Cohabitation With Atlantic Salmon (Salmo salar) Affects Brain Neuromodulators But Not Welfare Indicators in Lumpfish (Cyclopterus lumpus).

Lumpfish are utilized to combat ectoparasitic epidemics in salmon farming. Research gaps on both cleaning behavior and client preferences in a natural environment, emphasizes the need to investigate the physiological impacts on lumpfish during cohabitation with piscivorous Atlantic salmon. Lumpfish (39.9 g, S.D ± 8.98) were arranged in duplicate tanks (n = 40 per treatment) and exposed to Live Atlantic salmon (245.7 g, S.D ± 25.05), salmon Olfaction or lifelike salmon Models for 6 weeks. Growth and health scores were measured every second week. In addition, the final sampling included measurements of neuromodulators, body color, and plasma cortisol. A stimulation and suppression test of the hypothalamic-pituitary-interrenal (HPI) axis was used for chronic stress assessment. Results showed that growth, health scores, and body color remained unaffected by treatments. Significant reductions in levels of brain dopamine and norepinephrine were observed in Live compared to Control. Plasma cortisol was low in all treatments, while the stimulation and suppression test of the HPI axis revealed no indications of chronic stress. This study presents novel findings on the impact on neuromodulators from Atlantic salmon interaction in the lumpfish brain. We argue that the downregulation of dopamine and norepinephrine indicate plastic adjustments to cohabitation with no negative effect on the species. This is in accordance with no observed deviations in welfare measurements, including growth, health scores, body color, and stress. We conclude that exposure to salmon or salmon cues did not impact the welfare of the species in our laboratory setup, and that neuromodulators are affected by heterospecific interaction.

Is Habituation Measurable in Lumpfish Cyclopterus lumpus When Used as Cleaner Fish in Atlantic Salmon Salmo salar Aquaculture?

To investigate how lumpfish interact in Atlantic salmon aquaculture, physiological stress responses and changes in behaviour were analysed in experienced and naive lumpfish. Experienced lumpfish (30.2 ± 7.93 g, mean ± SD) coexisted with a commercial scale production unit of Atlantic salmon (1258.5 ± 152.12 g) for 30 to 60 days, while naive lumpfish (38.2 ± 12.37 g) were kept with conspecifics only. Ten trials from each background were tested. For each trial, 10 lumpfish were tagged and transferred to a video monitored experimental tank (2 × 2 × 0.7 m). In each trial, swimming behaviour was mapped for all lumpfish every 60 s in 20 min, 10 min before, and 10 min after the introduction of four Atlantic salmon. Naive lumpfish expressed significantly increased burst swimming activity and maintained longer interspecific distance to Atlantic salmon in comparison with experienced fish. In addition, mean plasma cortisol levels were significantly elevated in naive fish after exposure to Atlantic salmon. We argue that naive lumpfish expressed innate physiological and behavioural stress responses during first encounter with Atlantic salmon, while reduced responses in experienced individuals indicated habituation. The effect from behavioural and physiological stress in newly deployed naive lumpfish-before and during habituation-should be taken account for when lumpfish are introduced in commercial sea cages to improve welfare for the species. In addition, we suggest that habituation could be applicable during the rearing phase to moderate the transition from a simple tank environment with conspecifics only to interspecies interaction with Atlantic salmon in sea cages.

Fish sperm competition in hatcheries and between wild and hatchery origin fish in nature.

Males compete pre- and post-mating to fertilize the maximum number of eggs. In polyandry, sperm competition occurs when sperm from two or more males compete to fertilize eggs from a female. Here we review how sperm competition from hatchery origin fish can cause loss of genetic variability in fish populations kept in captivity and in wild populations. In fish hatchery practices, sperm competition occurs in mass spawners that release gametes in tanks, and in artificial fertilizations when pooled semen is used. In mass spawnings sperm competition is difficult to tease apart from pre-mating competition and other post-mating selective mechanisms, whereas, studies focused on the use of pooled semen in different fish species have shown a clear relationship between sperm motility parameters and precedence in fertilization. In both situations, sperm competition will result in a loss of genetic variability that accumulates over generations, but hatchery protocols can be adjusted to mitigate it. Another source of concern regarding sperm competition for hatchery produced fish is the spatial and temporal overlap in spawning with wild individuals, either via aquaculture escapees or purposeful stocking programs. This may result in sperm competition between hatchery origin and wild males and impact natural populations. Our review suggests that in order to give every adult selected as broodstock an equal opportunity to produce offspring in captivity, mass spawning and the use of pooled semen should be limited.

A mitochondrial long noncoding RNA in atlantic cod harbors complex heteroplasmic tandem repeat motifs.

A heteroplasmic tandem repeat (HTR) array occupies 100 to 300 bp of the mitochondrial DNA control region in the Atlantic cod, and recently we noted that the repeat appeared integrated in a polyadenylated mitochondrial long noncoding RNA. Here we provide a more detailed analysis of the mitochondrial HTR in the mitochondrial genome of 134 Atlantic cod specimens. We report all specimens to harbor mitochondrial HTRs in the control region, and identified 26 distinct variants among the 402 repeat motifs assessed. Whereas most specimens contained HTR profiles of 2-5 copies consisting of the same 40-bp motif, 22 specimens showed compound HTR arrays of at least two types of motifs present in the same mitochondrial DNA molecule. We found HTR profiles to be highly conserved between different tissue types of a single individual, and strictly maternally inherited in a mating experiment between parental Atlantic cod expressing different HTR profiles and array motifs. We conclude that mitochondrial heteroplasmy in the control region is very common in Atlantic cod, and results in length heterogenity of the long noncoding RNA lncCR-H.

Spawning behavior of Arctic charr (Salvelinus alpinus): Spawning synchrony, vibrational communication, and mate guarding.

A mismatch in synchrony between male and female gamete release in external fertilizers can result in reduced or failed fertilization, sperm competition, and reduced paternity. In Arctic charr (Salvelinus alpinus), males can adopt either a guard or sneak tactic resulting in both pre- and postcopulatory competition between males with alternative reproduction tactics. Here, spawning behavior of free-living Arctic charr was video-recorded, and their reproductive behavior was analyzed. From evaluating 157 spawning events, we observed that females mainly spawned with a guarding male and that the female and the guarding male synchronized timing of gamete release under sperm competition. Although sneakers spawned with higher synchrony than the guarding male in single-male spawning events, the average sneaker released his milt less synchronized with the female than the guarding male under sperm competition. Approximately 50% of the recorded spawning events occurred under sperm competition, where each event included an average of 2.7 males. Additionally, sneakers were more exposed to sperm competition than guarding males. An influx of males, in close proximity to the female, occurred during the behavioral sequences leading up to egg release, but this influx seemed not dependent on egg release, suggesting that something else than gonadal product attracts sneaker males to the spawning female. Just before and during the actual release of gametes, the spawning couple vibrates their bodies in close contact and it seems likely that this vibrational communication between the spawning couple, which results in a larger amplitude sound wave than seen under regular courting, reveals time of gamete release to sneaker males. Thus, vibrational communication may enable synchrony between the guarding male and the female, and this might be traded against the cost of higher detectability from surrounding sneaker males, eavesdropping in close proximity.

Mitochondrial genome variation of Atlantic cod.

OBJECTIVE: The objective of this study was to analyse intraspecific sequence variation of Atlantic cod mitochondrial DNA, based on a comprehensive collection of completely sequenced mitochondrial genomes. RESULTS: We determined the complete mitochondrial DNA sequence of 124 cod specimens from the eastern and western part of the species' distribution range in the North Atlantic Ocean. All specimens harboured a unique mitochondrial DNA haplotype. Nine hundred and fifty-two polymorphic sites were identified, including 109 non-synonymous sites within protein coding regions. Eighteen variable sites were identified as indels, exclusively distributed in structural RNA genes and non-coding regions. Phylogeographic analyses based on 156 available cod mitochondrial genomes did not reveal a clear structure. There was a lack of mitochondrial genetic differentiation between two ecotypes of cod in the eastern North Atlantic, but eastern and western cod were differentiated and mitochondrial genome diversity was higher in the eastern than the western Atlantic, suggesting deviating population histories. The geographic distribution of mitochondrial genome variation seems to be governed by demographic processes and gene flow among ecotypes that are otherwise characterized by localized genomic divergence associated with chromosomal inversions.

No support for cryptic choice by ovarian fluid in an external fertilizer.

Whether the ovarian fluid (OF) represents a selective environment influencing cryptic female choice was tested using an external fertilizer experiencing intense sperm competition and large effects of OF on sperm swimming behavior-the Arctic charr (Salvelinus alpinus). We physically separated the OF from the eggs of reproductively active females and reintroduced either their own OF or fluid from another female to the eggs. The eggs were then fertilized in vitro in a replicated split-brood design with sperm from two males under synchronized sperm competition trials, while also measuring sperm velocity of the individual males in the individual OFs. We found large effects of males, but no effect of females (i.e., eggs) on paternity, determined from microsatellites. More important, we found no effect of OF treatments on the relative paternity of the two competing males in each pair. This experimental setup does not provide support for the hypothesis that OF plays an important role as medium for cryptic female choice in charr. Power analyses revealed that our sample size is large enough to detect medium-sized changes in relative paternity (medium-sized effect sizes), but not large enough to detect small changes in relative paternity. More studies are needed before a conclusion can be drawn about OF's potential influence on paternity under sperm competition-even in charr.

Mitogenome sequence variation in migratory and stationary ecotypes of North-east Atlantic cod.

Sequencing of mitochondrial gene fragments from specimens representing a wide range of geographical locations has indicated limited population structuring in Atlantic cod (Gadus morhua). We recently performed whole genome analysis based on next-generation sequencing of two pooled ecotype samples representing offshore migratory and inshore stationary cod from the North-east Atlantic Ocean. Here we report molecular features and variability of the 16.7kb mitogenome component that was collected from the datasets. These sequences represented more than 25 times coverage of each individual and more than 1100 times coverage of each ecotype sample. We estimated the mitogenome to have evolved 14 times more rapidly than the nuclear genome. Among the 365 single nucleotide polymorphism (SNP) sites identified, 121 were shared between ecotypes, and 151 and 93 were private within the migratory and stationary cod, respectively. We found 323 SNPs to be located in protein coding genes, of which 29 were non-synonymous. One synonymous site in ND2 was likely to be under positive selection. FST measurements indicated weak differentiation in ND1 and ND2 between ecotypes. We conclude that the Atlantic cod mitogenome and the nuclear genome apparently evolved by distinct evolutionary constraints, and that the reproductive isolation observed from whole genome analysis was not visible in the mtDNA sequences.

Genomic divergence between the migratory and stationary ecotypes of Atlantic cod.

Atlantic cod displays a range of phenotypic and genotypic variations, which includes the differentiation into coastal stationary and offshore migratory types of cod that co-occur in several parts of its distribution range and are often sympatric on the spawning grounds. Differentiation of these ecotypes may involve both historical separation and adaptation to ecologically distinct environments, the genetic basis of which is now beginning to be unravelled. Genomic analyses based on recent sequencing advances are able to document genomic divergence in more detail and may facilitate the exploration of causes and consequences of genome-wide patterns. We examined genomic divergence between the stationary and migratory types of cod in the Northeast Atlantic, using next-generation sequencing of pooled DNA from each of two population samples. Sequence data was mapped to the published cod genome sequence, arranged in more than 6000 scaffolds (611 Mb). We identified 25 divergent scaffolds (26 Mb) with a higher than average gene density, against a backdrop of overall moderate genomic differentiation. Previous findings of localized genomic divergence in three linkage groups were confirmed, including a large (15 Mb) genomic region, which seems to be uniquely involved in the divergence of migratory and stationary cod. The results of the pooled sequencing approach support and extend recent findings based on single-nucleotide polymorphism markers and suggest a high degree of reproductive isolation between stationary and migratory cod in the North-east Atlantic.

RNA deep sequencing of the Atlantic cod transcriptome.

The Atlantic cod (Gadus morhua) is an emerging aquaculture species. Efforts to develop and characterize its genomic recourses, including draft-grade genome sequencing, have been initiated by the research community. The transcriptome represents the whole complement of RNA transcripts in cells and tissues and reflects the expressed genes at various life stages, tissue types, physiological states, and environmental conditions. We are investigating the Atlantic cod transcriptome by Roche 454, Illumina GA, and ABI SOLiD deep sequencing platforms and corresponding bioinformatics. Both embryonic developmental stages and adult tissues are studied. Here we summarize our recent progress in the analyses of nuclear and mitochondrial polyA mRNAs, non-protein-coding intermediate RNAs, and regulatory microRNAs.

Large-scale sequence analyses of Atlantic cod.

The Atlantic cod (Gadus morhua) is a key species in the North Atlantic ecosystem and commercial fisheries, with increasing aquacultural production in several countries. A Norwegian effort to sequence the complete 0.9Gbp genome by the 454 pyrosequencing technology has been initiated and is in progress. Here we review recent progress in large-scale sequence analyses of the nuclear genome, the mitochondrial genome and genome-wide microRNA identification in the Atlantic cod. The nuclear genome will be de novo sequenced with 25 times oversampling. A total of 120 mitochondrial genomes, sampled from several locations in the North Atlantic, are being completely sequenced by Sanger technology in a high-throughput pipeline. These sequences will be included in a new database for maternal marker reference of Atlantic cod diversity. High-throughput 454 sequencing, as well as Evolutionary Image Array (EvoArray) informatics, is used to investigate the complete set of expressed microRNAs and corresponding mRNA targets in various developmental stages and tissues. Information about microRNA profiles will be essential in the understanding of transcriptome complexity and regulation. Finally, developments and perspectives of Atlantic cod aquaculture are discussed in the light of next-generation high-throughput sequence technologies.