Salmon Embryo Research Articles

The application of plasmid DNA constructs encoding antiviral proteins as potential therapeutics against viral infections in salmonid aquaculture

Treatments to control infectious diseases in aquacultured fish commonly focus on the use of antibiotics (which are ineffective against viruses) and inorganic disinfectants. Currently, antiviral treatments are not available for use in aquaculture. The use of plasmid vectors to overexpress antiviral proteins in fish has been reported, with positive results against viral infections. The aim of this work was to overexpress the antiviral proteins interferon-induced protein with tetratricopeptide repeats 5 (IFIT5) and natural killer enhancing factor (NKEF) using plasmid vectors (pIFIT5 and pNKEF) as novel therapeutic agents against viral infections. The antiviral activity of pIFIT5 against viral hemorrhagic septicemia virus (VHSV) and infectious pancreatic necrosis virus (IPNV) was evaluated in vitro in epithelioma papulosum cyprinid and Chinook salmon embryo cell lines. Moreover, pNKEF antiviral activity against IPNV was also evaluated. Results showed that pIFIT5 had antiviral activity against both viruses, but pNKEF did not protect against IPNV. In addition, we evaluated the use of pIFIT5 and pNKEF constructs as therapeutics in rainbow trout. Biodistribution assays demonstrated systemic distribution of the IFIT5-TFP1 and NKEF-TFP1 proteins in rainbow trout injected with the plasmids, and pIFIT5 and pNKEF triggered an immune response in the injected fish. In a pilot study, we evaluated the level of protection conferred by pIFIT5 and pNKEF against VHSV in rainbow trout, and we observed 10 % percent survival for pIFIT5 compared to pTFP. Together, these results suggest the potential of pIFIT5 as therapeutic agent in aquaculture.

Identification of a novel circovirus associated with turbot (Scophthalmus maximus) acute hemorrhage disease

Turbot (Scophthalmus maximus) is an economically important farming fish in China. However, an emerging disease named turbot acute hemorrhage disease (TAHD) has been affecting turbot farms since November 2019. TAHD is characterized by severe bleeding in the fish fins and may lead to significant mortality in one or two weeks, with accumulated mortality exceeding 90 %. The TAHD spread rapidly across the major turbot farming regions in China and resulted in significant economic losses. Virome sequencing was utilized in the diseased fish to discover a novel turbot circovirus (TCV). The TCV particle is ∼30 nm in size and is located in the fish spleen and kidney. The TCV genome is 1774 bp long, with three open reading frames (ORFs) encoding the replication protein (Rep), capsid protein (Cap), and ORF3 of unknown function. The identity of the TCV Rep sequence was <53 % compared to the reported circovirus sequences. Phylogenetic analysis of the Rep and Cap protein sequences revealed that TCV is a novel circovirus. The polymerase chain reaction detection method was used to analyze the clinical TAHD samples from 2019, which were all TCV-positive. Various cell lines, including turbot kidney, epithelioma papilloma cyprinid, Chinook salmon embryo, and spotted halibut kidney, were used for TCV isolation. However, no cytopathic effect or replication was observed. The diseased fish tissue homogenate filtrate was used for experimental infection. As a result, all of the infected turbot showed signs of natural infection, with increasing numbers of cap gene copies post-infection. Based on these results, it was hypothesized that the novel circovirus TCV is closely associated with TAHD. Our research also indicated that the circovirus represents a significant threat to fish and more attention should be paid to it in aquaculture.

Dose rate dependent genotoxic and metabolic effects predict onset of impaired development and mortality in Atlantic salmon (S. salar) embryos exposed to chronic gamma radiation

Release of radionuclides to the environment from either nuclear weapon and fuel cycles or from naturally occurring radionuclides (NORM) may cause long term contamination of aquatic ecosystems and chronic exposure of living organisms to ionizing radiation, which in turn could lead to adverse effects compromising the sustainability of populations. To address the effects of chronic ionizing radiation on the development of fish, Atlantic salmon embryos were exposed from fertilization until hatching (88 days, 550 day-degree) to dose rates from 1 to 30 mGy·h−1 gamma radiation (60Co). The lowest adopted dose rate was similar to the highest doses measured in some water bodies right after the Chernobyl accident (1 mGy·h−1), however, well above current environmentally realistic scenarios (20 μGy·h−1), or the threshold assumed for significant effects on fish population (40 μGy·h−1). Dose dependent effects were observed on survival, hatching, morbidity, DNA damage, antioxidant defenses, and metabolic status. Histopathological analysis showed dose rate dependent impairment of eye and brain tissues development and establishment of epidermal mucus cell layers accompanied by increased DNA damage at doses ≥1.3 Gy (dose rates ≥1 mGy·h−1).At ≥32.8 Gy (dose rates ≥20 mGy·h−1) deformities and developmental growth defects resulted in respective 46 and 95 % pre-hatch mortality. The 10 mGy·h−1 exposure (≥ 12 Gy total dose) caused significantly increased DNA damage, impaired eye development, and both premature and delayed hatching, while no deformities or effect on survival were observed. We observed a dose rate dependent reduction from dose rate ≥ 20 mGy·h−1 (≥ 27 Gy total dose) on antioxidant SOD, catalase and glutathione reductase enzyme activities. The reduction of antioxidant enzyme activities was in line with observed developmental delay and disturbance to time of hatching. Metabolomic profiles showed a clear shift at dose rates ≥10 mGy·h−1 (≥ 12 Gy total dose) in pathways related to oxidative stress, detoxification, DNA damage and repair. Due to gamma radiation exposure, a switch of central metabolism from glycolysis, citric acid cycle and lactate production towards pentose phosphate pathway indicated a rewiring mechanism for increased production of reductive equivalents to maintain redox homeostasis at the expense of energy output and thus embryonic development.

Leukolectins support lifelong innate immunity in lower vertebrates, and reveal dichotomies of several leukocytic lineages

Innate immunity is vital for animal homeostasis and survival. First-line immuno-defense for fish larvae involves mucus enriched with leukolectin (LL) secreted by dermal lectocytes. Later during the critical transition from yolk-nutrition to feeding, additional larval immuno-protection in zebrafish (zF) is provided by macrophages containing LL (lectophages). This work investigated new LL-expression in embryos and in blood, structures of fish leukocytic LL and LL-genes, and LL-presence in chicken leukocytes. In zF-embryos, lectophages appear ∼10 hpf, while later, cells co-expressing myeloperoxidase- and LL-mRNA were detected (∼19 hpf). Furthermore, protein-extracts of Atlantic salmon (Ssal) leukocytes contained LL-proteins, compartmentalized in the cytosol. Cloning and sequencing revealed 94 % nt-sequence identity between variants of Ssal-leukolectins. Highly conserved LLs allowed production of epitope-specific anti-LL IgGs. Immuno-fluorescence-analysis demonstrated that most Ssal-bloodcells were LL-negative, but both some large cells with protrusions and some small, rounded cells did express LL. Immunoperoxidase-staining method confirmed LL-expression in some Ssal-leukocytes, identified as macrophages, PMN-leukocytes, thrombocytes and dendritic cells. However, closer examination revealed a dichotomy of these cell-categories into either LL-positive, or LL-negative variants. In situ hybridization demonstrated profuse LL-expression in Ssal head kidney interstitial tissue, while LL-transcripts were absent in large kidney tubules. Both hematopoietic (non-pigmented) marrow cells and melano-macrophages expressed LL-mRNA, implying that leukolectins provide lifelong innate immuno-protection. PCR-amplification using Ssal-leukocytic DNA as template, and direct sequencing yielded a leukocytic ll-gene. Some cells in salmon, cod, halibut, oikopleura and zebrafish embryos express LL-proteins and/or LL-mRNA, and LL-mRNA is detected in salmon, cod and chicken leukocytes. However, current genomes for these species lack recognizable LL-loci except the Ssal_v3.1 Genome-assembly. The data demonstrate an unexpected dichotomy of some leukocyte lineages into LL-positive or LL-negative cell-variants. Such dichotomies suggest exploring differential impacts from the duplicated leukocyte-lineages in health and disease.

Preparation and extraction of chorion proteins from Salmo salar embryos at the pigmented eye stage for electrophoresis with SDS-polyacrylamide gel

The chorion fulfills important functions in fish embryos, including protecting the embryo during development. The characterization of the protein profile of this envelope could be used as a bioindicator in the evaluation of the quality of embryonic development. The object of this work was to validate a standardized protocol for protein extraction from chorion of Salmo salar embryos at 280 accumulated thermal units (ATU) by comparing and combining existing methods. The protocol consists of consecutive washing of the chorion samples followed by protein extraction with the solution that was named SDS solution (Tris–HCl 100 mM (pH 8), Urea 8 M, 1% SDS, β-mercaptoethanol 300 mM and EGTA 10 Mm, and 1% protease inhibitor cocktail) and mechanical methods. Protein extraction is enhanced by a working temperature of 75 °C and use of a disperser. The protein concentration was quantified by Bradford Assay. After extraction, the samples were diluted (dilution factor 10) before reading against the calibration curve. After gel electrophoresis with a load of 3 µg of protein, staining showed more than 4 bands, with molecular weights between 25 kDa and 180 kDa.•The protein profile of fish chorion was between 25 kDa and 180 kDa.•Solution containing 1% SDS allows a higher extraction of proteins from the chorion of Atlantic salmon embryos with 280 ATU.•Chorion protein identification is a valuable tool in determining gamete and embryo quality in fish.

Highly efficient in vivo C-to-T base editing in Atlantic salmon (Salmo salar) – A step towards aquaculture precision breeding

The salmon industry has the potential to provide nutritious food to the expanding world population. However, the industry faces several sustainability challenges related to environmental impact and fish welfare. Genome editing presents a promising approach for precision breeding, where small, targeted changes to the fish genome may facilitate the introduction of advantageous traits like disease resistance, delayed maturation, or enhanced growth. This study presents the first application of base editing in an aquaculture fish species. By co-injecting cytidine base editor AncBE4max mRNA and gRNA targeting the pigmentation gene solute carrier family 45 member 2 in Atlantic salmon embryos, we were able to induce a specific C-to-T conversion, thereby introducing a premature stop codon and achieving an albino phenotype. High-throughput sequencing revealed an average of 50–66% correct conversion of the target base depending on the base editor mRNA concentration and up to 89% in a single individual. Our results suggest that AncBE4max provides a simple and efficient approach to making precise single nucleotide edits in the Atlantic salmon genome. Additionally, we used conventional CRISPR/Cas9 combined with oligonucleotide templates to insert point mutations at various positions up- and downstream of the Cas9 cleavage site, demonstrating that the insertion efficiency was affected by the position of the mutation. The application of genome editing tools that allow for precise changes in this species is a step towards aquaculture precision breeding and the introduction of traits promoting a more sustainable industry.

Effects of winter water quality conditions on Atlantic Salmon embryo mortality and deformity rates in the Serpentine River (New Brunswick, Canada)

In-stream incubation of Atlantic Salmon (Salmo salar) embryos within artificial redds using Jordan/Scotty fish egg incubator boxes is an alternative to traditional hatchery-based incubation methods. Winter water quality conditions, such as temperature and dissolved oxygen vary within natural redds and influence wild embryo development and survival. The differences in landscape characteristics and in-stream habitat features at catchment-, reach-, and site spatial scales were investigated to determine how they influenced water quality conditions within the Serpentine River, New Brunswick. A total of 14,400 hatchery-fertilized salmon embryos were incubated within river substrates from November 2020 to January 2021. The proportion of embryo mortalities within our experiment was high (44.9%), but still within the range observed in the literature from other incubation experiments, and the proportion of embryo deformities between our sites was very low (2.7%). Despite variations in average water temperature, cumulative degree days, and coefficient of variation in dissolved oxygen (DO) concentration, none were statistically significant for explaining embryo mortalities or deformities. Patterns were, however, evident across sites showing trends towards a decrease in mortality with cumulative degree days, and an increase in deformities with greater variation in DO. Improving our knowledge on Atlantic Salmon incubation habitat supports future research and restoration opportunities for adaptation under a changing climate.

DNA extraction from recently fertilised Atlantic salmon embryos for use in microsatellite validation of triploidy.

The current methods used for producing triploid Atlantic salmon are generally reliable but not infallible, and each batch of triploids must be validated to ensure consumer trust and licensing compliance. Microsatellites have recently been shown to offer a cheaper and more convenient alternative to traditional flow cytometry for triploidy validation in a commercial setting. However, incubating eggs to at least the eyed stage for microsatellite validation poses challenges, such as reduced quality and performance of triploids produced from later eggs in the stripping season. To address these issues, we propose another option: extracting DNA from recently fertilised eggs for use in conjunction with microsatellite validation. To achieve this, we have developed an optimized protocol for HotSHOT extraction that can rapidly and cheaply extract DNA from Atlantic salmon eggs, which can then be used for triploidy validation through microsatellites. Our approach offers a simpler and more cost-effective way to validate triploidy, without the need for skilled dissection or expensive kits.

In vivo CRISPR/LbCas12a-mediated knock-in and knock-out in Atlantic salmon (Salmo salar L.)

Genome editing using the CRISPR/Cas system offers the potential to enhance current breeding programs and introduce desirable genetic traits, including disease resistance, in salmon aquaculture. Several nucleases are available using this system, displaying differences regarding structure, cleavage, and PAM requirement. Cas9 is well established in Atlantic salmon, but Cas12a has yet to be tested in vivo in this species. In the present work, we microinjected salmon embryos with LbCas12a ribonucleoprotein complexes targeting the pigmentation gene solute carrier family 45 member 2 (slc45a2). Using CRISPR/LbCas12a, we were able to knock-out slc45a2 and knock-in a FLAG sequence element by providing single-stranded DNA templates. High-throughput sequencing revealed perfect HDR rates up to 34.3% and 54.9% in individual larvae using either target or non-target strand template design, respectively. In this work, we demonstrate the in vivo application of CRISPR/LbCas12a in Atlantic salmon, expanding the toolbox for editing the genome of this important aquaculture species.

Bioretention filtration prevents acute mortality and reduces chronic toxicity for early life stage coho salmon (Oncorhynchus kisutch) episodically exposed to urban stormwater runoff

As the human population of western North America continues to expand, widespread patterns of urban growth pose increasingly existential threats to certain wild stocks of Pacific salmon and steelhead (Oncorhynchus sp.). Rainfall previously absorbed into the soils of forests and grasslands falls instead on pavement and other hardened surfaces. This creates stormwater runoff that carries toxic metals, oil, and many other contaminants into salmon-bearing habitats. These include freshwater streams where coho salmon (O. kisutch) spawn in gravel beds. Coho salmon embryos develop within a thick eggshell (chorion) for weeks to months before hatching as alevins and ultimately emerging from the gravel as fry. Untreated urban runoff is highly toxic to older coho salmon (freshwater-resident juveniles and adult spawners), but the vulnerability of the earliest life stages remains poorly understood. To address this uncertainty, we fertilized eggs and raised them under an episodic stormwater exposure regimen, using runoff collected from a high-traffic arterial roadway from 15 discrete storm events. We monitored survival and morphological development, as well as molecular markers for contaminant exposure and cardiovascular stress. We also evaluated the benefit of treating runoff with green infrastructure (bioretention filtration) on coho salmon health and survival. Untreated runoff caused subtle sublethal toxicity in pre-hatch embryos with no mortality, followed by high rates of mortality from exposure at hatch. Bioretention filtration removed most measured contaminants (bacteria, dissolved metals, and polycyclic aromatic hydrocarbons), and the treated effluent was considerably less toxic – notably preventing mortality at the alevin stage. Our findings indicate that untreated urban runoff poses an important threat to early life stage coho salmon, in terms of both acute and delayed-in-time mortality. Moreover, while inexpensive management strategies involving bioinfiltration are promising, future green infrastructure effectiveness research should emphasize sublethal metrics for contaminant exposure and adverse health outcomes in salmonids.

Tire-Derived Transformation Product 6PPD-Quinone Induces Mortality and Transcriptionally Disrupts Vascular Permeability Pathways in Developing Coho Salmon.

Urban stormwater runoff frequently contains the car tire transformation product 6PPD-quinone, which is highly toxic to juvenile and adult coho salmon (Onchorychus kisutch). However, it is currently unclear if embryonic stages are impacted. We addressed this by exposing developing coho salmon embryos starting at the eyed stage to three concentrations of 6PPD-quinone twice weekly until hatch. Impacts on survival and growth were assessed. Further, whole-transcriptome sequencing was performed on recently hatched alevin to address the potential mechanism of 6PPD-quinone-induced toxicity. Acute mortality was not elicited in developing coho salmon embryos at environmentally measured concentrations lethal to juveniles and adults, however, growth was inhibited. Immediately after hatching, coho salmon were sensitive to 6PPD-quinone mortality, implicating a large window of juvenile vulnerability prior to smoltification. Molecularly, 6PPD-quinone induced dose-dependent effects that implicated broad dysregulation of genomic pathways governing cell-cell contacts and endothelial permeability. These pathways are consistent with previous observations of macromolecule accumulation in the brains of coho salmon exposed to 6PPD-quinone, implicating blood-brain barrier disruption as a potential pathway for toxicity. Overall, our data suggests that developing coho salmon exposed to 6PPD-quinone are at risk for adverse health events upon hatching while indicating potential mechanism(s) of action for this highly toxic chemical.

Assessing the occurrence of egg stranding for trout and salmon in a regulated river

AbstractA key challenge in many regulated rivers is to define adequate flow levels to protect aquatic organisms. Provisioning of suitable flow can be pivotal bottlenecks for fishes such as salmon and trout that use the riverbed as an incubation habitat. Additionally, the locations where females spawn will define the probability that embryos will be dewatered during the incubation period in a regulated flow regime. We investigated the water flow, dewatering, and incubation mortality in Atlantic salmon and brown trout in natural nests over 19 years in the regulated Bjoreio River in western Norway. During the study period, different flow strategies were applied to mitigate the dewatering of incubating salmon and trout embryos. Average survival in nests sampled in late winter ranged from 54% to 92% among years and was significantly correlated with the minimum water flow occurring during the incubation period. Mortality was significantly higher in nests in shallow areas, reflecting nests exposed to dewatering. The results demonstrate a strong link between incubation mortality and managed flow regimes for river spawning salmonids. Using detailed information on the nest location and incubation mortality, we estimate minimum flow requirements for this river and demonstrate an approach to effectively mitigate the impact of river regulations on embryo survival in Atlantic salmon and brown trout.

Early developmental toxicity of Atlantic salmon exposed to conventional and unconventional oils

Atlantic salmon is an important species for Canadian culture and economy and its importance extends beyond Canada to Scandinavia and Western Europe. However, it is a vulnerable species facing decline due to habitat contamination and destruction. Existing and new Canadian pipeline projects pose a threat to salmonid habitat. The effects of diluted bitumen (dilbit), the main oil circulating in pipelines, are less studied than those of conventional oils, especially during the critical early embryonic developmental stage occurring in freshwater ecosystems. Therefore, this study aimed to compare the effects of water-accommodated fractions (WAF) of the Clearwater McMurray dilbit and the Lloydminster Heavy conventional oil on Atlantic salmon embryos exposed either from fertilization or from eyed stage. The dilbit contained the highest concentrations of low molecular weight (LMW) compounds (including BTEX and C6-C10), while the conventional oil contained the highest concentrations of PAHs. The Clearwater dilbit caused a higher percentage of mortality and malformations than the conventional oil at similar WAF concentrations. In addition, the embryos exposed from fertilization suffered a higher mortality rate, more developmental delays, and malformations than embryos exposed from the eyed stage, suggesting that early development is the most sensitive developmental stage to oil exposure. Gene expression and enzymatic activity of the detoxification phase I and II enzymes (CYP1A and GST) were measured. Data showed increases in both cyp1a expression and GST activity with increasing WAF concentrations, while gst expression was not affected by the exposures. Also, gene expression of proteins involved in the biotransformation of vitamin A and DNA damage repair were modified by the oil exposures. Overall, this study indicates that Atlantic salmon is mostly affected by oil exposure at the beginning of its development, during which embryos accumulate deformities that may impact their survival at later life stages.

Collective behavior and virulence arsenal of the fish pathogen Piscirickettsia salmonis in the biofilm realm.

Piscirickettsiosis is a fish disease caused by the Gram-negative bacterium Piscirickettsia salmonis. This disease has a high socio-economic impact on the Chilean salmonid aquaculture industry. The bacterium has a cryptic character in the environment and their main reservoirs are yet unknown. Bacterial biofilms represent a ubiquitous mechanism of cell persistence in diverse natural environments and a risk factor for the pathogenesis of several infectious diseases, but their microbiological significance for waterborne veterinary diseases, including piscirickettsiosis, have seldom been evaluated. This study analyzed the in vitro biofilm behavior of P. salmonis LF-89T (genogroup LF-89) and CA5 (genogroup EM-90) using a multi-method approach and elucidated the potential arsenal of virulence of the P. salmonis LF-89T type strain in its biofilm state. P. salmonis exhibited a quick kinetics of biofilm formation that followed a multi-step and highly strain-dependent process. There were no major differences in enzymatic profiles or significant differences in cytotoxicity (as tested on the Chinook salmon embryo cell line) between biofilm-derived bacteria and planktonic equivalents. The potential arsenal of virulence of P. salmonis LF-89T in biofilms, as determined by whole-transcriptome sequencing and differential gene expression analysis, consisted of genes involved in cell adhesion, polysaccharide biosynthesis, transcriptional regulation, and gene mobility, among others. Importantly, the global gene expression profiles of P. salmonis LF-89T were not enriched with virulence-related genes upregulated in biofilm development stages at 24 and 48 h. An enrichment in virulence-related genes exclusively expressed in biofilms was also undetected. These results indicate that early and mature biofilm development stages of P. salmonis LF-89T were transcriptionally no more virulent than their planktonic counterparts, which was supported by cytotoxic trials, which, in turn, revealed that both modes of growth induced important and very similar levels of cytotoxicity on the salmon cell line. Our results suggest that the aforementioned biofilm development stages do not represent hot spots of virulence compared with planktonic counterparts. This study provides the first transcriptomic catalogue to select specific genes that could be useful to prevent or control the (in vitro and/or in vivo) adherence and/or biofilm formation by P. salmonis and gain further insights into piscirickettsiosis pathogenesis.

Choriolytic and non-choriolytic proteases during hatching of Atlantic Salmon embryos.

Fish embryonic hatching glands (HGs) secrete choriolysin-zymogens, which when activated degrade the chorion, allowing larval exit. Thus, hatching encompasses two dissimilar choriolysin-processes: pre-choriolysis including activated choriolysins accessing the perivitelline space (PVS), followed by choriolysis. Discovery of serine-proteolytic zonase in Atlantic salmon hatching fluid (HF) raises questions concerning its participation in hatching. This work aims to identify salmon choriolysins, and evaluate their role and that of zonase during hatching. Precocious salmon hatching occurs under alkaline conditions, producing an HF containing similar metallo- and serine- proteolytic activities. Purified zonase is selectively inhibited by peFabloc, whose MW (580Da) allows diffusion through the chorion into the PVS. Without apparent toxicity, brief peFabloc-pretreatment (2mM) of salmon eggs reduced precocious hatching substantially, compatible with a zonase-relevance for hatching. Atlantic salmon differed from other fishes since their HGs were not immuno-stained by polyclonal antibodies against pike choriolysins. However, cloning and sequencing experiments revealed a single low choriolytic enzyme (LCE) of 69% identity to pike LCE. Similar experiments with high choriolytic enzymes (HCEs) revealed two types (HCE-1 and HCE-2) with respectively 71% and 91% identity to pike HCE-1 & HCE-2. In situ hybridization revealed typical HGs. However, zebrafish-choriolysis is achieved by HCE-class choriolysins alone. Another zebrafish choriolysin (HE2) was not expressed. Peptide-bond hydrolysis by non-choriolytic zonase mimicks HCE-action generating hydrophilic sites for LCE-choriolytic catalysis. Ultimately, precise definitions of choriolytic and pre-choriolytic catalysis requires developmental genetics. Our data are compatible with a complex pre-choriolytic hatching-stage in Atlantic salmon, before LCE-choriolysis degrades the chorion.

A rapid classification tool for deformities in Atlantic salmon (Salmo salar) embryos.

Deformities in fish embryos are important for their survival in later life stages. However, a consistent way to refer to and classify salmonid embryo deformities does not exist. Expanding on reports of alevin deformities, we developed a classification tool for distinguishing the deformities observed in a collection of preserved Atlantic salmon (Salmo salar) embryos. Deformities were classified based on the deformed body part and a qualitative subtype. This classification tool uses external morphology, requires minimal equipment and can be applied from the first appearance of optic vesicles to hatch.

Effects of In-Channel Structure on Chinook Salmon Spawning Habitat and Embryo Production

Adult salmonids are frequently observed building redds adjacent to in-channel structure, including boulders and large woody debris. These areas are thought to be preferentially selected for a variety of reasons, including energy and/or predation refugia for spawners, and increased hyporheic exchange for incubating embryos. This research sought to quantify in-channel structure effects on local hydraulics and hyporheic flow and provide a mechanistic link between these changes and the survival, development, and growth of Chinook salmon Oncorhynchus tshawytscha embryos. Data were collected in an eight-kilometer reach, on the regulated lower Mokelumne River, in the California Central Valley. Nine paired sites, consisting of an area containing in-channel structure paired with an adjacent area lacking in-channel structure, were evaluated. Results indicated that in-channel structure disrupts surface water velocity patterns, creating pressure differences that significantly increase vertical hydraulic gradients within the subsurface. Overall, in-channel structure did not significantly increase survival, development, and growth of Chinook salmon embryos. However, at several low gradient downstream sites containing in-channel structure, embryo survival, development, and growth were significantly higher relative to paired sites lacking such features. Preliminary data indicate that adding or maintaining in-channel structure, including woody material, in suboptimal spawning reaches improves the incubation environment for salmonid embryos in regulated reaches of a lowland stream. More research examining temporal variation and a full range of incubation depths is needed to further assess these findings.

Quorum sensing in Aliivibrio wodanis 06/09/139 and its role in controlling various phenotypic traits.

BackgroundQuorum Sensing (QS) is a cell-to-cell communication system that bacteria utilize to adapt to the external environment by synthesizing and responding to signalling molecules called autoinducers. The psychrotrophic bacterium Aliivibrio wodanis 06/09/139, originally isolated from a winter ulcer of a reared Atlantic salmon, produces the autoinducer N-3-hydroxy-decanoyl-homoserine-lactone (3OHC10-HSL) and encodes the QS systems AinS/R and LuxS/PQ, and the master regulator LitR. However, the role of QS in this bacterium has not been investigated yet.ResultsIn the present work we show that 3OHC10-HSL production is cell density and temperature-dependent in A. wodanis 06/09/139 with the highest production occurring at a low temperature (6 °C). Gene inactivation demonstrates that AinS is responsible for 3OHC10-HSL production and positively regulated by LitR. Inactivation of ainS and litR further show that QS is involved in the regulation of growth, motility, hemolysis, protease activity and siderophore production. Of these QS regulated activities, only the protease activity was found to be independent of LitR. Lastly, supernatants harvested from the wild type and the ΔainS and ΔlitR mutants at high cell densities show that inactivation of QS leads to a decreased cytopathogenic effect (CPE) in a cell culture assay, and strongest attenuation of the CPE was observed with supernatants harvested from the ΔlitR mutant.ConclusionA. wodanis 06/09/139 use QS to regulate a number of activities that may prove important for host colonization or interactions. The temperature of 6 °C that is in the temperature range at which winter ulcer occurs, plays a role in AHL production and development of CPE on a Chinook Salmon Embryo (CHSE) cell line.

Ultrastructural insights into the replication cycle of salmon pancreas disease virus (SPDV) using salmon cardiac primary cultures (SCPCs).

Salmon pancreas disease virus (SPDV) has been affecting the salmon farming industry for over 30years, but despite the substantial amount of studies, there are still a number of recognized knowledge gaps, for example in the transmission of the virus. In this work, an ultrastructural morphological approach was used to describe observations after infection by SPDV of an ex vivo cardiac model generated from Atlantic salmon embryos. The observations in this study and those available on previous ultrastructural work on SPDV are compared and contrasted with the current knowledge on terrestrial mammalian and insect alphaviral replication cycles, which is deeper than that of SPDV both morphologically and mechanistically. Despite their limitations, morphological descriptions remain an excellent way to generate novel hypotheses, and this has been the aim of this work. This study has used a target host, ex vivo model and resulted in some previously undescribed features, including filopodial membrane projections, cytoplasmic stress granules or putative intracytoplasmic budding. The latter suggests a new hypothesis that warrants further mechanistic research: SPDV in salmon may have retained the capacity for non-cytolytic (persistent) infections by intracellular budding, similar to that noted in arthropod vectors of other alphaviruses. In the notable absence of a known intermediate host for SPDV, the presence of this pattern suggests that both cytopathic and persistent infections may coexist in the same host. It is our hope that the ultrastructural comparison presented here stimulates new research that brings the knowledge on SPDV replication cycle up to a similar level to that of terrestrial alphaviruses.

High-resolution remote sensing and multistate occupancy estimation identify drivers of spawning site selection in fall chum salmon (Oncorhynchus keta) across a sub-Arctic riverscape

Groundwater upwellings provide warmer, stable overwinter temperatures for developing salmon embryos, which may be particularly important in cold, braided, gravel-bed sub-Arctic rivers. We used a three-year time series of aerial counts and remote sensing to estimate the distribution of low and high aggregations of spawning fall chum salmon (Oncorhynchus keta), classify approximately 0.5 km long river segments by geomorphic channel type, and map thermal variability along a 25.4 km stretch of the Teedriinjik River, Alaska. We used a dynamic multistate occupancy model to estimate detectability, occupancy, and the dynamics of spawning aggregations among river segments. Detectability was higher for large (&gt;150) relative to smaller aggregations. Unoccupied segments were likely to remain so from year to year; low abundance spawning segments were dynamic and rarely remained in that state for multiple years, while ∼20%–35% of high abundance segments remained stable, indicating the presence of high-quality spawning habitat. Spawning habitat use was associated with warmer water temperatures likely caused by groundwater upwellings. We identified spawning habitat characteristics and trends in usage by fall chum salmon, which will inform land management decisions and assist in evaluating impacts of shifting climate conditions and resource management on Arctic salmon populations.