortality rate of post-released fish is their modified swimming behaviour due to the hatchery rearing practice. To investigate one of the possible causes for modified swimming behaviour, Acipenser baerii larvae were exposed to surface- and bottom-feeding applications with day and night light regimes in a factorial design. We also analysed the effect of 5 and 10 days of starvation after different feeding applications on sturgeon swimming behaviour. The surface-feeding application was previously expected to promote the frequent Siberian sturgeon swim up to the mid- and top-water layers in our rearing facilities. However, our results indicated that the modified behaviour of the Siberian sturgeon in our study was caused by fish starvation and a possible predator-free environment rather than by the method of feed application or the day/night light regimes. These results may be used to improve the implementation of restocking programmes either through modified hatchery rearing practice or the training of foraging skills with predator stimuli.

Fluorescence Properties of a Novel Isoquinoline Derivative Tested in an Invertebrate Chordate, Ciona intestinalis.

3-Hydroxyisoquinolines (ISOs) and their tautomeric isoquinolin-3-ones are heterocycles with attractive biological properties. Here we reported the revisited synthesis of a highly functionalized ISO that showed blue fluorescence and the characterization of its biological properties in an invertebrate animal model, the ascidian Ciona intestinalis. Larvae exposed to ISO at concentrations higher than 1 µM showed an intense fluorescence localized in the cell nuclei of all tissues. Moreover, exposure to ISO interfered with larval ability to swim; this neuromuscular effect was reversible. Overall, these results suggested that ISOs can have promising applications as novel fluorescent dyes of the cell nuclei.

Toxicity and risk assessment of six widely used pesticides on embryo-larval development of the Pacific oyster, Crassostrea gigas.

This study aims to assess the toxic effects and the potential risk of widely used agricultural pesticides on the development (malformations and developmental arrest), growth and swimming activity of oyster D-larvae (Crassostrea gigas). Freshly fertilized oyster embryos were exposed for 24 h at 24 °C to different concentrations (0, 0.01, 0.1, 1 and 10 µg.L-1) of six different pesticides: Glyphosate and its commercial solution (Roundup), Isoproturon, Nicosulfuron, Chlortoluron and Boscalid. The six pesticides tested induced a significant increase in larval malformations and developmental arrests. All pesticides except Glyphosate and Isoproturon affected larval growth. Roundup, Nicosulfuron, Chlortoluron and Boscalid also affected the swimming behaviour of the D-larvae, with a significant decrease recorded in their maximum swimming speed. Comparison of the LOEC (Lowest-Observed-Effect Concentration) of each compound led to the following toxicity classification: Boscalid > Chlortoluron = Nicosulfuron > Glyphosate > Roundup > Isoproturon, with respectively LOEC of 0.0028; 0.015; 0.017; 0.11; 0.3 and 0.78 µg.L-1. By comparison of the maximum concentrations in the Pertuis Charentais (South West, France) and LOEC of each pesticide, the following risk scale was obtained: Chlortoluron > Boscalid > Glyphosate > Roundup > Nicosulfuron > Isoproturon. Our results revealed that Chlortoluron, Boscalid and to a lesser extent Glyphosate represent a potential threat to early life stages of oyster living in the Pertuis Charentais marine area.

First description and diagnostics of disease caused by Piscirickettsia salmonis in farmed European sea bass (Dicentrarchus labrax Linnaeus) from Croatia.

During the winter of 2013 and 2016, several Croatian fish farms experienced mortalities in the fry of European sea bass, Dicentrarchus labrax. Affected fish showed abnormal swimming behaviour and reduced appetite, and death ensued several days after the onset of clinical signs of disease. Necropsy revealed pale liver, empty digestive tract, distended gall bladder, and hyperaemia and congestion of the meninges. Routine bacteriological examination tested negative, and virological examination ruled out nodavirus infection. Histological examination revealed multifocal necrosis and extensive inflammation in the brain with abundant cellular debris in the ventricles. Inflammatory cells displayed intra-cytoplasmic basophilic vacuoles leading to suspicion of Piscirickettsia salmonis infection. Fluorescent in situ hybridization using an oligonucleotide probe targeting Domain Bacterium applied to tissue sections tested positive. The pathogen was identified by 16S rRNA gene sequencing of brain material, and the sequence showed 99% similarity with P. salmonis. This result enabled the design of an oligonucleotide probe specifically targeting P. salmonis. In 2016, P. salmonis was successfully isolated on CHAB from the brain of an affected specimen and identified using 16S rRNA gene sequencing and MALDI-TOF. This study describes the first outbreak of disease caused by P. salmonis in sea bass in Croatia, while new diagnostic tools will enable further research on its epidemiology and pathogenicity.

Population variation in the thermal response to climate change reveals differing sensitivity in a benthic shark.

Many species with broad distributions are exposed to different thermal regimes which often select for varied phenotypes. This intraspecific variation is often overlooked but may be critical in dictating the vulnerability of different populations to environmental change. We reared Port Jackson shark (Heterodontus portusjacksoni) eggs from two thermally discrete populations (i.e. Jervis Bay and Adelaide) under each location's present-day mean temperatures, predicted end-of-century temperatures and under reciprocal-cross conditions to establish intraspecific thermal sensitivity. Rearing temperatures strongly influenced MO2 Max and critical thermal limits, regardless of population, indicative of acclimation processes. However, there were significant population-level effects, such that Jervis Bay sharks, regardless of rearing temperature, did not exhibit differences in MO2 Rest , but under elevated temperatures exhibited reduced maximum swimming activity with step-wise increases in temperature. In contrast, Adelaide sharks reared under elevated temperatures doubled their MO2 Rest , relative to their present-day temperature counterparts; however, maximum swimming activity was not influenced. With respect to reciprocal-cross comparisons, few differences were detected between Jervis Bay and Adelaide sharks reared under ambient Jervis Bay temperatures. Similarly, juveniles (from both populations) reared under Adelaide conditions had similar thermal limits and swimming activity (maximum volitional velocity and distance) to each other, indicative of conserved acclimation capacity. However, under Adelaide temperatures, the MO2 Rest of Jervis Bay sharks was greater than that of Adelaide sharks. This indicates that the energetics of cooler water population (Adelaide) is likely more thermally sensitive than that of the warmer population (Jervis Bay). While unique to elasmobranchs, these data provide further support that by treating species as static, homogeneous populations, we ignore the impacts of thermal history and intraspecific variation on thermal sensitivity. With climate change, intraspecific variation will manifest as populations move, demographics change or extirpations occur, starting with the most sensitive populations.

Structure and function of the nervous system in nectophores of the siphonophore Nanomia bijuga.

Although the bell-shaped nectophores of the siphonophore Nanomia bijuga are clearly specialized for locomotion, their complex neuroanatomy described here testifies to multiple subsidiary functions. These include secretion, by the extensively innervated 'flask cells' located around the bell margin, and protection, by the numerous nematocytes that line the nectophore's exposed ridges. The main nerve complex consists of a nerve ring at the base of the bell, an adjacent column-shaped matrix plus two associated nerve projections. At the top of the nectophore the upper nerve tract appears to have a sensory role; on the lower surface a second nerve tract provides a motor input connecting the nectophore with the rest of the colony via a cluster of nerve cells at the stem. N. bijuga is capable of both forward and backward jet-propelled swimming. During backwards swimming the water jet is redirected by the contraction of the Claus' muscle system, part of the muscular velum that fringes the bell aperture. Contractions can be elicited by electrical stimulation of the nectophore surface, even when both upper and lower nerve tracts have been destroyed. Epithelial impulses elicited there, generate slow potentials and action potentials in the velum musculature. Slow potentials arise at different sites around the bell margin and give rise to action potentials in contracting Claus' muscle fibres. A synaptic rather than an electrotonic model more readily accounts for the time course of the slow potentials. During backward swimming, isometrically contracting muscle fibres in the endoderm provide the Claus' fibres with an immobile base.

Heatwave effects on the swimming behaviour of a Mediterranean freshwater fish, the Iberian barbel Luciobarbus bocagei.

Heatwaves, which can be defined as increases of at least 5 °C in air temperature for more than five consecutive days for a specified reference period, are expected to become more frequent under the ongoing climate change, with freshwater organisms being particularly vulnerable to high temperature fluctuations. In Mediterranean-climate areas, depending on the extent of summer droughts and loss of longitudinal connectivity, river segments may become isolated, maintaining fish populations confined to a series of disconnected pools, with no possibility to move to thermal refugia and thus becoming more prone to thermal stress. In this study, we evaluated the effect of a simulated heatwave on the swimming behaviour of juvenile stages of a potamodromous native cyprinid fish, the Iberian barbel Luciobarbus bocagei, under experimental mesocosm conditions. Behavioural traits included fish activity, boldness and shoal cohesion and were continuously measured at a constant flow velocity of 18 cm s-1, which is typical of riffle habitats. Overall, results show that the behaviour of juvenile Iberian barbel is likely to be affected by heatwaves, with fish displaying lower activity and boldness, while no clear difference was observed in shoal cohesion. This study highlights the importance of managing thermal refugia that are crucial for fish to persist in intermittent rivers. Future studies should focus on the interaction of heatwaves with other stressors, such as oxygen depletion, for a broader understanding of the perturbation affecting freshwater fishes under a changing climate.

Experimental ingestion of fluorescent microplastics by pacific oysters, Crassostrea gigas, and their effects on the behaviour and development at early stages.

Plastics are persistent synthetic polymers that accumulate as waste in the marine environment. Microplastics (MPs, <5 mm) can be found either as microbeads in body care and some industrial products or as plastic debris through degradation. Plastic microbeads (1-5 µm, fluorescent, Cospheric) were used to characterise the MP ingestion and determine their potential harmful effects on both the swimming behaviour and development of oyster D-larvae (Crassostrea gigas). For 24 h, embryos were first exposed to MPs at a temperature of 24 °C. In addition, 3 day-old D-larvae were exposed to the same temperature for 1, 3 and 5 h. Three concentrations of MPs were used: 0.1, 1 and 10 mg MP. L-1. After a 24-h period of embryonic exposure, we noted that MP agglomerates were stuck to the D-larvae coat and locomotor eyelashes. We also observed a significant increase in severe malformations and developmental arrests for larvae exposed to MPs ranging from 1 mg MP. L-1. In terms of swimming behaviour, the maximum speed recorded was lower for larvae exposed at 0.1 and 1 mg MP. L-1. After an acute exposure to MPs, particles were found in the digestive tract of 3 dpf (days post fertilisation) D-larvae. After 1-h exposure, the concentrations tested (0.1, 1 and 10 mg MP. L-1) resulted in respectively 38%, 86% and 98%. The larvae swimming behaviour was recorded and analysed. Unlike the results observed at the embryo-larval stage, 3-dpf larvae showed significant impacts with no dose-response effect.

Quantification of the influence of drugs on zebrafish larvae swimming kinematics and energetics.

The use of zebrafish larvae has aroused wide interest in the medical field for its potential role in the development of new therapies. The larvae grow extremely quickly and the embryos are nearly transparent which allows easy examination of its internal structures using fluorescent imaging techniques. Medical treatment of zebrafish larvae can directly influence its swimming behaviours. These behaviour changes are related to functional changes of central nervous system and transformations of the zebrafish body such as muscle mechanical power and force variation, which cannot be measured directly by pure experiment observation. To quantify the influence of drugs on zebrafish larvae swimming behaviours and energetics, we have developed a novel methodology to exploit intravital changes based on observed zebrafish locomotion. Specifically, by using an in-house MATLAB code to process the recorded live zebrafish swimming video, the kinematic locomotion equation of a 3D zebrafish larvae was obtained, and a customised Computational Fluid Dynamics tool was used to solve the fluid flow around the fish model which was geometrically the same as experimentally tested zebrafish. The developed methodology was firstly verified against experiment, and further applied to quantify the fish internal body force, torque and power consumption associated with a group of normal zebrafish larvae vs. those immersed in acetic acid and two neuroactive drugs. As indicated by our results, zebrafish larvae immersed in 0.01% acetic acid display approximately 30% higher hydrodynamic power and 10% higher cost of transport than control group. In addition, 500 µM diphenylhydantoin significantly decreases the locomotion activity for approximately 50% lower hydrodynamic power, whereas 100 mg/L yohimbine has not caused any significant influences on 5 dpf zebrafish larvae locomotion. The approach has potential to evaluate the influence of drugs on the aquatic animal's behaviour changes and thus support the development of new analgesic and neuroactive drugs.

High density polyethylene (HDPE) microplastics impair development and swimming activity of Pacific oyster D-larvae, Crassostrea gigas, depending on particle size.

Understanding the effects of plastic debris on marine ecosystems is essential in encouraging decision-makers to take action. The present study investigates the effect of a 24 h experimental exposure to high density polyethylene (HDPE) microplastics (MPs) of different sizes (4-6, 11-13 and 20-25 µm) and at three concentrations (0.1, 1 and 10 mg MP.L-1) on the development and locomotor activity of early stages of Pacific oyster, Crassostrea gigas. The bivalve embryo-larval assay (NF ISO 17244, 2015) was used in this study but with additional toxicity criteria: developmental arrests, abnormal D-larvae, maximum speed and swimming trajectory. Copper (Cu), was used as a positive control. Our results show that smaller MPs (4-6 and 11-13 µm) induced higher rates of malformations and developmental arrests than the larger ones (20-25 µm). In addition, a dose-dependent decrease of maximum swimming speed was observed for larvae exposed to MPs of 4-6 and 11-13 µm. On the other hand, there was no significant difference in swimming speed with the largest MPs size tested (20-25 µm). For all three sizes of MPs, there was a decrease in straight-line swimming trajectories, and an increase in circular trajectories. This abnormal swimming behaviour could affect larvae survival as well as colonization of new habitats.