The Atlantic wolffish (Anarhichas lupus) is a cold-water species with the potential to diversify aquaculture in Northern countries. Few studies have investigated the stress physiology of Atlantic wolffish, and the current knowledge on stress in wolffish species is largely derived from the closely related spotted wolffish (Anarhichas minor). In the current study, Atlantic wolffish were exposed to handling stress mimicking common husbandry conditions in aquaculture such as repeated air exposure and net-chasing. Samples were taken prior to stress exposure (pre-stress; control) as well as 5- and 24-h post-stress. A series of primary and secondary acute stress response parameters were assessed: plasma cortisol, glucose and lactate levels, hematological indices (hemoglobin, Hb; hematocrit, Hct; mean corpuscular hemoglobin concentration, MCHC), and osmoregulatory capacity through plasma osmolality and gill Na+/K+ATPase (NKA) activity. Other secondary stress responses with implications for fish health and welfare are intestinal integrity and transport functions. These parameters were assessed using the Ussing chamber technique. The cortisol peak values were low in comparison to other fish species studied after acute handling stress and occurred as late as 24 h post-stress, suggesting that Atlantic wolffish is a slow and low cortisol responder. Plasma glucose remained stable, whereas lactate concentrations significantly decreased between 5 and 24 h after stress. There was no effect on pH, Hb, or Hct, although a significant increase in MCHC was found after 5 h and 24 h, originating from a small increase in Hb. This result suggests a minor increase in Hb synthesis after stress exposure. The intestinal integrity and transport functions as well as gill NKA-activity remained unchanged after stress exposure. In conclusion, Atlantic wolffish appears to exhibit a relatively moderate stress response, characterized by a slow and low primary stress response and minimal secondary effects following husbandry-related acute stress. These findings contribute to the understanding of the species’ potential for development as a candidate for marine, cold-water aquaculture.

The present study investigates the mitigation of the stress response induced by a bivalent vaccine against vibriosis and pasteurellosis in sea bass through treatment with thymol-carvacrol oil extracts, known as FishEaseC (FEC) and FishEase-L (FEL). Plasma and skin mucus cortisol as well as mucosal transcript stress-immune response were measured at 1 and 24 h post-vaccination (hpv). Our results indicate that FEL but not FEC was capable of reducing plasmatic cortisol secretion by 24 hpv, in contrast to vaccinated and vaccinated plus treated with FEC groups. A similar pattern was noted in the skin mucus though secretion of cortisol was prevented by the FEL. The expression of stress-immune related gene transcripts (gr1, gr2, crh, hsp70, cox2, il1(1, il6, il8, il10, tgf(11, lysozyme, igm, igt and igd/igt) revealed that stress-immune gene regulation was not homogenous among mucosae. Gills were more responsive to the stress/inflammation induced by FEC, as shown by crh, hsp70, cox2, il1(1 and il6 enhanced expression, suggesting higher absorption of FEC via this mucosae. The gills also appeared to be more resilient to the stressful effect of the vaccine, while higher regulation was observed in skin and intestine. In summary, administration of FEL was able to mitigate the stress response at physological (e.g., cortisol) and the stress-immune genes activation in skin and intestine(hsp70, il1(1, il6), mainly at 24 hpv. We recommend FEL as a customized formulation to farmers for use on sea bass prior to vaccination, in order to mitigate negative effect of vaccination procedure.

A nutritional bottleneck in salmonid aquaculture is the procurement of marine-derived compounds, such as essential amino and fatty acids, including omega-3 fatty acids, lysine, and methionine. Therefore, insects containing these compounds are highly promising as feed ingredients. The present study evaluates larvae of a "marine" insect (Coelopa frigida, the bristly-legged seaweed fly larvae, SWFL) reared on brown algae side streams as a feed ingredient for rainbow trout (Oncorhynchus mykiss). SWFL contained, on a dry matter basis, 60% crude protein (CP), 3.5% lysine, and 1.5% methionine, as well as 17% lipids, including 4% eicosapentaenoic acid and docosahexaenoic acid. Four isoenergetic (ca. 23 MJ kg-1 gross energy) and isoproteic (ca. 45% CP) feeds were evaluated in a 10-week feeding trial. The diets included a control containing 25% fish meal, a commercial reference, and two diets substituting 40% fish meal with either SWFL or partially defatted black soldier fly larvae (BSFL) meal (Hermetia illucens). SWFL-fed fish displayed higher specific growth rates (SGR) compared to BSFL-fed fish and similar SGR compared to control and reference diet-fed fish. Feed intake in SWFL-fed fish was higher than for the control and BSFL diets and similar to the reference diet. The feed conversion ratio of fish fed the BSFL and SWFL diets was higher compared to the control, suggesting lower bioavailability of both insect meals compared to fish meals. No difference in intestinal health parameters was noted between the insect diets and the control diet, indicating good intestinal health across all treatments. However, changes in electrogenic intestinal transport were observed between the SWFL and BSFL diets, illustrating the heterogeneous effect of different insect products. Overall, SWFL meal is a promising alternative marine feed ingredient, compatible with circular production systems, as it can be efficiently cultivated using marine side streams.

Aquaculture is essential for meeting the growing global demand for fish consumption. However, the widespread use of plastic and the presence of microplastics in aquaculture systems raise concerns about their impact on fish health and the safety of aquaculture products. This study focused on the Nile tilapia (Oreochromis niloticus), one of the most important aquaculture fish species globally. The aim of this study was to investigate the effects of dietary exposure to a mixture of four conventional fossil fuel-based polymers (microplastics) on the health of adult and juvenile Nile tilapia. Two experiments were conducted, with 36 juvenile tilapia (10- 40 g weight) exposed for 30 days and 24 adult tilapia (600- 1000 g) exposed for 7 days, the former including a natural particle (kaolin) treatment. In the adult tilapia experiment, no significant effects on intestinal health (Ussing chamber method), oxidative stress, or inflammatory pathways (enzymatic and genetic biomarkers) were observed after exposure to the microplastic mixture. However, in the juvenile tilapia experiment, significant alterations in inflammatory pathways were observed following 30 days of exposure to the microplastic mixture, indicating potential adverse effects on fish health. These results highlight the potential negative impacts of microplastics on fish health and the economics and safety of aquaculture.

AbstractMicroplastic (MP) ingestion through fish consumption is a concern for human exposure. While the presence of plastic particles in fish tissues has been documented worldwide, information on microplastic concentrations in edible tissues, especially those smaller than 10 µm, remains scarce. Spectrometric techniques provide a complementary analytical tool to measure MP mass for human exposure studies without intrinsic size limitations; however, their application to fish analysis is limited. In this study, we utilized pyrolysis gas chromatography–mass spectrometry (Py-GC–MS) for the identification and quantification of MPs in fish muscle tissues. Two sample preparation methods, pressurized liquid extraction, and chemical digestion, were tested for compatibility with Py-GC–MS analysis. An analytical method using chemical digestion was validated for analyzing particles ≥ 0.7 µm for 4 polymer types: polypropylene, polyethylene, polystyrene, and polymethyl methacrylate. The developed method was applied to 24 adult Nile tilapia (Oreochromis niloticus) samples from a recirculating aquaculture system. MPs were detected in 42% of the samples, with an average concentration of 0.14 ± 0.32 µg/g, while high variations within subsamples were observed. Our findings reveal trace amounts of MPs in edible fish tissues from aquaculture, highlighting the potential risk of microplastic ingestion through fish consumption. This underscores the need for further risk assessments to evaluate the impact on human health and to develop appropriate mitigation measures. Graphical Abstract

Microplastics research has gained momentum in the 21st century but lags behind the long-standing research on clastic sediment. An interdisciplinary review paper was conducted, comparing microplastics with natural sediments in terms of particle properties, transport processes, sampling techniques, and ecotoxicology. The paper identifies seven research goals to enhance our understanding of microplastics in freshwater environments while learning from sediment research. This extended abstract presents the core message of the review paper, emphasizing the need to improve descriptions of microplastic particles, understand their transport processes, develop standardized sampling methods, and study their ecotoxicological effects. The research goals outline specific tasks to achieve these objectives and emphasize the importance of comparing microplastics to sediments to gain insights into their toxicity. Addressing these research goals will contribute to a comprehensive understanding of microplastics and their impact on freshwater ecosystems. For detailed insights, the original paper should be consulted.

The mechanisms underpinning ionic transport and barrier function have been relatively well characterised in amphibians and fish. In teleost fish, these processes have mostly been characterised in the gill and intestine. In contrast, these processes remain much less clear for the trunk skin of fish. In this study, we measured barrier function and active transport in the trunk skin of the rainbow trout, using the Ussing chamber technique. The effects of epithelial damage, skin region, salinity, and pharmacological inhibition were tested. Skin barrier function decreased significantly after the infliction of a superficial wound through the removal of scales. Wound healing was already underway after 3 h and, after 24 h, there was no significant difference in barrier function towards ions between the wounded and control skin. In relation to salinity, skin permeability decreased drastically following exposure to freshwater, and increased following exposure to seawater. Changes in epithelial permeability were accompanied by salinity-dependent changes in transepithelial potential and short-circuit current. The results of this study support the idea that barrier function in rainbow trout trunk skin is regulated by tight junctions that rapidly respond to changes in salinity. The changes in transepithelial permeability and short circuit current also suggest the presence of an active transport component. Immunostaining and selective inhibition suggest that one active transport component is an apical V-ATPase. However, further research is required to determine the exact role of this transporter in the context of the trunk skin.

Many studies have now reported adverse effects of exposure to microplastics in aquatic organisms. Still, relatively few studies have compared the effects of MPs with those of natural particle controls, which makes it difficult to separate particle effects from chemical effects. In this study, we carry out a meta-analysis of 26 studies to compare the effects of MPs and natural particles on aquatic animals using three different endpoints - growth, reproduction, and mortality. This analysis showed that MPs have the capacity to induce more adverse effects on growth, reproduction, and mortality for some taxonomic groups. However, the effects of exposure to MPs are not consistent across each endpoint or between taxonomic groups. We were not able to clearly discern differing impacts resulting from exposure to specific polymer types or shapes, though more negative effects were associated with polylactic acid and polyethylene, as well as fragments as opposed to beads or fibres. Additionally, meta-regression indicated that larger MP sizes, higher experimental temperatures, and longer exposure periods were all generally associated with more adverse effects. Future studies should continue to make use of negative particle controls to allow for better risk assessment of microplastics and nanoplastics in aquatic ecosystems.

Size-selective harvesting of intertidal molluscs is a common practice. However, the effects of long-term traditional harvesting remain unclear. Changes in mean shell size are generally taken as evidence of changes in harvesting intensity. However, mean shell size is also influenced by environmental pressures, which may confound the analysis of size variation over time. In this study, we apply geometric morphometrics to historical data from two shell middens, in an attempt to classify Littorina littorea shells to their environmental origins, prior to carrying out shell size analysis. Using this method, shell shape was found to be consistent within and between the midden sites. Based on comparison with modern populations from shores of known wave exposure, the midden shells were found to be more consistent with sheltered shores, and to differ most from the very exposed shore sites, the latter of which are located adjacent to the midden remains. The mean shell size was significantly smaller in the more recent midden site. We hypothesize this reduction is caused by an increase in harvesting intensity over this period. It is also possible, given the very slight reduction in shell size, coupled with certain life-history traits of L. littorea, that the change in shell size was caused by slight differences in environmental conditions. The use of midden shells and morphometrics has the potential to provide an insight into previous environmental conditions and past harvesting practices, which may be used to inform current harvesting practices.

Although the study of microplastics in the aquatic environment incorporates a diversity of research fields, it is still in its infancy in many aspects while comparable topics have been studied in other disciplines for decades. In particular, extensive research in sedimentology can provide valuable insights to guide future microplastics research. To advance our understanding of the comparability of natural sediments with microplastics, we take an interdisciplinary look at the existing literature describing particle properties, transport processes, sampling techniques and ecotoxicology. Based on our analysis, we define seven research goals that are essential to improve our understanding of microplastics and can be tackled by learning from natural sediment research, and identify relevant tasks to achieve each goal. These goals address (1) the description of microplastic particles, (2) the interaction of microplastics with environmental substances, (3) the vertical distribution of microplastics, (4) the erosion and deposition behaviour of microplastics, (5) the impact of biota on microplastic transport, (6) the sampling methods and (7) the microplastic toxicity. When describing microplastic particles, we should specifically draw from the knowledge of natural sediments, for example by using shape factors or applying methods for determining the principal dimensions of non-spherical particles. Sediment transport offers many fundamentals that are transferable to microplastic transport, and could be usefully applied. However, major knowledge gaps still exist in understanding the role of transport modes, the influence of biota on microplastic transport, and the importance and implementation of the dynamic behaviour of microplastics as a result of time-dependent changes in particle properties in numerical models. We give an overview of available sampling methods from sedimentology and discuss their suitability for microplastic sampling, which can be used for creating standardised guidelines for future application with microplastics. In order to comprehensively assess the ecotoxicology of microplastics, a distinction must be made between the effects of the polymers themselves, their physical form, the plastic-associated chemicals and the attached pollutants. This review highlights areas where we can rely on understanding and techniques from sediment research - and areas where we need new, microplastic-specific knowledge - and synthesize recommendations to guide future, interdisciplinary microplastic research.

Microplastic ingestion by intertidal fauna is a well-documented phenomenon, with emphasis on the physiological consequences of microplastic exposure. However, the behavioural effects of microplastic ingestion have not been explored to the same degree, even in species with documented microplastic ingestion. In this study, the predator-avoidance emergence response of Littorina littorea was assessed and related to microplastic levels within the samples. This is a novel approach to microplastic behavioural experiments, whereby current environmental L. littorea microplastic levels are assessed, rather than levels vastly in excess of those recorded under field conditions. The results showed no difference in emergence likelihood or emergence latency related to microplastic abundance, sex, or treatment. However, L. littorea size did have a significant effect on emergence likelihood and emergence latency, with smaller individuals emerging faster and more frequently. This study shows that microplastics, at their current environmental levels, do not seem to affect L. littorea emergence behaviour.

20 Microplastics (MPs) are an environmental pollutant of increasing concern, particularly within 21 intertidal habitats. However, little research has been carried out to assess MP levels in intertidal 22 fauna. Despite being at particular risk of encountering MPs through grazing, rocky shore 23 gastropods are one such groups that have been relatively understudied in relation to MP 24 pollution. The authors explored MP abundance in the gastropod Littorina littorea from four 25 sites within Galway Bay in West Ireland. To do this, 50 L. littorea individuals were collected 26 from four rocky shores of varying wave exposure, with two sites located in the north of the bay 27 and two in the south. An additional 50 individuals were taken from a commercial exporter, to 28 determine MP levels in L. littorea intended for human consumption. MPs were recovered from 29 60.4% of the samples and the average MP level was 2.14 MPs/gram. MP levels were found to 30 differ significantly between sites, with the two northern sites having the greatest MP 31 abundances. Results are discussed in relation to differences in environmental conditions and 32 the possible implications of MP levels for selection of harvesting sites. 33

Microplastics (MPs) are an environmental pollutant of increasing concern. However, little research has assessed MP levels in intertidal gastropods. The authors explored MP abundance in the gastropod Littorina littorea from four sites within Galway Bay, West Ireland. To do this, 50 L. littorea were collected from four rocky shores of varying wave exposure, with two sites located in the north of the bay and two in the south. An additional 50 individuals were taken from a commercial exporter, to determine MP levels in L. littorea intended for human consumption. MPs were recovered from 60.4% of the individuals and the average MP level was 2.14 MPs/gram of wet soft body mass. MP levels differed significantly between sites, with the two northern sites having the highest levels. Oceanographic conditions within Galway Bay are likely responsible for the observed variation in MP abundance, with the North Sound of the bay potentially facilitating deposition.

The study of morphology is a common means of biological grouping and classification. In recent years, morphometric studies have been dominated by quantitative geometric-morphometric methods of data extraction such as outline or landmark-based analysis. These methods are often used in conjunction with various classification methods such as linear discriminant analysis (LDA) and random forests (RF) in order to achieve interand intraspecific grouping based on environmental factors. Despite numerous studies incorporating these data-extraction and classification methods, comparisons of the effectiveness of these methods are largely lacking, especially for species which display low morphological variation. The aim of this study was to compare the effectiveness of two data-extraction methods, elliptic Fourier analysis (EFA) and generalized Procrustes analysis, and two classification methods, LDA and RF, using Littorina littorea as the study organism. The results show that the principal component scores derived from EFA, provided the optimal data input for classification while the greatest percentage of successfully classified individuals was achieved using LDA. However, based on this study RF is the recommended classification method as it is resistant to overfitting, makes no assumptions about the data, is well suited to morphometric data and produces similar rates of classification to LDA. The results are discussed in a biological context for L. littorea, based on the environmental factors of zonation and shore exposure.