Embryonic exposures to chemicals acting on brain aromatase lead to different locomotor effects in zebrafish larvae.

Pathways underlying neurodevelopmental effects of endocrine disruptors (EDs) remain poorly known. Expression of brain aromatase (aroB), responsible for estrogen production in the brain of teleosts, is regulated by estrogenic EDs and could play a role in their behavioral effects. We exposed zebrafish eleutheroembryos (0-120 h post-fertilization) to various concentrations of 16 estrogenic chemicals (incl. bisphenols and contraceptives), and of 2 aroB inhibitors. Behavior was monitored using a photomotor response test procedure. Both aroB inhibitors (clotrimazole and prochloraz) and a total of 6 estrogenic EDs induced significant behavioral alterations, including DM-BPA, BPC and BPS-MPE, three bisphenol substitutes which behavioral effects were, to our knowledge, previously unknown. However, no consensus was reported on the effects among tested substances. It appears that behavioral changes could not be linked to groups of substances defined by their specificity or potency to modulate aroB expression, or by their structure. Altogether, behavioral effects of estrogenic EDs in 120 h post-fertilization larvae appear unrelated to aroB but are nonetheless not to be neglected in the context of environmental safety.

Temperature and feeding frequency impact the survival, growth, and metamorphosis success of Solea solea larvae.

Human-induced climate change impacts the oceans, increasing their temperature, changing their circulation and chemical properties, and affecting marine ecosystems. Like most marine species, sole has a biphasic life cycle, where one planktonic larval stage and juvenile/adult stages occur in a different ecological niche. The year-class strength, usually quantified by the end of the larvae stage, is crucial for explaining the species' recruitment. We implemented an experimental system for rearing larvae under laboratory conditions and experimentally investigated the effects of temperature and feeding frequencies on survival, development (growth), and metamorphosis success of S. solea larvae. Specific questions addressed in this work include: what are the effects of feeding regimes on larvae development? How does temperature impact larvae development? Our results highlight that survival depends on the first feeding, that the onset of metamorphosis varies according to rearing temperature and that poorly fed larvae take significantly longer to start (if they do) metamorphosing. Moreover, larvae reared at the higher temperature (a +4°C scenario) showed a higher incidence in metamorphosis defects. We discuss the implications of our results in an ecological context, notably in terms of recruitment and settlement. Understanding the processes that regulate the abundance of wild populations is of primary importance, especially if these populations are living resources exploited by humans.

Thermal imprinting during embryogenesis modifies skin repair in juvenile European sea bass (Dicentrarchus labrax).

Fish skin is a multifunctional tissue that develops during embryogenesis, a developmental stage highly susceptible to epigenetic marks. In this study, the impact of egg incubation temperature on the regeneration of a cutaneous wound caused by scale removal in juvenile European sea bass was evaluated. Sea bass eggs were incubated at 11, 13.5 and 16 °C until hatching and then were reared at a common temperature until 9 months when the skin was damaged and sampled at 0, 1 and 3 days after scale removal and compared to the intact skin from the other flank. Skin damage elicited an immediate significant (p < 0.001) up-regulation of pcna in fish from eggs incubated at higher temperatures. In fish from eggs incubated at 11 °C there was a significant (p < 0.001) up-regulation of krt2 compared to fish from higher thermal backgrounds 1 day after skin damage. Damaged epidermis was regenerated after 3 days in all fish irrespective of the thermal background, but in fish from eggs incubated at 11 °C the epidermis was significantly (p < 0.01) thinner compared to other groups, had less goblet cells and less melanomacrophages. The thickness of the dermis increased during regeneration of wounded skin irrespective of the thermal background and by 3 days was significantly (p < 0.01) thicker than the dermis from the intact flank. The expression of genes for ECM remodelling (mmp9, colXα, col1α1, sparc, and angptl2b) and innate immunity (lyg1, lalba, sod1, csf-1r and pparγ) changed during regeneration but were not affected by egg thermal regime. Overall, the results indicate that thermal imprinting of eggs modifies the damage-repair response in juvenile sea bass skin.

Exposure of zebrafish to an environmental mixture of persistent organic pollutants triggers an increase in anxiety-like syndrome but does not affect boldness in unexposed offspring.

Polychlorinated biphenyls (PCBs) and polybrominated diphenyl ethers (PBDEs) are persistent organic pollutants (POPs) that are present as complex mixtures in all environmental compartments, including aquatic ecosystems. However, little is known about the effects of such complex mixtures on teleost behaviour. In this study, zebrafish (Danio rerio) were chronically exposed to an environmentally relevant mixture (MIX) containing 22 PCB and 7 PBDE congeners through diet from 5 days post fertilization onwards. MIX-exposed F0 fish produced offspring (F1 and F2 generations) that were fed using plain food and grown until adulthood. In each generation, five behavioural traits (i.e. boldness, activity, sociality, exploration and anxiety) were evaluated by the mean of different experimental set-ups. Two distinct behavioural syndromes were identified: boldness, positively correlated to activity and exploration; and anxiety, associated with low sociality. F0 fish did not display any behavioural disruption resulting from POP exposure whereas F1 MIX fish were bolder than fish from other generations but did not differ significantly from F1 controls. F2 MIX fish displayed a higher anxiety syndrome than F2 controls. This is of particular importance since such behavioural changes in offspring generations may have persistent ecological consequences, may affect fitness and hence cause detrimental effects on wild fish populations exposed to POP mixtures.

Environmental microplastics disrupt swimming activity in acute exposure in Danio rerio larvae and reduce growth and reproduction success in chronic exposure in D. rerio and Oryzias melastigma.

Microplastics (MPs), widely present in aquatic ecosystems, can be ingested by numerous organisms, but their toxicity remains poorly understood. Toxicity of environmental MPs from 2 beaches located on the Guadeloupe archipelago, Marie Galante (MG) and Petit-Bourg (PB) located near the North Atlantic gyre, was evaluated. A first experiment consisted in exposing early life stages of zebrafish (Danio rerio) to MPs at 1 or 10 mg/L. The exposure of early life stages to particles in water induced no toxic effects except a decrease in larval swimming activity for both MPs exposures (MG or PB). Then, a second experiment was performed as a chronic feeding exposure over 4 months, using a freshwater fish species, zebrafish, and a marine fish species, marine medaka (Oryzias melastigma). Fish were fed with food supplemented with environmentally relevant concentrations (1% wet weight of MPs in food) of environmental MPs from both sites. Chronic feeding exposure led to growth alterations in both species exposed to either MG or PB MPs but were more pronounced in marine medaka. Ethoxyresorufin-O-deethylase (EROD) and acetylcholinesterase (AChE) activities were only altered for marine medaka. Reproductive outputs were modified following PB exposure with a 70 and 42% decrease for zebrafish and marine medaka, respectively. Offspring of both species (F1 generation) were reared to evaluate toxicity following parental exposure on unexposed larvae. For zebrafish offspring, it revealed premature mortality after parental MG exposure and parental PB exposure produced behavioural disruptions with hyperactivity of F1 unexposed larvae. This was not observed in marine medaka offspring. This study highlights the ecotoxicological consequences of short and long-term exposures to environmental microplastics relevant to coastal marine areas, which represent essential habitats for a wide range of aquatic organisms.

The extensive transgenerational transcriptomic effects of ocean acidification on the olfactory epithelium of a marine fish are associated with a better viral resistance.

BACKGROUND: Progressive CO2-induced ocean acidification (OA) impacts marine life in ways that are difficult to predict but are likely to become exacerbated over generations. Although marine fishes can balance acid-base homeostasis efficiently, indirect ionic regulation that alter neurosensory systems can result in behavioural abnormalities. In marine invertebrates, OA can also affect immune system function, but whether this is the case in marine fishes is not fully understood. Farmed fish are highly susceptible to disease outbreak, yet strategies for overcoming such threats in the wake of OA are wanting. Here, we exposed two generations of the European sea bass (Dicentrarchus labrax) to end-of-century predicted pH levels (IPCC RCP8.5), with parents (F1) being exposed for four years and their offspring (F2) for 18 months. Our design included a transcriptomic analysis of the olfactory rosette (collected from the F2) and a viral challenge (exposing F2 to betanodavirus) where we assessed survival rates. RESULTS: We discovered transcriptomic trade-offs in both sensory and immune systems after long-term transgenerational exposure to OA. Specifically, RNA-Seq analysis of the olfactory rosette, the peripheral olfactory organ, from 18-months-old F2 revealed extensive regulation in genes involved in ion transport and neuronal signalling, including GABAergic signalling. We also detected OA-induced up-regulation of genes associated with odour transduction, synaptic plasticity, neuron excitability and wiring and down-regulation of genes involved in energy metabolism. Furthermore, OA-exposure induced up-regulation of genes involved in innate antiviral immunity (pathogen recognition receptors and interferon-stimulated genes) in combination with down-regulation of the protein biosynthetic machinery. Consistently, OA-exposed F2 challenged with betanodavirus, which causes damage to the nervous system of marine fish, had acquired improved resistance. CONCLUSION: F2 exposed to long-term transgenerational OA acclimation showed superior viral resistance, though as their metabolic and odour transduction programs were altered, odour-mediated behaviours might be consequently impacted. Although it is difficult to unveil how long-term OA impacts propagated between generations, our results reveal that, across generations, trade-offs in plastic responses is a core feature of the olfactory epithelium transcriptome in OA-exposed F2 offspring, and will have important consequences for how cultured and wild fish interacts with its environment.

Transcriptomic profiles of consistent risk-taking behaviour across time and contexts in European sea bass.

Bolder individuals have greater access to food sources and reproductive partners but are also at increased risk of predation. Boldness is believed to be consistent across time and contexts, but few studies have investigated the stability of this trait across variable environments, such as varying stress loads or long periods of time. Moreover, the underlying molecular components of boldness are poorly studied. Here, we report that boldness of 1154 European sea bass, evaluated using group risk-taking tests, is consistent over seven months and for individuals subjected to multiple environments, including a chronically stressful environment. Differences in risk-taking behaviour were further supported by differences observed in the responses to a novel environment test: shy individuals displayed more group dispersion, more thigmotaxic behaviour and lower activity levels. Transcriptomic analyses performed on extreme phenotypes revealed that bold individuals display greater expression for genes involved in social and exploration behaviours, and memory in the pituitary, and genes involved in immunity and responses to stimuli in the head kidney. This study demonstrates that personality traits come with an underpinning molecular signature, especially in organs involved in the endocrine and immune systems. As such, our results help to depict state-behaviour feedback mechanisms, previously proposed as key in shaping animal personality.

Familiarity reduces aggression but does not modify acoustic communication in pairs of Nile tilapia (Oreochromis niloticus) and black-chinned tilapia (Sarotherodon melanotheron).

Reproduction involves multiple complex behaviours, and the effects of familiarity on such social interactions are seldom described in fish. This is particularly true for sound production and communication within aggressive or non-aggressive context. This study explores the effects of a common garden rearing without parental care of two closely related cichlid species (Nile tilapia Oreochromis niloticus and black-chinned tilapia Sarotherodon melanotheron) on their sound production features and social interactions. After 9 months in common garden rearing, from embryonic stage to first maturity, sound production and associated behaviours were recorded on specimens of the two species in intraspecific and interspecific pairings. The authors found that fish were able to produce the same kind of sounds as those recorded in similar context for their parents. Drum sounds were associated to chasing, lateral attack and courtship in O. niloticus and only to fleeing or avoidance in S. melanotheron. Specific grunts were produced in chasing, after biting and in nest building by O. niloticus, and specific rolling sounds were associated to courtship in S. melanotheron. Sound production and behaviours were not correlated to sex steroid levels, but the number of sounds recorded in aggressive context was correlated to dominance in O. niloticus. The authors conclude that one generation of common garden rearing does not modify sound features, which remain specific and innate in the two cichlids. Despite the familiarity, O. niloticus remained dominant on S. melanotheron, but the aggressiveness between the two species decreased.

Chronic feeding exposure to virgin and spiked microplastics disrupts essential biological functions in teleost fish.

Toxicity of polyethylene (PE) and polyvinyl chloride (PVC) microplastics (MPs), either virgin or spiked with chemicals, was evaluated in two short-lived fish using a freshwater species, zebrafish, and a marine species, marine medaka. Exposures were performed through diet using environmentally relevant concentrations of MPs over 4 months. No modification of classical biomarkers, lipid peroxidation, genotoxicity or F0 behaviour was observed. A significant decrease in growth was reported after at least two months of exposure. This decrease was similar between species, independent from the type of MPs polymer and the presence or not of spiked chemicals, but was much stronger in females. The reproduction was evaluated and it revealed a significant decrease in the reproductive output for both species and in far more serious numbers in medaka. PVC appeared more reprotoxic than PE as were MPs spiked with PFOS and benzophenone-3 compared to MPs spiked with benzo[a]pyrene. Further, PVC-benzophenone-3 produced behavioural disruption in offspring larvae. These results obtained with two species representing different aquatic environments suggest that microplastics exert toxic effects, slightly different according to polymers and the presence or not of sorbed chemicals, which may lead in all cases to serious ecological disruptions.

Analysis across diverse fish species highlights no conserved transcriptome signature for proactive behaviour.

BACKGROUND: Consistent individual differences in behaviour, known as animal personalities, have been demonstrated within and across species. In fish, studies applying an animal personality approach have been used to resolve variation in physiological and molecular data suggesting a linkage, genotype-phenotype, between behaviour and transcriptome regulation. In this study, using three fish species (zebrafish; Danio rerio, Atlantic salmon; Salmo salar and European sea bass; Dicentrarchus labrax), we firstly address whether personality-specific mRNA transcript abundances are transferrable across distantly-related fish species and secondly whether a proactive transcriptome signature is conserved across all three species. RESULTS: Previous zebrafish transcriptome data was used as a foundation to produce a curated list of mRNA transcripts related to animal personality across all three species. mRNA transcript copy numbers for selected gene targets show that differential mRNA transcript abundance in the brain appears to be partially conserved across species relative to personality type. Secondly, we performed RNA-Seq using whole brains from S. salar and D. labrax scoring positively for both behavioural and molecular assays for proactive behaviour. We further enriched this dataset by incorporating a zebrafish brain transcriptome dataset specific to the proactive phenotype. Our results indicate that cross-species molecular signatures related to proactive behaviour are functionally conserved where shared functional pathways suggest that evolutionary convergence may be more important than individual mRNAs. CONCLUSIONS: Our data supports the proposition that highly polygenic clusters of genes, with small additive effects, likely support the underpinning molecular variation related to the animal personalities in the fish used in this study. The polygenic nature of the proactive brain transcriptome across all three species questions the existence of specific molecular signatures for proactive behaviour, at least at the granularity of specific regulatory gene modules, level of genes, gene networks and molecular functions.

Chemicals sorbed to environmental microplastics are toxic to early life stages of aquatic organisms.

Microplastics are ubiquitous in aquatic ecosystems, but little information is currently available on the dangers and risks to living organisms. In order to assess the ecotoxicity of environmental microplastics (MPs), samples were collected from the beaches of two islands in the Guadeloupe archipelago, Petit-Bourg (PB) located on the main island of Guadeloupe and Marie-Galante (MG) on the second island of the archipelago. These samples have a similar polymer composition with mainly polyethylene (PE) and polypropylene (PP). However, these two samples are very dissimilar with regard to their contamination profile and their toxicity. MPs from MG contain more lead, cadmium and organochlorine compounds while those from PB have higher levels of copper, zinc and hydrocarbons. The leachates of these two samples of MPs induced sublethal effects on the growth of sea urchins and on the pulsation frequency of jellyfish ephyrae but not on the development of zebrafish embryos. The toxic effects are much more marked for samples from the PB site than those from the MG site. This work demonstrates that MPs can contain high levels of potentially bioavailable toxic substances that may represent a significant ecotoxicological risk, particularly for the early life stages of aquatic animals.

An environmentally relevant mixture of polychlorinated biphenyls (PCBs) and polybrominated diphenylethers (PBDEs) disrupts mitochondrial function, lipid metabolism and neurotransmission in the brain of exposed zebrafish and their unexposed F2 offspring.

Polychlorinated biphenyls (PCBs) and polybrominated diphenyl ethers (PBDEs) are persistent organic pollutants still present in aquatic environments despite their total or partial ban. Previously, we observed that an environmentally realistic mixture of these compounds affects energy balance, growth, and reproduction in exposed zebrafish (F0), and behavior in their unexposed offspring (F1-F4). In the present work, we performed lipidomic and transcriptomic analyses on brains of zebrafish (F0-F2) from exposed and control lineages to identify molecular changes that could explain the observed phenotypes. The use of both technologies highlighted that F0 zebrafish displayed impaired mitochondrial function and lipid metabolism regulation (depletion in triacylglycerols and phospholipids) which can explain disruption of energy homeostasis. A subset of the regulated biological pathways related to energetic metabolism and neurotransmission were inherited in F2. In addition, there were increasing effects on epigenetic pathways from the F0 to the F2 generation. Altogether, we show that the effects of an environmental exposure to PCBs and PBDEs on energetic metabolism as well as neurotransmission extend over 2 generations of zebrafish, possibly due to transgenerational epigenetic inheritance.

Acoustic signals produced by Nile tilapia Oreochromis niloticus and black-chinned tilapia Sarotherodon melanotheron during intra- and interspecific pairings.

We characterised, for the first-time, the sound production of black-chinned tilapia Sarotherodon melanotheron and show differences with that of Nile tilapia Oreochromis niloticus in a hybridization pairing context. Although both species were able to produce drum sounds, they showed different acoustic features. Drum sounds were produced in aggressive (chasing or lateral attack) and non-aggressive (courtship) contexts by O. niloticus but only in aggressive situations (fleeing or avoidance) by S. melanotheron. The second type of sounds produced by O. niloticus were grunts, produced in both aggressive (chasing and after biting) and non-aggressive contexts (nest building). The second type of sound produced by S. melanotheron was a rolling sound, produced only during courtship. Each species was able to produce common sounds (drum) and species-specific sounds (grunts and rolling). This implies that species can communicate without being able to understand each other because the sounds emitted may probably have different significance. Drumming corresponded only to aggressivity in S. melanotheron, whereas this was not true for O. niloticus. 11-ketotestosterone (11-kt) levels were significantly higher in male O. niloticus than male S. melanotheron, but there was no significant correlation between 11-kt or estradiol concentrations and the number of sounds produced in aggressive or non-aggressive behavioural contexts in either species. During interspecies interactions, O. niloticus drum sounds are likely considered to be aggressive by S. melanotheron and could potentially constitute a reproductive barrier between the two species.

Microplastics and sorbed contaminants - Trophic exposure in fish sensitive early life stages.

The present study evaluated very small microplastic particle (MPs) transfer to zebrafish and marine medaka larvae via prey experimentally exposed to MPs from the onset of feeding. Larvae were fed Paramecium or Artemia nauplii loaded with fluorescent 1-5 or 10-20 µm MP. Pollutant accumulation was analyzed by optically tracking of benzo[a]pyrene (BaP) and recording cyp1a transcription. Paramecium transferred 1-5 µm particles only, whereas Artemia efficiently transferred both MPs. Although zebrafish and medaka larvae fed from the onset of active food intake (2-3 dph, respectively) on Paramecium and from days 6-7 post-hatch on Artemia nauplii, neither MP accumulation nor translocation to tissues was detected. MP egestion started within few hours after ingestion. Cyp1a induction and fluorescent analyses proved BaP bioavailability after transfer via Paramecium and Artemia. Unicellular or plankton organisms ingest contaminants via MPS and transfer effectively these to sensitive early life-stages of vertebrates, giving rise to whole-life exposure.

Organic contaminants sorbed to microplastics affect marine medaka fish early life stages development.

The role of polyethylene microplastics 4-6 µm size (MPs) in the toxicity of environmental compounds to fish early life stages (ELS) was investigated. Marine medaka Oryzias melastigma embryos and larvae were exposed to suspended MPs spiked with three model contaminants: benzo(a)pyrene (MP-BaP), perfluorooctanesulfonic acid (MP-PFOS) and benzophenone-3 (MP-BP3) for 12 days. There was no evidence of MPs ingestion but MPs agglomerated on the surface of the chorion. Fish ELS exposed to virgin MPs did not show toxic effects. Exposure to MP-PFOS decreased embryonic survival and prevented hatching. Larvae exposed to MP-BaP or MP-BP3 exhibited reduced growth, increased developmental anomalies and abnormal behavior. Compared to equivalent waterborne concentrations, BaP and PFOS appeared to be more embryotoxic when spiked on MPs than when alone in seawater. These results suggest a relevant pollutant transfer by direct contact of MPs to fish ELS that should be included in the ecotoxicological risk assessment of MPs.

Zebrafish Danio rerio shows behavioural cross-context consistency at larval and juvenile stages but no consistency between stages.

Coping style is defined as a set of individual physiological and behavioural characteristics that are consistent across time and context. In the zebrafish Danio rerio, as well as in many other animals, several covariations have been established among behavioural, physiological and molecular responses. Nonetheless, not many studies have addressed the consistency in behavioural responses over time starting at the larval stage. Therefore, this study aimed to improve the understanding of behavioural consistency across contexts and over time in zebrafish from the larval to juvenile stages. Two distinct experiments were conducted: a larval stage experiment (from 8 to 21 days post fertilization, dpf) and a juvenile stage experiment (from 21 to 60 dpf). On one hand, the larval experiment allows to focus on the transition between 8 and 21 dpf, marked by significant morphological changes related to the end of larval stage and initiation of metamorphosis. On the other hand, the juvenile experiment allows to properly cover the period extending from the end of larval stage to the juvenile stage (60 dpf), including metamorphosis which is itself completed around 45 dpf. Within each experiment, boldness was determined using a group risk-taking test to identify bold and shy individuals. A novel environment test was then performed at the same age to evaluate consistency across contexts. Groups of fish (either bold or shy) were bathed in an alizarin red S solution for later identification of their initially determined coping style to evaluate behavioural consistency over time. Fish were then reared under common garden conditions and challenged again with the same behavioural tests at a later age (21 and 60 dpf in the larval and juvenile experiments, respectively). Behavioural consistency was observed across contexts, with bold fish being more active and expressing higher thigmotaxis regardless of age. There was, however, little behavioural consistency over age, suggesting behavioural plasticity during development. Moreover, the use of alizarin red S to conduct this experiment provides new perspectives for the further study of the longitudinal evolution of various traits, including behaviour, over life stages in fish.

Physiological responses during acute stress recovery depend on stress coping style in European sea bass, Dicentrarchus labrax.

Individual stress coping style (reactive, intermediate and proactive) was determined in 3 groups of 120 pit tagged European seabass using the hypoxia avoidance test. The same three groups (no change in social composition) were then reared according to the standards recommended for this species. Then, 127 days later, individuals initially characterized as reactive, intermediate or proactive were submitted to an acute confinement stress for 30 min. Blood samples were taken to measure plasma cortisol levels 30 min (Stress30) or 150 min (Stress150) after the end of the confinement stress. Individuals were then sacrificed to sample the telencephalon in order to measure the main monoamines and their catabolites (at Stress30 only). Individuals from Stress150 were sampled for whole brain for a transcriptomic analysis. The main results showed that reactive individuals had a lower body mass than intermediate individuals which did not differ from proactive individuals. The physiological cortisol response did not differ between coping style at Stress30 but at Stress150 when intermediate and proactive individuals had recovered pre stress levels, reactive individuals showed a significant higher level illustrating a modulation of stress recovery by coping style. Serotonin turnover ratio was higher in proactive and reactive individuals compared to intermediate individuals and a significant positive correlation was observed with cortisol levels whatever the coping style. Further, the confinement stress led to a general increase in the serotonin turnover comparable between coping styles. Stress150 had a significant effect on target mRNA copy number (Gapdh mRNA copy number decreased while ifrd1 mRNA copy number increased) and such changes tended to depend upon coping style.

Low temperature has opposite effects on sex determination in a marine fish at the larval/postlarval and juvenile stages.

Temperature-dependent sex determination (TSD) can be observed in multiple reptile and fish species. It is adaptive when varying environmental conditions advantage either males or females. A good knowledge of the thermosensitive period is key to understand how environmental changes may lead to changes in population sex ratio. Here, by manipulating temperature during development, we confirm that cold temperature (16°C) increases the proportion of fish that develop as females in European sea bass (Dicentrarchus labrax) until 56 days posthatching, but show that it has an opposite effect at later stages, with the proportion of males reaching ~90% after 230 days at 16°C. This is the first observation of opposite effects of temperature at different time periods on the sex ratio of a vertebrate. Our results highlight the potential complexity of environmental effects on sex determination.

Prospective severity classification of scientific procedures in cephalopods: Report of a COST FA1301 Working Group survey.

Cephalopods are the first invertebrate class regulated by the European Union (EU) under Directive 2010/63/EU on the protection of animals used for scientific purposes, which requires prospective assessment of severity of procedures. To assist the scientific community in establishing severity classification for cephalopods, we undertook a web-based survey of the EU cephalopod research community as represented by the participants in the European COoperation on Science and Technology (COST) Action FA1301, CephsInAction'. The survey consisted of 50 scenarios covering a range of procedures involving several cephalopod species at different life stages. Respondents (59 people from 15 countries) either allocated a severity classification to each scenario or indicated that they were unable to decide (UTD). Analyses evaluated score distributions and clustering. Overall, the UTD scores were low (7.0 ± 0.6%) and did not affect the severity classification. Procedures involving paralarvae and killing methods (not specified in Annexe IV) had the highest UTD scores. Consensus on non-recovery procedures was reached consistently, although occasionally non-recovery appeared to be confused with killing methods. Scenarios describing procedures above the lower threshold for regulation, including those describing behavioural studies, were also identified and allocated throughout the full range of severity classifications. Severity classification for scenarios based on different species (e.g. cuttlefish vs. octopus) was consistent, comparable and dependent on potentially more harmful interventions. We found no marked or statistically significant differences in the overall scoring of scenarios between the demographic subgroups (age, sex, PhD and cephalopod experience). The COST Action FA1301 survey data provide a basis for a prospective severity classification for cephalopods to serve as guide for researchers, project assessors and regulators.