Acute and Sublethal Effects of Deltamethrin Discharges from the Aquaculture Industry on Northern Shrimp (Pandalus borealis Krøyer, 1838): Dispersal Modeling and Field Investigations.

Pharmaceutical deltamethrin (Alpha Max), used as delousing treatments in aquaculture, has raised concerns due to possible negative impacts on the marine environment. A novel approach combining different scientific disciplines has addressed this topic. Acute (mortality) and sublethal effects (i.e., fitness, neurological, immunological, and oxidative responses) of exposure of northern shrimp (Pandalus borealis) were studied in laboratory experiments. Passive water sampling combined with sediment analyses revealed environmental concentrations. Finally, dispersal modeling was performed to predict environmental concentrations. Ecotoxicological analyses showed mortality in shrimp after 1 h of exposure to 2 ng L-1 (1000-fold dilution of treatment dose), revealing a high sensitivity to deltamethrin. Sublethal effects included induction of acetylcholinesterase and acyl CoA oxidase activities and oxidative impairment, which may be linked to neurotoxic responses. Field concentrations of 10-200 ng L-1 in water (100 m from the pens) and

Peritrophin-like Genes Are Associated with Delousing Drug Response and Sensitivity in the Sea Louse Caligus rogercresseyi.

Caligus rogercresseyi is the main ectoparasite that affects the salmon industry in Chile. The mechanisms used by the parasite to support its life strategy are of great interest for developing control strategies. Due to the critical role of insect peritrophins in host-parasite interactions and response to pest control drugs, this study aimed to identify and characterize the peritrophin-like genes present in C. rogercresseyi. Moreover, the expression of peritrophin-like genes was evaluated on parasites exposed to delousing drugs such as pyrethroids and azamethiphos. Peritrophin genes were identified by homology analysis among the sea louse transcriptome database and arthropods peritrophin-protein database obtained from GenBank and UniProt. Moreover, the gene loci in the parasite genome were located. Furthermore, peritrophin gene expression levels were evaluated by RNA-Seq analysis in sea louse developmental stages and sea lice exposed to delousing drugs deltamethrin, cypermethrin, and azamethiphos. Seven putative peritrophin-like genes were identified in C. rogercresseyi with high homology with other crustacean peritrophins. Differences in the presence of signal peptides, the number of chitin-binding domains, and the position of conserved cysteines were found. In addition, seven peritrophin-like gene sequences were identified in the C. rogercresseyi genome. Gene expression analysis revealed a stage-dependent expression profile. Notably, differential regulation of peritrophin genes in resistant and susceptible populations to delousing drugs was found. These data are the first report and characterization of peritrophin genes in the sea louse C. rogercresseyi, representing valuable knowledge to understand sea louse biology. Moreover, this study provides evidence for a deeper understanding of the molecular basis of C. rogercresseyi response to delousing drugs.

Warm water treatment increased mortality risk in salmon.

Thermal treatment is a controversial method to control sea lice in the Atlantic salmon farming industry. This study aimed to complement the growing evidence base to document the impact of thermal treatments on salmon welfare, behaviour, physiology and health. Here, fish were treated two times (four weeks apart) for 30 s in either 27, 30, or 33 °C warm water, and parameters were compared to a procedural control (exposed to their holding temperature of 14 °C) or a negative control (where no treatments were applied). The fish had a clear behavioural response to the warm water, despite low difference between treatment and holding temperature (Δt = 13, 16 or 19 °C). Eye damages were more prevalent in the warm water treated groups than in the controls. Little difference was recorded between treatment groups in their growth and condition factor, blood plasma values, organ health, and long-term coping ability. There was, however, a significant increase in mortality as a function of temperature after the first treatment (14 °C: 6.5%, 27 °C: 5.3%, 30 °C: 12.4% and 33 °C: 18.9% mortality). The first treatment was performed only two weeks after the fish had been tagged and moved into the experimental holding tanks, while the fish had been allowed to recover for four weeks without any handling before the second treatment. The group of fish that were not subjected to any treatments (the negative control) had no mortality throughout the entire experimental period.

Transcriptome profiling of the early developmental stages in the giant mussel Choromytilus chorus exposed to delousing drugs.

The giant mussel Choromytilus chorus is a marine bivalve commonly collected in central - southern Chile from fishery zones shared with the salmon industry. These economically relevant areas are also affected by the use of pesticides for controlling sea lice infestations in salmon aquaculture. Their main target is the sea louse Caligus rogercresseyi. However, other than some physiological impacts, the molecular effects of delousing drugs in non-target species such as C. chorus remain largely understudied. This study aimed to explore the transcriptome modulation of Trochophore and D larvae stages of C. chorus after exposure to azamethiphos and deltamethrin drugs. Herein, RNA-seq analyses and mRNA-lncRNAs molecular interactions were obtained. The most significant changes were found between different larval development stages exposed to delousing drugs. Notably, significant transcriptional variations were correlated with the drug concentrations tested. The biological processes involved in the development, such as cell movement and transcriptional activity, were mainly affected. Long non-coding RNAs (lncRNAs) were also identified in this species, and the transcription activity showed similar patterns with coding mRNAs. Most of the significantly expressed lncRNAs were associated with genes annotated to matrix metalloproteinases, collagenases, and transcription factors. This study suggests that exposure to azamethiphos or deltamethrin drugs can modulate the transcriptome signatures related to the early development of the giant mussel C. chorus.

qPCR screening for Yersinia ruckeri clonal complex 1 against a background of putatively avirulent strains in Norwegian aquaculture.

Although a number of genetically diverse Yersinia ruckeri strains are present in Norwegian aquaculture environments, most if not all outbreaks of yersiniosis in Atlantic salmon in Norway are associated with a single specific genetic lineage of serotype O1, termed clonal complex 1. To investigate the presence and spread of virulent and putatively avirulent strains in Norwegian salmon farms, PCR assays specific for Y. ruckeri (species level) and Y. ruckeri clonal complex 1 were developed. Following extensive screening of water and biofilm, the widespread prevalence of putatively avirulent Y. ruckeri strains was confirmed in freshwater salmon hatcheries, while Y. ruckeri clonal complex 1 was found in fewer farms. The formalin-killed bacterin yersiniosis vaccine was detected in environmental samples by both PCR assays for several weeks post-vaccination. It is thus important to interpret results from recently vaccinated fish with great care. Moreover, field studies and laboratory trials confirmed that stressful management procedures may result in increased shedding of Y. ruckeri by sub-clinically infected fish. Analysis of sea water sampled throughout thermal delousing procedures proved effective for detection of Y. ruckeri in sub-clinically infected populations.

Comprehensive Transcriptome Analyses in Sea Louse Reveal Novel Delousing Drug Responses Through MicroRNA regulation.

The role of miRNAs in pharmacological responses through gene regulation related to drug metabolism and the detoxification system has recently been determined for terrestrial species. However, studies on marine ectoparasites have scarcely been conducted to investigate the molecular mechanisms of pesticide resistance. Herein, we explored the sea louse Caligus rogercresseyi miRNome responses exposed to delousing drugs and the interplaying with coding/non-coding RNAs. Drug sensitivity in sea lice was tested by in vitro bioassays for the pesticides azamethiphos, deltamethrin, and cypermethrin. Ectoparasites strains with contrasting susceptibility to these compounds were used. Small-RNA sequencing was conducted, identifying 2776 novel annotated miRNAs, where 163 mature miRNAs were differentially expressed in response to the drug testing. Notably, putative binding sites for miRNAs were found in the ADME genes associated with the drugs' absorption, distribution, metabolism, and excretion. Interactions between the miRNAs and long non-coding RNAs (lncRNAs) were also found, suggesting putative molecular gene regulation mechanisms. This study reports putative miRNAs correlated to the coding/non-coding RNAs modulation, revealing novel pharmacological mechanisms associated with drug resistance in sea lice species.

Establishment and Characterization of a Novel Gill Cell Line, LG-1, from Atlantic Lumpfish (Cyclopterus lumpus L.).

The use of lumpfish (Cyclopterus lumpus) as a cleaner fish to fight sea lice infestation in farmed Atlantic salmon has become increasingly common. Still, tools to increase our knowledge about lumpfish biology are lacking. Here, we successfully established and characterized the first Lumpfish Gill cell line (LG-1). LG-1 are adherent, homogenous and have a flat, stretched-out and almost transparent appearance. Transmission electron microscopy revealed cellular protrusions and desmosome-like structures that, together with their ability to generate a transcellular epithelial/endothelial resistance, suggest an epithelial or endothelial cell type. Furthermore, the cells exert Cytochrome P450 1A activity. LG-1 supported the propagation of several viruses that may lead to severe infectious diseases with high mortalities in fish farming, including viral hemorrhagic septicemia virus (VHSV) and infectious hematopoietic necrosis virus (IHNV). Altogether, our data indicate that the LG-1 cell line originates from an epithelial or endothelial cell type and will be a valuable in vitro research tool to study gill cell function as well as host-pathogen interactions in lumpfish.

Estimating cage-level mortality distributions following different delousing treatments of Atlantic salmon (salmo salar) in Norway.

This retrospective descriptive study estimates cage-level mortality distributions after six immediate delousing methods: thermal, mechanical, hydrogen peroxide, medicinal, freshwater and combination of medicinal treatments. We investigated mortality patterns associated with 4 644 delousing treatment of 1 837 cohorts of farmed Atlantic salmon (Salmo salar) stocked in sea along the Norwegian coast between 2014 and 2017. The mortality is expressed as mortality rates. We found distributions of delta mortality rate within 1, 7 and 14 days after all six delousing treatments, using mortality rate within 7 days before treatments as baseline. The results show that we can expect increased mortality rates after all six delousing methods. The median delta mortality rates after thermal and mechanical delousing are 5.4 and 6.3 times higher than medicinal treatment, respectively, for the 2017 year-class. There is a reduction in the delta median mortality for thermal and freshwater delousing from 2015 to 2019. There is a wide variability in the mortality rates, in particular for thermal delousing. Our results suggest that the variability in delta mortality for thermal delousing has been reduced from the 2014 to 2017 year-class, indicating an improvement of the technique. However, a significant increase in the number of thermal treatments from 14 in 2015 to 738 in 2018 probably contributes to the overall increased mortality in Norwegian salmon farming.

Salmon lice survive the straight shooter: A commercial scale sea cage trial of laser delousing.

Ectoparasitic salmon louse (Lepeophtheirus salmonis) infestations are costly for Atlantic salmon (Salmo salar) farmers in Norway. As a result, there is a strong desire for solutions to prevent and control infestations, and new technologies are typically developed and commercialised rapidly, without rigorous validation. Here, we tested the efficacy of a new commercially available control measure-delousing by underwater lasers-using a replicated design at full commercial scale. Laser delousing was used in combination with a preventive method (snorkel cages), with laser nodes deployed in 3 of the 6 sea cages at the site. The trial ran for 54 days, after which time there was no difference in infestation density of mobile salmon louse stages (pre-adult, adult male or adult female) in cages with or without laser nodes installed. By the end of the trial, adult female lice numbers in all cages were close to the legislated trigger for mandatory delousing (0.5 adult female lice per fish). The laser nodes delivered a large number of pulses relative to the number of lice in the cages, indicating that a lack of lethality rather than a lack of target detection was the limiting factor. If all pulses had been effective, they should have removed between 4-38 % of mobile lice each day. There was no effect on salmon welfare indicators such as skin condition or eye status. Our results highlight the importance of rigorous validation of new technologies across a range of conditions before widespread implementation by industry.

How are the salmon lice (Lepeophtheirus salmonis Krøyer, 1837) in Atlantic salmon farming affected by different control efforts: A case study of an intensive production area with coordinated production cycles and changing delousing practices in 2013-2018.

The aims of the present study were to describe the salmon lice (Lepeophtheirus salmonis Krøyer, 1837) situation in an intensive salmon production area in mid-Norway and to consider implications of changing practices of how salmon lice infestation can be controlled. The results in this study suggest that there are steps that can be carried out to keep salmon lice under control even during years when the temperature facilitates a quick salmon lice development. The present work indicates that the use of cleaner fish can delay the time it takes adult female lice to reach 0.1 per salmon in the beginning of a production cycle. It suggests that the timing of cleaner fish deployment into salmon cages can influence its effectiveness in controlling salmon lice. It also gives caution to letting salmon lice develop unchecked, even at levels far below the current lice limit, because of the difficulties to control salmon lice when the external infection pressure is too high. This study took place during a rapid change in delousing methods, in an area with coordinated salmon production. Despite its exploratory nature, this study offers insights into the salmon lice fluctuations in relation to efforts aimed at controlling it.

Sudden exposure to warm water causes instant behavioural responses indicative of nociception or pain in Atlantic salmon.

Thermal treatment has become the most used delousing method in salmonid aquaculture. However, concerns have been raised about it being painful for the fish. We studied the behavioural response of Atlantic salmon acclimated to 8 °C when transferred to temperatures in the range 0-38 °C. Exposure time was 5 min or until they reached the endpoint of losing equilibrium and laying on their side, a sign of imminent death. At temperatures below 28 °C, none of the fish reached endpoint within the 5-min maximum. At 28 °C four of five fish reached endpoint, and fish reached endpoint more rapidly as temperature increased further. Fish transferred to temperatures above 28 °C had higher swimming speed immediately after transfer and maintained a high swimming speed until just before loss of equilibrium. Their behaviour was from the start characterised by collisions into tank walls and head shaking. Just before loss of equilibrium they started breaking the surface of the water, swimming in a circle pattern and in some instances displayed a side-wise bending of their body. In other words, salmon transferred to temperatures above 28 °C showed instant behavioural responses indicative of nociception or pain.

Thermal injuries in Atlantic salmon in a pilot laboratory trial.

Thermal delousing is a new method for removing sea lice from farmed Atlantic salmon (Salmo salar L). We investigated thermally-related tissue injuries in Atlantic salmon in a pilot laboratory trial to describe the acute effect of high water temperatures (34-38 °C). Acute tissue injuries in gills, eyes, brain and possible also nasal cavity and thymus were seen in salmon exposed to water temperatures of 34 - 38 °C in 72 to 140 s. This implies that exposing salmon to such water temperatures is a welfare risk, not only due to the direct tissue injuries that may also be dependent on exposure time, but also due to risk of thermal pain and aversion, including flight reactions.

It works! Lumpfish can significantly lower sea lice infestation in large-scale salmon farming.

To assess the efficacy of lumpfish grazing on attached sea lice on Atlantic salmon, six large-scale sea cages, (130 m circumference, 37,688 m3 volume) each stocked with approximately 200,000 salmon 0+ smolts, were stocked with a 4, 6 and 8% density (8000, 12,000 and 16,000, respectively) of lumpfish. The sea cages without lumpfish acted as controls. Sea lice infestation levels on the salmon were monitored weekly and bi-weekly from 6 October to 17 May the subsequent year. Mortality of the lumpfish rose with decreasing sea temperatures to around 0.8% week-1 and did not vary between the lumpfish groups. There were clear signs of lumpfish grazing on sea lice, with significantly lower average levels of chalimus, pre-adult and adult female Lepeophtheirus salmonis and Caligus elongatus sea lice per salmon. Lumpfish in the high density (8%) group reduced the mature female L. salmonis to levels equal to or lower than the counts recorded prior to the start of the study. Overall, the present results indicate that lumpfish are a suitable cold-water option for biological delousing of Atlantic salmon in large-scale production conditions.

Changing Demographics and Prevalence of Body Lice among Homeless Persons, Marseille, France.

The prevalence of body lice among 2,288 sheltered homeless persons in the city of Marseille during 2000-2017 was 12.2% and significantly decreased over time. We report a positive association between body lice infestations and older age, duration of stays in France for migrants, frequent consumption of alcohol, and tobacco smoking.

Time-to-response toxicity analysis as a method for drug susceptibility assessment in salmon lice.

The salmon louse Lepeophtheirus salmonis (Krøyer, 1837) is an ectoparasite causing infections of wild and farmed Atlantic salmon (Salmo salar L.) in the Northern hemisphere. While L. salmonis control at commercial mariculture sites increasingly employs non-medicinal approaches, such as cage designs reducing infection rates and biological control through cleaner fish, anti-parasitic drugs are still a requirement for effective fish health care. With only a limited range of salmon delousing agents available, all of which have been in use for more than a decade, drug resistance formation has been reported for different products. Successful resistance management requires reliable susceptibility assessment, which is usually achieved through L. salmonis bioassays. These tests involve the exposure of parasites to different drug concentrations and require significant numbers of suitable L. salmonis stages. The present study reports an alternative bioassay that is based on time-to-response toxicity analyses and can be carried out with limited parasite numbers. The assay determines the median effective time (ET50), i.e., the time required until impaired swimming and/or attachment behaviour becomes apparent in 50% of parasites, by conducting repeated examinations of test animals starting at the time point where exposure to a set drug concentration commences. This experimental approach further allows the estimation of the apparent drug susceptibility of individual L. salmonis by determining their time to response, which may prove useful in experiments designed to elucidate associations between genetic factors and the drug susceptibility phenotype of parasites. Three laboratory strains of L. salmonis differing in susceptibility to emamectin benzoate were characterised using standard 24 h bioassays and time-to-response toxicity assays. While both the median effective concentration (EC50) and the ET50 showed variability between experimental repeats, both types of bioassay consistently discriminated susceptible and drug-resistant L. salmonis laboratory strains. STATEMENT OF RELEVANCE: Infections by sea lice cause significant costs to the global salmon farming industry, which have been estimated to exceed €300 million per year worldwide. Control of sea lice still relies to a significant extent on chemical delousing; however, chemical control is threatened by resistance formation. Resistance can be combated by rotation between different drugs and strategic implementation of non-medicinal strategies. However, resistance management requires reliable and feasible methods of susceptibility assessment. The present study is a technical note introducing a novel approach to susceptibility assessments in sea lice. The method can be applied in susceptibility assessments on farms, where it offers the advantage of a reduced requirement of parasites for testing. In addition, the novel method allows deriving the times of parasite require to show a response after drug treatment has started, thus providing a variable characterizing the drug susceptibility phenotype of individual parasites. Accordingly, the bioassay approach presented here will be useful for studies aiming at unravelling the genetic determinants of drug resistance.

Delousing efficiency of farmed ballan wrasse (Labrus bergylta) against Lepeophtheirus salmonis infecting Atlantic salmon (Salmo salar) post-smolts.

BACKGROUND: Cleaner-fish (wrasse, Labridae) are increasingly deployed within the Atlantic salmon (Salmo salar L.) industry as a biological control against sea-lice (Lepeophtheirus salmonis Krøyer). Two tank-based trials were performed to test the effect of farmed ballan wrasse (Labrus bergylta Ascanius) body mass and supplementary feeding on the delousing of Atlantic salmon post-smolts with an initial infection level of ∼12 lice salmon(-1) and a ∼5% wrasse:salmon ratio. RESULTS: Sea-louse levels below 0.5 lice salmon(-1) were obtained within 84 h, and preferential preying upon larger motile stages was found. The wrasse body mass and the availability of fresh, opened blue mussels (Mytilus edulis L.) did not significantly affect delousing efficiency. The functional predator response was linear, showing no minimum prey density threshold for sea-louse foraging and no satiation plateau, in spite of the high consumption rates measured. Sea-louse infection levels declined following a one-phase exponential decay model, with a standardised decline time constant of 0.8-1.3% h(-1) for each wrasse stocked per 100 salmon. CONCLUSIONS: Farmed ballan wrasse are confirmed as highly effective therapeutic and preventive biological controls against sea-lice. The study supports the current minimum hatchery size target (10 mm total length) and the use of supplementary feeding to sustain the wrasse stocks in operation. The functional predator response and the standardised decline time constant of sea-louse abundance are proposed as useful indicators of delousing efficiency.