Divergent bacterial landscapes: unraveling geographically driven microbiomes in Atlantic cod.

Establishing microbiome signatures is now recognized as a critical step toward identifying genetic and environmental factors shaping animal-associated microbiomes and informing the health status of a given host. In the present work, we prospectively collected 63 blood samples of the Atlantic cod population of the Southern Gulf of Saint Lawrence (GSL) and characterized their 16S rRNA circulating microbiome signature. Our results revealed that the blood microbiome signature was dominated at the phylum level by Proteobacteria, Bacteroidetes, Acidobacteria and Actinobacteria, a typical signature for fish populations inhabiting the GSL and other marine ecosystems. At the genus level, however, we identified two distinct cod groups. While the microbiome signature of the first group was dominated by Pseudoalteromonas, a genus we previously found in the microbiome signature of Greenland and Atlantic halibut populations of the GSL, the second group had a microbiome signature dominated by Nitrobacter and Sediminibacterium (approximately 75% of the circulating microbiome). Cods harboring a Nitrobacter/Sediminibacterium-rich microbiome signature were localized in the most southern part of the GSL, just along the northern coast of Cape Breton Island. Atlantic cod microbiome signatures did not correlate with the weight, length, relative condition, depth, temperature, sex, and salinity, as previously observed in the halibut populations. Our study provides, for the first time, a unique snapshot of the circulating microbiome signature of Atlantic cod populations and the potential existence of dysbiotic signatures associated with the geographical distribution of the population, probably linked with the presence of nitrite in the environment.

Fish species mislabelling in China: evidence from a survey of frozen, dried, and surimi-based fish products marketed as cod-related species.

'Cod'-related species are among the most appreciated marine fish resources around the world, but are also prone to species mislabelling. In the present study, a total of 76 frozen, dried, and surimi-based fish products, sold as 'Cod' (59 products), 'Atlantic authentic Cod' (11 products), and 'Authentic Cod' (6 products), were collected in China. A species-specific LAMP (loop-mediated isothermal amplification) method was used to screen for the presence of Atlantic cod (Gadus morhua), Pacific cod (G. macrocephalus), Alaska pollock (G. chalcogrammus), Southern hake (Merluccius australis), which was cross-confirmed using real-time PCR and DNA sequencing methods. The results highlighted the greatest species diversity for 'Cod' products, and the identified species were from nine different families. It appears that the practice of assigning a specific type or category of species to the common name 'Cod' has not been widely advocated, and the misuse of this ambiguous common name has been a common practice for species adulteration, negatively impacting consumers' rights and marine conservation. To rebuild consumers' confidence, retail fish suppliers have differentiated their products by adding specific qualifiers in front of the common name 'Cod' on the label, such as 'Authentic cod' and 'Atlantic authentic cod'. The endeavour is highly meaningful, since Gadus morhua was identified as the species for a significant majority of 'Atlantic authentic cod' and 'Authentic cod' products (64.7%, 11/17), with the remaining six products identified as Alaskan pollock (G. chalcogrammus), Pacific cod (G. macrocephalus) and North Pacific hake (Merluccius productus). Despite the positive effort to reverse species mislabelling from retail on-line fish suppliers, a standardized fish nomenclature stipulated by the responsible authorities remains crucial for enhancing transparency and continuing to reduce species mislabelling.

The involvement of Pseudoterranova decipiens fish infestation on the shelf-life of fresh Atlantic cod (Gadus morhua) fillet.

Zoonotic nematodes of the family Anisakidae are highly common in many marine fish species, which act as paratenic hosts for the third larval stage. In the fish, these parasites may migrate from the fish's gastro-intestinal tract (GI-tract) further to the coelomic cavity and muscles, making them a possible contamination source of bacteria they carry on their cuticle and in their GI-tract. A previous study revealed no apparent effect of Anisakis simplex on spoilage of fish, but the equally common anisakid species Pseudoterranova decipiens has a larger body surface potentially increasing the bacterial load brought into the fish muscle upon migration. As the presence of shelf-life reducing spoilage bacteria in the microbiome of this anisakid species has been demonstrated, the objective of the present study was to assess the potential shelf-life reducing effect of P. decipiens in fresh fish fillets stored in a domestic refrigerator. Atlantic cod was used as a model since members of the cod family are the third most consumed marine fish globally and it has the highest prevalence of P. decipiens infections. Infected and non-infected codfish fillet portions were collected and microbiologically analyzed at day 0 and day 4 of storage in a domestic fridge. Three isolation media were used to enhance maximum bacterial recovery and isolates were identified using MALDI-TOF MS and 16S rRNA gene sequencing. In parallel to the microbiological examination, sensory analysis was performed daily on the cod fillets to evaluate the freshness of the fish. Results revealed the presence of typical spoilage bacteria (e.g., Pseudomonas sp., Photobacterium sp.) in all fish, but based on the total viable counts, total H2S-producing bacteria, and sensory analysis, there were no objective indications to assume an increased fish spoilage rate by the presence and migration P. decipiens. Additionally, a beta-diversity comparison revealed no significant differences in microbiota composition between infected and non-infected fish parts, though individual heterogeneity in microbiome composition among Atlantic codfish individuals was found. As total viable counts did, however, exceed the guideline limits for fresh fish, further research should now focus on the role of the candling step as a potential source of post-harvest contamination. As such, anisakid infection might still accelerate fish spoilage, though now in an indirect way.

Diverse Transcriptome Responses to Salinity Change in Atlantic Cod Subpopulations.

Adaptation to environmental variation caused by global climate change is a significant aspect of fisheries management and ecology. A reduction in ocean salinity is visible in near-shore areas, especially in the Baltic Sea, where it is affecting the Atlantic cod population. Cod is one of the most significant teleost species, with high ecological and economical value worldwide. The population of cod in the Baltic Sea has been traditionally divided into two subpopulations (western and eastern) existing in higher- and lower-salinity waters, respectively. In recent decades, both Baltic cod subpopulations have declined massively. One of the reasons for the poor condition of cod in the Baltic Sea is environmental factors, including salinity. Thus, in this study, an oligonucleotide microarray was applied to explore differences between Baltic cod subpopulations in response to salinity fluctuations. For this purpose, an exposure experiment was conducted consisting of salinity elevation and reduction, and gene expression was measured in gill tissue. We found 400 differentially expressed genes (DEGs) involved in the immune response, metabolism, programmed cell death, cytoskeleton, and extracellular matrix that showed a subpopulation-dependent pattern. These findings indicate that osmoregulation in Baltic cod is a complex process, and that western and eastern Baltic cod subpopulations respond differently to salinity changes.

Genetic assignment predicts depth of benthic settlement for 0-group Atlantic cod.

Atlantic cod is a keystone species that remains among the most economically important demersal fish in the North Atlantic. Throughout its distribution range, Atlantic cod is composed of populations with varying environmental preferences and migratory propensities. This life-history variation is likely to have contributed to the niche width and large population sizes of Atlantic cod, and its relative resilience to environmental change and exploitation. The Icelandic cod stock is currently managed as a single unit, but early research indicates population variation by depth and temperature and distinct offshore and inshore spawning components. Pelagic 0-group juveniles from different spawning grounds coexist in nursery areas around Iceland, but their genetic composition or habitat partitioning had not been examined post benthic settlement. In the current study we examine the genetic composition of Atlantic cod juvenile aggregations at nearshore nursery grounds in NW-Iceland and report distinct segregation by the depth of offshore and inshore juvenile cod. The physiological mechanism of this segregation is not known, but the pattern demonstrates the need to consider population structure at nursery grounds in the application of marine spatial planning and other area-based conservation tools.

Nasal responses to elevated temperature and Francisella noatunensis infection in Atlantic cod (Gadus morhua).

We report the histological and transcriptomic changes in the olfactory organ of Atlantic cod exposed to Francisella noatunensis. Experimental infection was performed at either 12 °C or 17 °C. Infected fish presented the classic gross pathologies of francisellosis. Nasal morpho-phenotypic parameters were not significantly affected by elevated temperature and infection, except for the number of mucus cells in the 12 °C group seven weeks after the challenge. A higher number of genes were altered through time in the group reared at 17 °C. At termination, the nasal transcriptome of infected fish in both groups was similar to the control. When both infected groups were compared, 754 DEGs were identified, many of which were involved in signalling, defence, transmembrane and enzymatic processes. In conclusion, the study reveals that elevated temperature could trigger responses in the olfactory organ of Atlantic cod and shape the nasal response to F. noatunensis infection.

Intergenerational effects of early life-stage temperature modulation on gene expression and DNA methylation in Atlantic cod (Gadus morhua).

After suffering several collapses, the cod farming industry is now in the process of trying to re-establish itself. We have used material from Norway's National Cod Breeding Program to study how different early life-stage temperature regimes affect DNA methylation and gene expression. Long-term effects were detected by sampling fish several weeks after the end of differential treatments, and offspring from the different exposure groups was also sampled. Many overlapping genes were found between the different exposure groups and generations, coupled with genes associated with differential CpG methylation levels. Genes involved in muscle fibre development, general metabolic processes and formation of deformities were significantly affected, and genes relevant for intergenerational transfer of epigenetic marks were also detected. We believe the use of environmental cues can be a useful strategy for improving the production of Atlantic cod.

Movement diversity and partial sympatry of coastal and Northeast Arctic cod ecotypes at high latitudes.

Movement diversity within species represent an important but often neglected, component of biodiversity that affects ecological and genetic interactions, as well as the productivity of exploited systems. By combining individual tracking data from acoustic telemetry with novel genetic analyses, we describe the movement diversity of two Atlantic cod Gadus morhua ecotypes in two high-latitude fjord systems: the highly migratory Northeast Arctic cod (NEA cod) that supports the largest cod fishery in the world, and the more sedentary Norwegian coastal cod, which is currently in a depleted state. As predicted, coastal cod displayed a higher level of fjord residency than NEA cod. Of the cod tagged during the spawning season, NEA cod left the fjords permanently to a greater extent and earlier compared to coastal cod, which to a greater extent remained resident and left the fjords temporarily. Despite this overall pattern, horizontal movements atypical for the ecotypes were common with some NEA cod remaining within the fjords year-round and some coastal cod displaying a low fjord fidelity. Fjord residency and exit timing also differed with spawning status and body size, with spawning cod and large individuals tagged during the feeding season more prone to leave the fjords and earlier than non-spawning and smaller individuals. While our results confirm a lower fjord dependency for NEA cod, they highlight a movement diversity within each ecotype and sympatric residency between ecotypes, previously undetected by population-level monitoring. This new knowledge is relevant for the management, which should base their fisheries advice for these interacting ecotypes on their habitat use and seasonal movements.

Closed-tube visual detection of Atlantic cod (Gadus morhua) using self-quenched primer coupled with a designed loop-mediated isothermal amplification vessel.

BACKGROUND: Species adulteration has been widely revealed around the world, and the possible reasons include declining stocks in most source areas of the world, poor transparency in the global supply chain, and difficulty in distinguishing the features of processed products. The present work selected Atlantic cod (Gadus morhua) as a case study, and developed a novel loop-mediated isothermal amplification (LAMP) assay for Atlantic cod authentication, where a self-quenched primer and a newly designed reaction vessel were used to realize the endpoint visual detection of the target-specific products. RESULTS: A novel LAMP primer set was designed for Atlantic cod, and the inner primer BIP was selected to label the self-quenched fluorogenic element. The fluorophore's dequenching only occurred along with LAMP elongation for the target species. No fluorescence could be observed with both single-stranded DNA and partially complementary dsDNA of the non-target species. With the novel reaction vessel, both amplification as well as detection were operated in an enclosed device, and visual differentiation of Atlantic cod, negative control, and false positive generated from primer dimers was achieved. The novel assay has proved its specificity and applicability, and could detect as little as 1 pg of Atlantic cod DNA. Moreover, Atlantic cod adulteration as low as 10% could be detected in haddock (Melanogrammus aeglefinus), and no cross-reactivity was observed. CONCLUSION: The established assay could be a useful tool to detect mislabeling incidents involving Atlantic cod considering the advantages of speed, simplicity and accuracy. © 2023 Society of Chemical Industry.

Detecting parallel polygenic adaptation to novel evolutionary pressure in wild populations: a case study in Atlantic cod (Gadus morhua).

Populations can adapt to novel selection pressures through dramatic frequency changes in a few genes of large effect or subtle shifts in many genes of small effect. The latter (polygenic adaptation) is expected to be the primary mode of evolution for many life-history traits but tends to be more difficult to detect than changes in genes of large effect. Atlantic cod (Gadus morhua) were subjected to intense fishing pressure over the twentieth century, leading to abundance crashes and a phenotypic shift toward earlier maturation across many populations. Here, we use spatially replicated temporal genomic data to test for a shared polygenic adaptive response to fishing using methods previously applied to evolve-and-resequence experiments. Cod populations on either side of the Atlantic show covariance in allele frequency change across the genome that are characteristic of recent polygenic adaptation. Using simulations, we demonstrate that the degree of covariance in allele frequency change observed in cod is unlikely to be explained by neutral processes or background selection. As human pressures on wild populations continue to increase, understanding and attributing modes of adaptation using methods similar to those demonstrated here will be important in identifying the capacity for adaptive responses and evolutionary rescue. This article is part of the theme issue 'Detecting and attributing the causes of biodiversity change: needs, gaps and solutions'.

Protein phosphorylation profile of Atlantic cod (Gadus morhua) in response to pre-slaughter pumping stress and postmortem time.

This study aimed to evaluate the effect of pumping stress (pumping and pumping-resting) and postmortem time (before and after rigor mortis) on phosphorylation profiles of myofibrillar protein (MP) and sarcoplasmic protein (SP) of Atlantic cod (Gadus morhua) fillets. The result showed that MP had higher global phosphorylation levels than SP regardless of stress condition and postmortem time. The pumping process resulted in significant changes in phosphorylation of structural proteins including myosin heavy and light chains. Pumping also affected the phosphorylation status of heat shock proteins and metabolic enzymes involved in the glycolytic pathways, indicating the possible role of phosphorylation in regulating energy hemostasis of fish under stressful conditions. The pumping-induced phosphorylation changes mainly occurred before rigor mortis, and postmortem time affected the phosphorylation status to a less extent. This work contributes to a deeper understanding on protein phosphorylation affected by pre-slaughter stress and postmortem time of fish.

Aquaculture rearing systems induce no legacy effects in Atlantic cod larvae or their rearing water bacterial communities.

The microbial rearing quality influences the survival of marine larvae. Microbially matured water treatment systems (MMS) provide a more favourable rearing water microbiome than flow-through systems (FTS). It has previously been hypothesised, but not investigated, that initial rearing in MMS leaves a protective legacy effect in Atlantic cod larvae (Gadus morhua). We tested this hypothesis through a crossover 2 × 2 factorial experiment varying the rearing water treatment system (MMS vs FTS) and the microbial carrying capacity (+ /- added organic matter). At 9 days post-hatching, we switched the rearing water treatment system. By comparing switched and unswitched rearing tanks, we evaluated if legacy effects had been established in the larvae or their surrounding rearing water bacterial community. We analysed the bacterial communities with flow cytometry and 16S rRNA gene sequencing. We found no evidence that the initial rearing condition left a legacy effect in the communities by evaluating the bacterial community diversity and structure. Instead, the present rearing condition was the most important driver for differences in the rearing water microbiota. Furthermore, we found that MMS with high microbial carrying capacity appeared to seed a stable bacterial community to the rearing tanks. This finding highlights the importance of keeping a similar carrying capacity between the inlet and rearing water. Moreover, we reject the hypothesis that the initial rearing condition leaves a protective legacy effect in larvae, as the larval survival and robustness were linked to the present rearing condition. In conclusion, our results highlight the importance of maintaining a beneficial microbial rearing environment from hatching and throughout the larval rearing period.

Ancient DNA evidence for the ecological globalization of cod fishing in medieval and post-medieval Europe.

Understanding the historical emergence and growth of long-range fisheries can provide fundamental insights into the timing of ecological impacts and the development of coastal communities during the last millennium. Whole-genome sequencing approaches can improve such understanding by determining the origin of archaeological fish specimens that may have been obtained from historic trade or distant water. Here, we used genome-wide data to individually infer the biological source of 37 ancient Atlantic cod specimens (ca 1050-1950 CE) from England and Spain. Our findings provide novel genetic evidence that eleventh- to twelfth-century specimens from London were predominantly obtained from nearby populations, while thirteenth- to fourteenth-century specimens were derived from distant sources. Our results further suggest that Icelandic cod was indeed exported to London earlier than previously reported. Our observations confirm the chronology and geography of the trans-Atlantic cod trade from Newfoundland to Spain starting by the early sixteenth century. Our findings demonstrate the utility of whole-genome sequencing and ancient DNA approaches to describe the globalization of marine fisheries and increase our understanding regarding the extent of the North Atlantic fish trade and long-range fisheries in medieval and early modern times.

Warming Accelerates the Onset of the Molecular Stress Response and Increases Mortality of Larval Atlantic Cod.

Temperature profoundly affects ectotherm physiology. Although differential thermal responses influence fitness, thus driving population dynamics and species distributions, our understanding of the molecular architecture underlying these responses is limited, especially during the critical larval stage. Here, using RNA-sequencing of laboratory-reared Atlantic cod (Gadus morhua) larvae of wild origin, we find changes in gene expression in thousands of transcripts consistent with a severe cellular stress response at both ambient and projected (+2°C and +4°C) temperatures. In addition, specific responses to stress, heat, and hypoxia were commonly identified in gene ontology enrichment analyses and 33 of the 44 genes comprising the minimum stress proteome of all organisms were upregulated. Earlier onset of the stress response was evident at higher temperatures; concomitant increased growth and mortality suggests a reduction in fitness. Temporal differences in gene expression levels do not correspond to differences in growing degree days, suggesting negative physiological consequences of warming beyond accelerated development. Because gene expression is costly, we infer that the upregulation of thousands of transcripts in response to warming in larval cod might act as an energetic drain. We hypothesize that the energetically costly stress response, coupled with increased growth rate at warmer temperatures, leads to faster depletion of energy reserves and increased risk of mortality in larval cod. As sea surface temperatures continue to rise over the next century, reduced fitness of Atlantic cod larvae might lead to population declines in this ecologically and socioeconomically important species. Further, our findings expand our understanding of transcriptomic responses to temperature by ectothermic vertebrate larvae beyond the critical first-feeding stage, a time when organisms begin balancing the energetic demands of growth, foraging, development, and maintenance. Linking the molecular basis of a thermal response to key fitness-related traits is fundamentally important to predicting how global warming will affect ectotherms.

Lymphocyte subsets in Atlantic cod (Gadus morhua) interrogated by single-cell sequencing.

Atlantic Cod (Gadus morhua) has lost the major histocompatibility complex class II presentation pathway. We recently identified CD8-positive T cells, B cells, and plasma cells in cod, but further characterisation of lymphocyte subsets is needed to elucidate immune adaptations triggered by the absence of CD4-positive T lymphocytes. Here, we use single-cell RNA sequencing to examine the lymphocyte heterogeneity in Atlantic cod spleen. We describe five T cell subsets and eight B cell subsets and propose a B cell trajectory of differentiation. Notably, we identify a subpopulation of T cells that are CD8-negative. Most of the CD8-negative T lymphocytes highly express the homologue of monocyte chemotactic protein 1b, and another subset of CD8-negative T lymphocytes express the homologue of the scavenger receptor m130. Uncovering the multiple lymphocyte cell sub-clusters reveals the different immune states present within the B and T cell populations, building a foundation for further work.

Ancient DNA reveals a southern presence of the Northeast Arctic cod during the Holocene.

Climate change has been implicated in an increased number of distributional shifts of marine species during the last century. Nonetheless, it is unclear whether earlier climatic fluctuations had similar impacts. We use ancient DNA to investigate the long-term spawning distribution of the Northeast Arctic cod (skrei) which performs yearly migrations from the Barents Sea towards spawning grounds along the Norwegian coast. The distribution of these spawning grounds has shifted northwards during the last century, which is thought to be associated with food availability and warming temperatures. We genetically identify skrei specimens from Ruskeneset in west Norway, an archaeological site located south of their current spawning range. Remarkably, 14C analyses date these specimens to the late Holocene, when temperatures were warmer than present-day conditions. Our results either suggest that temperature is not the only driver influencing the spawning distribution of Atlantic cod, or could be indicative of uncertainty in palaeoclimate reconstructions in this region. Regardless, our findings highlight the utility of aDNA to reconstruct the historical distribution of economically important fish populations and reveal the complexity of long-term ecological interactions in the marine environment.

Supergenes promote ecological stasis in a keystone species.

Structural variation can create supergene architectures through tight genomic linkages that maintain traits in favourable combinations. A new study by Sodeland et al. links such supergenes in Atlantic cod with species persistence over millennia, despite the fisheries-induced decline in populations. This links intraspecific supergene diversity to ecological stasis, with significant consequences for ecosystem stability.

Unraveling the microbiota of the fish parasite Pseudoterranova decipiens in codfish (Gadus morhua) reveals a fish-related bacterial community.

Anisakidae, mainly represented by the species Anisakis simplex and Pseudoterranova decipiens, are one of the most commonly zoonotic nematodes present in marine fish species. Apart from public health risks directly linked to the parasite itself, little is known on the effects of the migrating nematodes on the hygienic quality of the fish fillet due to bacteria it carries. In the present study, the cultivated bacterial community on and in individual P. decipiens larvae deriving from codfish is reported. Four isolation media were included and evaluated to increase the bacterial diversity isolated, and identification of the bacterial growth was performed by a combination of Matrix-Assisted Laser Desorption Ionization Time-Of-Flight mass spectrometry and 16S rRNA gene sequencing. Results revealed that the microbiota of P. decipiens larvae comprises both potential spoilage bacteria and human opportunistic pathogens, and that a combined isolation on the general isolation medium tryptone soy agar and a medium supplemented with artificial seawater resulted in the highest bacterial recovery in terms of diversity and enumeration. Dissimilarity analysis also revealed similar, though unique, bacterial communities between nematodes originating from the same fish suggesting that anisakid microbiota compositions are reflections of the microbial assemblages in the fish host as an individual, and that the gut microbiome is diverse within gadoid fish species originating from the same geographical habitat. Future research should, based on the results in the present study, further elaborate on the comparison of the bacterial communities of both the larva and the codfish from which it was isolated, and, explore the extrapolation potential towards other fish and nematode species. Also, the actual degree of risk beyond the simple presence of the parasite due to carriage of opportunistic bacteria should be examined, as well as the nematode's true effect on spoilage.

Stabilizing selection on Atlantic cod supergenes through a millennium of extensive exploitation.

Life on Earth has been characterized by recurring cycles of ecological stasis and disruption, relating biological eras to geological and climatic transitions through the history of our planet. Due to the increasing degree of ecological abruption caused by human influences many advocate that we now have entered the geological era of the Anthropocene, or "the age of man." Considering the ongoing mass extinction and ecosystem reshuffling observed worldwide, a better understanding of the drivers of ecological stasis will be a requisite for identifying routes of intervention and mitigation. Ecosystem stability may rely on one or a few keystone species, and the loss of such species could potentially have detrimental effects. The Atlantic cod (Gadus morhua) has historically been highly abundant and is considered a keystone species in ecosystems of the northern Atlantic Ocean. Collapses of cod stocks have been observed on both sides of the Atlantic and reported to have detrimental effects that include vast ecosystem reshuffling. By whole-genome resequencing we demonstrate that stabilizing selection maintains three extensive "supergenes" in Atlantic cod, linking these genes to species persistence and ecological stasis. Genomic inference of historic effective population sizes shows continued declines for cod in the North Sea-Skagerrak-Kattegat system through the past millennia, consistent with an early onset of the marine Anthropocene through industrialization and commercialization of fisheries throughout the medieval period.

Supergene origin and maintenance in Atlantic cod.

Supergenes are sets of genes that are inherited as a single marker and encode complex phenotypes through their joint action. They are identified in an increasing number of organisms, yet their origins and evolution remain enigmatic. In Atlantic cod, four megabase-scale supergenes have been identified and linked to migratory lifestyle and environmental adaptations. Here we investigate the origin and maintenance of these four supergenes through analysis of whole-genome-sequencing data, including a new long-read-based genome assembly for a non-migratory Atlantic cod individual. We corroborate the finding that chromosomal inversions underlie all four supergenes, and we show that they originated at different times between 0.40 and 1.66 million years ago. We reveal gene flux between supergene haplotypes where migratory and stationary Atlantic cod co-occur and conclude that this gene flux is driven by gene conversion, on the basis of an increase in GC content in exchanged sites. Additionally, we find evidence for double crossover between supergene haplotypes, leading to the exchange of an ~275 kilobase fragment with genes potentially involved in adaptation to low salinity in the Baltic Sea. Our results suggest that supergenes can be maintained over long timescales in the same way as hybridizing species, through the selective purging of introduced genetic variation.