Effects of dietary L-tryptophan enrichment on stress response, growth and survival in lumpfish (Cyclopterus lumpus) following sea transfer

Characteristics of lumpfish (Cyclopterus lumpus) with high cleaning efficacy in commercial Atlantic salmon (Salmo salar) production

To mitigate salmon lice infestations in Atlantic salmon (Salmo salar) sea cages, deployment of cleaner fish have become a widespread strategy. However, species of cleaner fish may experience poor welfare in the highly fluctuating farm environment owing to differences in physiological adaptations and niche requirements. In particular, occurrences of reduced oxygen levels are common in salmon cages. The purpose of this study was therefore to compare hypoxia responses of Atlantic salmon and two commonly used cleaner fish species, the lumpfish (Cyclopterus lumpus) and the ballan wrasse (Labrus bergylta). We used respirometry to measure metabolic rates (MO2) during progressive hypoxia down to 20% oxygen saturation. In addition, we also measured key haematological parameters before, during and after hypoxia exposure. While all fish survived exposure down to 20% oxygen saturation, distinct differences in metabolic and haematological responses were found, reflecting species specific adaptations and lifestyles. In Atlantic salmon, MO2 was independent of ambient oxygen levels until 27% saturation, after which it decreased linearly. In lumpfish, MO2 steadily decreased throughout the hypoxia trial. In ballan wrasse, MO2 was notably lower than in the other species and unaffected by the levels of hypoxia encountered. Hypoxia induced changes in plasma cortisol, plasma lactate and plasma osmolality were substantially greater in Atlantic salmon compared to both cleaner fish species. This suggests that similar magnitudes of hypoxia exposure were more stressful to Atlantic salmon. Hence, neither cleaner fish species should be in immediate danger as long as hypoxia levels that are known to be detrimental to Atlantic salmon are avoided. However, lumpfish had markedly reduced activity levels at the early onset of progressive hypoxia, and is therefore likely to require near normoxic conditions to efficiently function as cleaner fish.

Lumpfish (Cyclopterus lumpus) are used as cleaner fish in Atlantic salmon (Salmo salar) aquaculture as one of multiple control measures against salmon lice (Lepeophtheirus salmonis). Numerous studies have documented weight as an important factor for characterising the cleaning efficacy of individual lumpfish. Hence, the efficacy of lumpfish in fish farms depends on the size at stocking and the weight development over time. Here, we aimed to quantify how weight developed over time for lumpfish deployed in salmon farming sea cages in Norway through modelling the daily growth rate. We found that the coefficient of variation for lumpfish weight increased over time, implying that the standard deviation increased more than the expected weight. The data thus showed heterogeneous growth for lumpfish in salmon farming cages, where some individuals exhibited no apparent growth, and others significant proliferation. We found that the daily growth rate was best described as bell-shaped functions of weight and temperature, consistent with a sigmoid-shaped growth curve and an optimal temperature around 10 °C. These results allow for more realistic modelling of the efficacy of lumpfish in controlling salmon lice in fish farms, as one can combine estimates of weight-dependent cleaning efficacy with weight development over time.

The cleaning efficacy of lumpfish (Cyclopterus lumpus L.) in Faroese salmon (Salmo salar L.) farming pens in relation to lumpfish size and seasonality

The commercial production of lumpfish Cyclopterus lumpus L. is expanding with the increased demand for their use as cleaner fish, to control sea-lice numbers, at marine Atlantic salmon Salmo salar L. aquaculture sites throughout Northern Europe. A new ranavirus has been isolated from lumpfish at multiple locations in the North Atlantic area. First isolated in 2014 in the Faroe Islands, the virus has subsequently been found in lumpfish from Iceland in 2015 and from Scotland and Ireland in 2016. The Icelandic lumpfish ranavirus has been characterized by immunofluorescent antibody test, optimal growth conditions and transmission electron microscopy. Partial sequences of the major capsid protein gene from 12 isolates showed 99.79-100% nt identity between the lumpfish ranaviruses. Complete genome sequencing from three of the isolates and phylogenetic analysis based on the concatenated 26 iridovirus core genes suggest these lumpfish ranavirus isolates form a distinct clade with ranaviruses from cod Gadus morhua L. and turbot Scophthalmus maximus L. isolated in Denmark in 1979 and 1999, respectively. These data suggest that these viruses should be grouped together as a new ranavirus species, European North Atlantic Ranavirus, which encompasses ranaviruses isolated from marine fishes in European North Atlantic waters.

European North Atlantic ranavirus (ENARV, Iridoviridae), is a ranavirus species recently isolated from lumpfish (Cyclopterus lumpus, L.), which are used as cleaner fish in Atlantic salmon (Salmo salar) farming in Northern Europe. This study aimed to investigate (1) the virulence of ENARV isolates from Ireland, Iceland and the Faroe Islands to lumpfish; (2) horizontal transmission between lumpfish; and (3) virulence to Atlantic salmon parr. Lumpfish were challenged in a cohabitation model using intraperitoneally (IP) injected shedders, and naïve cohabitants. IP challenge with isolates from Iceland (1.9× 107 TCID50 ml-1 ) and the Faroe Islands (5.9× 107 TCID50 ml-1 ) reduced survival in lumpfish, associated with consistent pathological changes. IP challenge with the Irish strain (8.6× 105 TCID50 ml-1 ) did not significantly reduce survival in lumpfish, but the lower challenge titre complicated interpretation. Horizontal transmission occurred in all strains tested, but no clinical impact was demonstrated in cohabitants. Salmon parr were challenged by IP injection with the Irish isolate, no virulence or virus replication were demonstrated. A ranavirus qPCR assay, previously validated for fish ranaviruses, was first used to detect ENARV in tissues of both in lumpfish and Atlantic salmon. This study provides the first data on the assessment of virulence of ENARV isolates to lumpfish and salmon, guidelines for the diagnosis of ENARV infection, and poses a basis for further investigations into virulence markers.

Lumpfish (Cyclopterus lumpus L.) develop amoebic gill disease (AGD) after experimental challenge with Paramoeba perurans and can transfer amoebae to Atlantic salmon (Salmo salar L.)

Effects of reduced water exchange rate and oxygen saturation on growth and stress indicators of juvenile lumpfish (Cyclopterus lumpus L.) in aquaculture

[This study examined cortisol release into the water by European sea bass, Dicentrarchus labrax . The time-course of plasma and water cortisol concentrations were determined in adult fish subjected to acute stress, by sampling blood and water at 0 h (before stress) and 0.5, 1, 2, 4, 8 and 24 h after stress. Sea bass showed a typical stress response, with plasma glucose and lactate concentrations peaking at 2 h, and plasma cortisol levels peaking at 1 h. Cortisol release rate into the water increased in response to stress and was positively correlated with plasma cortisol concentrations. In a further trial, juvenile fish were confined at densities of 20 and 50 kg/m 3 and water cortisol was evaluated over a 24 h period. Cortisol release rates peaked between 0–1 h in the high and 1–2 h in the low density group. In conclusion, these results provide strong evidence that cortisol release rate into the water can be used as a non-invasive method for the assessment of the stress response and that although sea bass presents a high blood stress response after exposure to acute husbandry stressors, it is releasing less cortisol into the water compared to other species previously examined., This study examined cortisol release into the water by European sea bass, Dicentrarchus labrax . The time-course of plasma and water cortisol concentrations were determined in adult fish subjected to acute stress, by sampling blood and water at 0 h (before stress) and 0.5, 1, 2, 4, 8 and 24 h after stress. Sea bass showed a typical stress response, with plasma glucose and lactate concentrations peaking at 2 h, and plasma cortisol levels peaking at 1 h. Cortisol release rate into the water increased in response to stress and was positively correlated with plasma cortisol concentrations. In a further trial, juvenile fish were confined at densities of 20 and 50 kg/m 3 and water cortisol was evaluated over a 24 h period. Cortisol release rates peaked between 0–1 h in the high and 1–2 h in the low density group. In conclusion, these results provide strong evidence that cortisol release rate into the water can be used as a non-invasive method for the assessment of the stress response and that although sea bass presents a high blood stress response after exposure to acute husbandry stressors, it is releasing less cortisol into the water compared to other species previously examined.]

Lumpfish, Cyclopterus lumpus, have historically been harvested throughout Atlantic Canada and are increasingly in demand as a solution to controlling sea lice in Atlantic salmon farms—a process which involves both the domestication and the transfer of lumpfish between geographic regions. At present, little is known regarding population structure and diversity of wild lumpfish in Atlantic Canada, limiting attempts to assess the potential impacts of escaped lumpfish individuals from salmon pens on currently at‐risk wild populations. Here, we characterize the spatial population structure and genomic‐environmental associations of wild populations of lumpfish throughout the Northwest Atlantic using both 70K SNP array data and whole‐genome re‐sequencing data (WGS). At broad spatial scales, our results reveal a large environmentally associated genetic break between the southern populations (Gulf of Maine and Bay of Fundy) and northern populations (Newfoundland and the Gulf of St. Lawrence), linked to variation in ocean temperature and ice cover. At finer spatial scales, evidence of population structure was also evident in a distinct coastal group in Newfoundland and significant isolation by distance across the northern region. Both evidence of consistent environmental associations and elevated genome‐wide variation in FST values among these three regional groups supports their biological relevance. This study represents the first extensive description of population structure of lumpfish in Atlantic Canada, revealing evidence of broad and fine geographic scale environmentally associated genomic diversity. Our results will facilitate the commercial use of lumpfish as a cleaner fish in Atlantic salmon aquaculture, the identification of lumpfish escapees, and the delineation of conservation units of this at‐risk species throughout Atlantic Canada.

First interaction with carnivorous clients induces stress responses even in the boldest of cleaner fishes observed in nature. This is relevant for the expanding use of lumpfish in aquaculture, where stress could impede the species ability to interact with Atlantic salmon. The study investigated how naive lumpfish (34.3 g, S.D. ± 6.48) responded to different heterospecific cues including (1) exposure to water from a tank with Atlantic salmon (“Olfaction”), (2) salmon lifelike models (“Model”) and (3) Atlantic salmon (“Live”). Experiments were repeated thrice, using duplicate tank replicates on each occasion (n = 36 per treatment). Behaviour was recorded 30 min before and 30 min after the introduction of each treatment. Responses measured included swimming activity, body colour and pigmentation, neurotransmitters, and plasma cortisol. Data demonstrated a significant increase in swimming activity upon introduction of Olfaction and Live salmon, but not from Models. After 30 min of interaction, swimming activity decreased towards levels observed in control groups. Body colour significantly increased in lumpfish exposed to Olfaction while body pigmentation significantly increased in both Olfaction and Model treatments. Neurotransmitters and plasma cortisol measurements did not differ between treatments and control, yet large individual variation was observed. Our findings revealed that lumpfish discriminated salmon cues, whereas Olfaction induced the overall strongest behavioural and physiological responses. The study suggested that acute change in skin colour and pigmentation can be impacted by salmon interaction, yet deployment of naive juvenile lumpfish with small Atlantic salmon is preferable based on the overall mild stress responses.

The management of sea lice is one of the major challenges in farming of Atlantic salmon (Salmo salar) in Norway. Different methods are in use to remove sea lice from farmed salmonids during the grow-out phase in the sea, including mechanical, medicinal, and biological treatments. The use of farmed lumpfish (Cyclopterus lumpus) and ballan wrasse (Labrus bergylta) as cleaner fish is considered less stressful for salmon compared to other treatments. Documenting cleaning behavior in commercial sea cages is difficult. In this study, we used artificial sea lice dummies and camera to study cleaning behavior of farmed ballan wrasse in commercial sea cages during summer and autumn of 2021. Artificial sea lice dummies, 11–12 mm in length, and 26–27 mm total length including egg-strings, were attached to fixed positions on a dead salmon and placed in the sea cage. A total of 74 h of video recordings from three different cages were analyzed. We recorded inspections of the salmon by ballan wrasse, and any bites made at sea lice dummies or the salmon. Ballan wrasse rapidly showed interest in the model after introduction, with most of the first observations of behavioral events occurring within the first 30 min after the salmon were placed in the sea cage. An inspection of salmon rarely resulted in a bite on sea lice dummies (8%) and ballan wrasse was also observed biting on the dead salmon (12%). Cleaning behavior (i.e., bite on sea lice dummies) was on average ≈1 bite hour−1 and lice dummies placed on anterior ventral area were most susceptible to be bitten. This study uses a non-invasive method to study cleaner fish behavior, and it can in the future be used to investigate how different environmental factors impact cleaner fish behavior in large sea cages.

The objective of this study was to describe the responses of the plasma progesterone and cortisol concentrations in ovariectomized lactating cows to low doses of adrenocorticotropic hormone (ACTH). The estrous cycles in 3 lactating cows were synchronized, and the cows were ovariectomized in the luteal phase. ACTH challenge tests were conducted at doses of 3, 6, 12 and 25 IU. Blood samples were collected at 30 min intervals, and the plasma progesterone and cortisol concentrations were analyzed by EIA. A concomitant rise in plasma progesterone and plasma cortisol was observed in cows treated with 12 IU or higher doses of ACTH. Significant increments in the plasma cortisol concentrations were observed at all doses of ACTH. The means (+/- SE) of the peak plasma progesterone concentrations after the 3, 6, 12 and 25 IU ACTH challenge tests were 0.6 +/- 0.1, 1.3 +/- 0.4, 1.5 +/- 0.3 and 2.4 +/- 0.3 ng/ml, respectively. The means of the peak plasma cortisol concentrations in the 3 cows after the ACTH challenge were 14.0 +/- 1.5, 17.0 +/- 2.5, 23.3 +/- 3.0, and 33.3 +/- 7.0 ng/ml, respectively. The effects of the doses, time after treatment, and their interaction on the plasma progesterone concentrations after the ACTH challenge were significant (P<0.01). Likewise, the effects of the doses, time after treatment, and their interaction on the plasma cortisol concentrations after the ACTH challenge were significant (P<0.01). The mean AUC values for the plasma progesterone and cortisol concentrations after the ACTH treatments were also significantly affected by the dose of ACTH (P<0.01 and P<0.05, respectively). A significantly positive correlation was obtained between the peak plasma progesterone and cortisol concentrations after different doses of ACTH (r=0.7, P<0.05). The results suggest that lactating dairy cows are capable of secreting a significant amount of adrenal progesterone, reaching up to the minimal concentration necessary to cause suppression of estrus in response to 12 IU ACTH (P<0.01). The concomitant plasma cortisol concentration was 23.3 ng/ml.

Cleaner fish are commonly used as a control measure against salmon lice infestations in salmonid farms. Lumpfish (Cyclopterus lumpus) is the most common cleaner fish species used in Norwegian farms. However, little is known about how different operational, including environmental, conditions affect the salmon lice grazing efficacy by lumpfish. In this paper, we analyse salmon lice (Lepeophtheirus salmonis) in the stomach contents of a large sample of more than 20,000 lumpfish from 80 different Norwegian farms. We investigate the proportion of lumpfish with salmon lice and the mean number of salmon lice in the stomach contents of the lumpfish. We further explore how the salmon lice contents vary with different factors like lumpfish weight, weight of salmonids, salmon lice abundance in the cage, cloud cover, and sea temperature. We find that 3.1% of the 24,693 lumpfish contained salmon lice. Most of the lumpfish with salmon lice in their stomach contents contained few (one or two) lice, while there were a few lumpfish which contained many salmon lice. We find more salmon lice in the stomach contents with increasing abundance of salmon lice in the sea cage, lower weight of the salmonids, and in clear weather. Interestingly, for the relationship between lumpfish weight and salmon lice in the stomach contents, we find increased salmon lice grazing from ca. 5 g up to an optimal weight of ca. 40 g, and then a decrease from 40 g. Surprisingly, we find no relationship between sea temperature and salmon lice per lumpfish. We find more salmon lice in the stomach contents of the lumpfish with comparatively high condition. By studying the factors associated with most efficient salmon lice grazing, our paper contributes to understanding how different operational factors affect salmon lice grazing by lumpfish. For controllable factors, the results thus contribute to guiding the best practice for use of lumpfish as a salmon lice control measure.

Lumpfish (Cyclopterus lumpus L.) are commercially important species used world-wide since 2008 as cleaner fish to control sea lice (Lepeophtheirus salmonis), one of the most significant parasites affecting Atlantic salmon (Salmo salar L.) and costing the industry over 500M € annually. However, lumpfish mortalities in sea cages can be as high as 100%, raising welfare concerns on whether their use continue to be acceptable. The industry is under scrutiny by both government agencies and non-governmental organisations to improve lumpfish welfare, but they do not provide much direction as welfare standards are not developed yet for this species. This thesis examined novel ways to monitor welfare in farmed lumpfish in order to increase the sustainability and ethical use of lumpfish in aquaculture. Chapter 1 reviewed the bases of fish welfare as well as lumpfish biology, use as cleaner fish and main challenges within the aquaculture industry. Chapter 2 developed and tested a practical, easy-to-use, validated and highly repeatable (ICC=0.83) scoring index combining six Operational Welfare Indicators to measure lumpfish welfare under farm conditions. Chapter 3 found welfare deterioration in sea cages to be dependent on time spent at sea and to be worse in smaller fish. Welfare monitoring should be particularly regular during the first months post-transfer at sea. Chapter 4 found differences in welfare, growth, feeding preferences and gut microbiota between different genetic stocks of lumpfish, with Icelandic lumpfish growing faster, showing better welfare and ingesting more formulated pellets than Scottish lumpfish. Significant associations in the gut microbiota were identified between compromised welfare and Candidatus branchiomonas and high plasma cortisol and Clostridium, suggesting these could be used as potential biomarkers. Chapter 5 investigated the effect of sea lice ingestion in lumpfish welfare and gut health and found that sea lice ingestion did not have any influence and is not detrimental for lumpfish welfare. Welfare investigations under commercial conditions proved that lumpfish welfare status (measured by using individual morphological indicators) deteriorates in time when stocked in salmon net pens, resulting in an increase of these scores, which also differ between commercial sites and populations under the same conditions. The ability of monitoring welfare in regular basis serves as an early warning for health and welfare issues and the application of the index along the knowledge withdrawn from this thesis will help farmers to identify critical periods where lumpfish welfare starts deteriorating, as well as provide scientific reference to policy developers, welfare organisations and NGOs and quality assurance schemes. In this sense, welfare standards for these novel species can be developed, and corrective actions can be taken before any issues progress into mortalities. This will enhance the sustainability of the lumpfish industry and will reduce economic costs of sea lice management, mitigating environmental impacts for not using chemotherapeutants at sea and improving salmon welfare overall.