The impact of dissolved oxygen concentration on the swimming performance and welfare of ballan wrasse (Labrus bergylta Ascanius, 1767).

Sea lice (Lepeophtheirus salmonis [Krøyer, 1838]) are a key issue for salmon aquaculture, contributing to increased mortality for both wild and farmed salmon if no action is taken. Using cleaner fish can be an effective, drug-free treatment method, and ballan wrasse (Labrus bergylta) is a hardy wrasse species that displays cleaning behavior. With concerns about the overharvest of wild ballan wrasse, many companies farm this species, but the optimal ranges of a wide variety of rearing parameters are still unknown. This study investigated the effect of 6-week exposure to four dissolved oxygen (DO) levels (125%, 100%, 85%, and 75% DO saturation as the percentage of air) on ballan wrasse. Survival; growth (specific growth rate, SGR); condition factor (CF); and primary (cortisol), secondary (glucose, lactate, magnesium), and tertiary stress indicators (swimming performance) were investigated. There were no differences in SGR, CF, survival, or cortisol level among the groups at the end of the 6 weeks. There was variation in the magnitude of the cortisol response to an acute stressor at the end of the 6-week period, with the 75% DO treatment exhibiting a 3.3-fold increase in cortisol compared to a 5.2-fold increase in the control group (100%), which could suggest chronic stress. Relative critical swimming speed (RUcrit) was measured to investigate swimming performance once all groups were returned to 100% DO saturation. The 75% RUcrit was lower than the 100% treatment (1.7 ± 0.18 body length [BL]/s compared to 2.5 ± 0.16 BL/s). Overall, these results suggest that DO levels of 75% trigger physiological changes and therefore may negatively affect welfare.

A new maximum age observed within the family Labridae, ballan wrasse Labrus bergylta.

Ballan wrasse Labrus bergylta is the largest species of wrasse inhabiting European waters and one of the longest-living species within the family Labridae. A large specimen was caught off the coast of Skjerjehamn, western Norway (total length = 410 mm; weight = 1274 g). The age of the specimen was determined to be 34 years old based on the analysis of its opercula bones. This specimen establishes a new maximum age for ballan wrasse, 5 years older than the previously observed maximum age.

Soya saponins and prebiotics alter intestinal functions in Ballan wrasse (Labrus bergylta).

A 5-week feeding trial was conducted in the cleaner fish Ballan wrasse (Labrus bergylta) for a better understanding of the basic biology of the intestinal functions and health in this stomach less species. During the trial, Ballan wrasse was fed either a reference diet, the reference diet supplemented with (i) a commercial prebiotic (Aquate™ SG, 0·4 %) expected to have beneficial effects, (ii) soya saponins (0·7 %) expected to induce inflammation or (iii) a combination of the prebiotics and the soya saponins to find a remedy for gut inflammation. Blood, intestinal tissue and gut content from four consecutive intestinal segments (IN1 - IN4) were collected. No significant differences in fish growth were observed between the four dietary groups. Saponin supplementation, both alone and in combination with prebiotics, increased weight index of IN2 and IN3 and decreased blood plasma glucose, cholesterol and total protein. Dry matter of intestinal content and activity of digestive enzymes were not affected by diet. Histomorphological analyses revealed a progressing inflammation with increased infiltration by immune cells particularly into the distal parts of the intestine in fish fed diets with saponins, both alone and in combination with prebiotics. Gene expression profiles obtained by RNA sequencing and quantitative PCR mirrored the histological and biochemical changes induced by the saponin load. The study demonstrated that Ballan wrasse gut health and digestive function may be markedly affected by feed ingredients containing antinutrients.

Efficacy testing of an immersion vaccine against Aeromonas salmonicida and immunocompetence in ballan wrasse (Labrus bergylta, Ascanius).

The development of effective vaccines is a critical step towards the domestication of emerging fish species for aquaculture. However, traditional vaccine delivery through intraperitoneal (i.p.) injection requires fish to reach a minimum size and age and therefore cannot provide protection at early developmental stages when infection may occur. This study investigated the effectiveness of immersion vaccination with respect to immunocompetence in a cleaner fish species (ballan wrasse, Labrus bergylta, Ascanius) used in Atlantic salmon farming as an alternative means to control sea lice. The species is susceptible to atypical strains of Aeromonas salmonicida (aAs) at early life stages (<15 g), when i.p. vaccination is not applicable. While immersion vaccination is currently used in commercial hatcheries, the optimal fish size for vaccination, and efficacy of the vaccine delivered by this route has not yet been established. Importantly, efficacy depends on the capability of the species immune system to recognise antigens and process antigens to trigger and produce an adaptive immune response, (process known as immunocompetence). In this study, the efficacy of a polyvalent autogenous vaccine administered by immersion in juvenile ballan wrasse and the subsequent immune response induced was investigated after prime and booster vaccination regimes. In addition, temporal expression (0-150 days post hatch) of adaptive immune genes including major histocompatibility complex (MHC II CD74 molecule) and immunoglobulin M (IgM) was assessed using quantitative PCR (qPCR). Prime and/or boost vaccination by immersion of juvenile ballan wrasse (0.5 g and 1.5 g corresponding to 80 and 170 days post hatch (dph), respectively) did not provide significant protection against aAs vapA V after bath challenge under experimental conditions. Despite no evident protection >80 dph, MHC II and IgM transcripts were first reported at 35 and 75 dph, respectively, suggesting a window of immunocompetence. The results provide important new information on the onset of adaptive immunity in ballan wrasse and highlight that immersion vaccination in the species for protection against aAs should be performed at later developmental stages (>1.5 g) in the hatchery.

Development of diagnostic assays for differentiation of atypical Aeromonas salmonicida vapA type V and type VI in ballan wrasse (Labrus bergylta, Ascanius).

Aeromonas salmonicida (As) is a highly heterogeneous bacterial species, and strains' host specificity has been reported. Ballan wrasse (Labrus bergylta Ascanius, 1767) is susceptible to atypical As (aAs) vapA type V and type VI in Scotland and Norway. Identification of the bacterium is achieved by culture and molecular techniques; however, the available methods used to distinguish the As types are costly and time-consuming. This paper describes the development of a PCR and a restriction enzyme assay for the detection of aAs vapA type V and type VI in ballan wrasse, respectively. Type V-specific primers were designed on conserved regions of the vapA gene, and the restriction enzyme assay was performed on the PCR products of the hypervariable region of vapA gene for the detection of type VI isolates. Amplification product was produced for type V (254 bp) and restriction bands (368 and 254 bp) for type VI isolates only. In addition, the assays detected type V and type VI isolates in spiked water samples and type V in diagnostic tissue samples. The assays are fast, specific and cost-effective and can be used as specific diagnostic tools for cleaner fish, to detect infectious divergence strains, and to manage and mitigate aAs disease outbreaks through vaccine development.

Digestive and immune functions in the intestine of wild Ballan wrasse (Labrus bergylta).

This study was carried out to profile key characteristics of intestinal functions and health in wild-caught Ballan wrasse. To describe functional variation along the intestine, samples were collected from four intestinal segments, named from the proximal to the distal segment: IN1, IN2, IN3 and IN4. The sections showed quite similar structure, i.e. regarding mucosal fold height and branching, lamina propria and submucosal width and cellular composition and thickness of the muscle layers. Leucine aminopeptidase and maltase capacity decreased from IN1 to IN4, suggesting a predominant role of IN1 in digestion. Gene expression levels of vitamin C transporter (slc23a1) and fatty acid transporters (cd36 and fabp2) were higher in IN1 than in IN4, indicating a more important role of the proximal intestine regarding transport of vitamins and fatty acids. Higher expression of the gene coding for IgM heavy chain constant region (ighm) was found in IN4 than in IN1, suggesting an important immune function of the distal intestine. Other immune related genes il1b, il6, cd40, showed similar expression in the proximal and the distal part of the intestine. Parasite infection, especially the myxozoan parasite Enteromyxum leei, coincided with infiltration of lymphocytic and eosinophilic granular cells in the submucosa and lamina propria. The present study established reference information necessary for interpretation of results of studies of intestinal functions and health in cultured Ballan wrasse.

Skeletal deformities in wild and farmed cleaner fish species used in Atlantic salmon Salmo salar aquaculture.

As a first attempt to assess bone health in cleaner fish production, wild and cultured ballan wrasse Labrus bergylta and lumpfish Cyclopterus lumpus were examined by radiology. In C. lumpus, wild fish (57%) had more vertebra deformities (≥1 deformed vertebrae) than cultured fish (2-16%). One wild C. lumpus had lordosis and another was missing the tail fin. In L. bergylta, wild fish (11%) had fewer vertebra deformities than cultured individuals (78-91%). Among the cultured L. bergylta, 17-53% of the fish had severe vertebra deformities (≥6 deformed vertebrae) with two predominate sites of location, one between vertebra 4 and 10 (S1) in the trunk, and one between 19 and 26 (S2) in the tail. Fusions dominated S1, while compressions dominated S2. Although wild L. bergylta had a low vertebra deformity level, 83% had calluses and 14% had fractures in haemal/neural spines and/or ribs. The site-specific appearance and pathology of fracture and callus in wild L. bergylta suggests these are induced by chronic mechanical stress, and a possible pathogenesis for fish hyperostosis is presented based on this notion. In conclusion, good bone health was documented in cultured C. lumpus, but cultured L. bergylta suffered poor bone health. How this affects survival, growth, swimming abilities and welfare in cultured wrasse should be further investigated. SIGNIFICANCE STATEMENT: Skeletal deformities were studied in ballan wrasse and lumpfish of both wild and cultured origin for the first time to identify potential welfare issues when deploying them as cleaner fish in salmon sea cages. While cultured lumpfish showed good bone health, cultured wrasse had a high occurrence of vertebra deformities, which is expected to impact lice eating efficiency and animal welfare negatively. These deformities are most likely induced early in development.

Atypical Aeromonas salmonicida vapA type V and Vibrio spp. are predominant bacteria recovered from ballan wrasse Labrus bergylta in Scotland.

Healthy and/or moribund farmed and wild ballan wrasse Labrus bergylta (>0.5 to 900 g) were sampled from hatcheries (n = 2) and Atlantic salmon Salmo salar cage sites (n = 8) in Scotland between February 2016 and October 2018. Less than half of the sampled individuals (n = 43; 32.3%) had been vaccinated (autogenous polyvalent vaccine; dip and/or injection) against atypical furunculosis (type V and VI), while 20 (15.0%) fish were not vaccinated, and the rest (70 individuals, 52.7%) were of unknown vaccination status. Swab samples from skin lesions, gill, liver, spleen and kidney were inoculated onto a variety of bacteriological agar plates, and bacteriology identification and sequencing analysis was performed on significant bacterial colonies. Atypical Aeromonas salmonicida (aAs) vapA type V was the predominant bacterial species (70/215 bacterial isolates, 32.5% of bacterial samples; 43/117 positive individual fish, 36.8%) isolated in this survey followed by Vibrio species, which were the most geographically prevalent bacteria. Photobacterium indicum/profundum was also isolated from L. bergylta for the first time during this study. The collection of these bacterial isolates provides useful information for disease management. Identifying the aAs isolates involved in disease in ballan wrasse could provide vital information for improving/updating existing autogenous vaccines.

Antibiotics modulate biofilm formation in fish pathogenic isolates of atypical Aeromonas salmonicida.

Atypical Aeromonas salmonicida causes furunculosis infections of non-salmonid fish, which requires antibiotic therapy. However, antibiotics may induce biofilm in some bacteria, which protects them against hostile conditions while allowing them to persist on surfaces, thus forming a reservoir for infection. The aim of this study was to determine whether atypical isolates of A. salmonicida increased biofilm in the presence of two antibiotics, florfenicol and oxytetracycline. A microtitre plate assay was used to quantify biofilm in the presence and absence of each antibiotic. Fifteen of 28 isolates formed biofilms under control conditions, while 23 of 28 isolates increased biofilm formation in the presence of at least one concentration of at least one antibiotic. For oxytetracycline, the most effective concentration causing biofilm to increase was one-quarter of that preventing visible bacterial growth, whereas for florfenicol it was one-half of this value. This is the first study to demonstrate that a bacterial pathogen of fish increases biofilm in response to antibiotics. Biofilm formation may increase the risk of re-infection in culture systems and this lifestyle favours the transmission of genetic material, which has implications for the dissemination of antibiotic-resistance genes and demonstrates the need for enhanced disease prevention measures against atypical A. salmonicida.

Health assessment of the cleaner fish ballan wrasse Labrus bergylta from the British south-west coast.

Wild-caught ballan wrasse Labrus bergylta are translocated en masse from the British south-west coast to Scotland for use as cleaner fish to tackle Atlantic salmon Salmo salar sea lice infestations; however, very little is known about the background health status of this species. This is the first health assessment of wild ballan wrasse from the British south-west. Wild-caught ballan wrasse (n = 75) from coastal populations off Dorset and Cornwall were subjected to a full health screen for viral, bacterial and parasitic infections and associated pathology. A range of metazoan and protozoan parasites were observed in histological sections, including copepods (sea lice Caligus centrodonti), nematodes, cestodes, digenean metacercariae, Cryptocaryon-like ciliates and an intestinal coccidian (Eimeria sp.) observed in 26.6% of the samples. The mycoplasma Acholeplasma laidlawii was associated with cytopathic effect in cell culture inoculated with tissue homogenates. The opportunistic pathogen Photobacterium damselae damselae was isolated from a single fish with a systemic infection. The isolate was confirmed to possess the virulence factors hlyAch and plpV, previously associated with cell toxicity and pathogenicity to fish. There are no immediate concerns for the continued mass translation of ballan wrasse, however careful monitoring of the population is recommended.

Interannual fine-scale site fidelity of male ballan wrasse Labrus bergylta revealed by photo-identification and tagging.

The site fidelity of ballan wrasse Labrus bergylta was studied using photo-identification and external tagging. Five male individuals were observed to defend the same small territory composed of a few rocks during several reproductive seasons spanning 2 to 15 years. These results provide one of the strongest indications of long-term very fine-scale site fidelity in marine fishes.

Loss of stomach, loss of appetite? Sequencing of the ballan wrasse (Labrus bergylta) genome and intestinal transcriptomic profiling illuminate the evolution of loss of stomach function in fish.

BACKGROUND: The ballan wrasse (Labrus bergylta) belongs to a large teleost family containing more than 600 species showing several unique evolutionary traits such as lack of stomach and hermaphroditism. Agastric fish are found throughout the teleost phylogeny, in quite diverse and unrelated lineages, indicating stomach loss has occurred independently multiple times in the course of evolution. By assembling the ballan wrasse genome and transcriptome we aimed to determine the genetic basis for its digestive system function and appetite regulation. Among other, this knowledge will aid the formulation of aquaculture diets that meet the nutritional needs of agastric species. RESULTS: Long and short read sequencing technologies were combined to generate a ballan wrasse genome of 805 Mbp. Analysis of the genome and transcriptome assemblies confirmed the absence of genes that code for proteins involved in gastric function. The gene coding for the appetite stimulating protein ghrelin was also absent in wrasse. Gene synteny mapping identified several appetite-controlling genes and their paralogs previously undescribed in fish. Transcriptome profiling along the length of the intestine found a declining expression gradient from the anterior to the posterior, and a distinct expression profile in the hind gut. CONCLUSIONS: We showed gene loss has occurred for all known genes related to stomach function in the ballan wrasse, while the remaining functions of the digestive tract appear intact. The results also show appetite control in ballan wrasse has undergone substantial changes. The loss of ghrelin suggests that other genes, such as motilin, may play a ghrelin like role. The wrasse genome offers novel insight in to the evolutionary traits of this large family. As the stomach plays a major role in protein digestion, the lack of genes related to stomach digestion in wrasse suggests it requires formulated diets with higher levels of readily digestible protein than those for gastric species.

Isolation of salmonid alphavirus subtype 6 from wild-caught ballan wrasse, Labrus bergylta (Ascanius).

The use of cleaner fish as a biological control for sea lice in Atlantic salmon aquaculture has increased in recent years. Wild-caught wrasse are commonly used as cleaner fish in Europe. In Ireland, samples of wrasse from each fishing area are screened for potential pathogens prior to their deployment into sea cages. Salmonid alphavirus was isolated from a pooled sample of ballan wrasse, showing no signs of disease, caught from the NW of Ireland. Partial sequencing of the E2 and nsP3 genes showed that it was closely related to the previously reported SAV subtype 6. This represents only the second isolation of this subtype and the first from a wild fish species, namely ballan wrasse.

Sustainable production and use of cleaner fish for the biological control of sea lice: recent advances and current challenges.

Currently, cleaner fish are one of the most widely used sea lice control strategies in Atlantic salmon aquaculture. Two species are currently being farmed in North Atlantic countries, ballan wrasse (Labrus bergylta) and lumpfish (Cyclopterus lumpus), and the sector in most countries is rapidly expanding towards self-sufficiency. The species are very different both in terms of their biology and life histories and, consequently, production and husbandry methods must be tailored to each species. There are numerous health challenges currently experienced in both species, with bacterial and parasitic diseases being the most prevalent, and cohabitation with salmon may increase the risk of disease. Good husbandry and routine health monitoring are essential, although treatment is often required when disease outbreaks occur. Ballan wrasse and lumpfish are both proven to be effective salmon delousers, although delousing efficacy can be variable in farmed fish; the provision of suitable habitat and acclimation to net-pen conditions may encourage natural behaviours, including delousing, and the use of operational welfare indicators can highlight potential welfare issues. Cleaner fish research is progressing rapidly, although much of the basic knowledge regarding the species' biology remains unknown. The simultaneous domestication of two new marine aquaculture species is a significant challenge demanding sustained effort and funding over a prolonged period of time. Research must focus on enhancing the robustness of the farmed stocks and increasing hatchery outputs to meet the urgent demands from the salmon sector and protect wild stocks from overfishing.

T Cell Receptor Alpha Chain Genes in the Teleost Ballan Wrasse (Labrus bergylta) Are Subjected to Somatic Hypermutation.

Previously, somatic hypermutation (SHM) was considered to be exclusively associated with affinity maturation of antibodies, although it also occurred in T cells under certain conditions. More recently, it has been shown that SHM generates diversity in the variable domain of T cell receptor (TCR) in camel and shark. Here, we report somatic mutations in TCR alpha chain genes of the teleost fish, Ballan wrasse (Labrus bergylta), and show that this mechanism adds extra diversity to the polymorphic constant (C) region as well. The organization of the TCR alpha/delta locus in Ballan wrasse was obtained from a scaffold covering a single copy C alpha gene, 65 putative J alpha segments, a single copy C delta gene, 1 J delta segment, and 2 D delta segments. Analysis of 37 fish revealed 6 allotypes of the C alpha gene, each with 1-3 replacement substitutions. Somatic mutations were analyzed by molecular cloning of TCR alpha chain cDNA. Initially, 79 unique clones comprising four families of variable (V) alpha genes were characterized. Subsequently, a more restricted PCR was performed to focus on a specific V gene. Comparison of 48 clones indicated that the frequency of somatic mutations in the VJ region was 4.5/1,000 base pairs (bps), and most prevalent in complementary determining region 2 (CDR2). In total, 45 different J segments were identified among the 127 cDNA clones, counting for most of the CDR3 diversity. The number of mutations in the C alpha chain gene was 1.76 mutations/1,000 bps and A nucleotides were most frequently targeted, in contrast to the VJ region, where G nucleotides appeared to be mutational hotspots. The replacement/synonymous ratios in the VJ and C regions were 2.5 and 1.85, respectively. Only 7% of the mutations were found to be linked to the activation-induced cytidine deaminase hotspot motif (RGYW/WRCY).

Is the ballan wrasse (Labrus bergylta) two species? Genetic analysis reveals within-species divergence associated with plain and spotted morphotype frequencies.

The ballan wrasse (Labrus bergylta) is a marine fish belonging to the family Labridae characterized by 2 main morphotypes that occur in sympatry: spotty and plain. Previous studies have revealed differences in their life-history traits, such as growth and maturation; however, the genetic relationship between forms is presently unknown. Using 20 recently developed microsatellite markers, we conducted a genetic analysis of 41 and 48 spotty and plain ballan wrasse collected in Galicia (northwest Spain). The 2 morphotypes displayed highly significant genetic differences to each other (FST = 0.018, P < 0.0001). A similar degree of genetic differentiation (FST = 0.025, P < 0.0001) was shown using the STRUCTURE clustering approach with no priors at K = 2. In this case, the frequency of spotty and plain morphotypes was significantly different (χ(2) = 9.46, P = 0.002). It is concluded that there is significant genetic heterogeneity within this species, which appears to be highly associated with the spotty and plain forms, but not completely explained by them. Given the previously demonstrated biological differences between morphotypes, and the present genetic analyses, we speculate about the convenience of a taxonomic re-evaluation of this species.

Allometric growth and development of organs in ballan wrasse (Labrus bergylta Ascanius, 1767) larvae in relation to different live prey diets and growth rates.

Small fish larvae grow allometrically, but little is known about how this growth pattern may be affected by different growth rates and early diet quality. The present study investigates how different growth rates, caused by start-feeding with copepods or rotifers the first 30 days post-hatch (dph), affect allometric growth and development of nine major organs in ballan wrasse (Labrus bergylta) larvae up to experimental end at 60 dph. Feeding with cultivated copepod nauplii led to both increased larval somatic growth and faster development and growth of organ systems than feeding with rotifers. Of the organs studied, the digestive and respiratory organs increased the most in size between 4 and 8 dph, having a daily specific growth rate (SGR) between 30 and 40% in larvae fed copepods compared with 20% or less for rotifer-fed larvae. Muscle growth was prioritised from flexion stage and onwards, with a daily SGR close to 30% between 21 and 33 dph regardless of treatment. All larvae demonstrated a positive linear correlation between larval standard length (SL) and increase in total tissue volume, and no difference in allometric growth pattern was found between the larval treatments. A change from positive allometric to isometric growth was observed at a SL close to 6.0 mm, a sign associated with the start of metamorphosis. This was also where the larvae reached postflexion stage, and was accompanied by a change in growth pattern for most of the major organ systems. The first sign of a developing hepatopancreas was, however, first observed in the largest larva (17.4 mm SL, 55 dph), indicating that the metamorphosis in ballan wrasse is a gradual process lasting from 6.0 to at least 15-17 mm SL.