In sea trout, the physiological response to salmon louse is stronger in female than in males.

The aims of this study were to compare male and female sea trout (Salmo trutta) with respect to their hypo-osmoregulatory ability over a simulated migration to seawater and their clinical and physiological response to salmon louse (Lepeophtheirus salmonis) infection in seawater and over a simulated pre-mature return to freshwater. For this purpose, 2-year-old hatchery-reared male and female brown trout (F1 offspring of wild caught anadromous fish) were infected with salmon lice and measured for changes in plasma ions, glucose, lactate and osmolality and relative heart, liver and gonad sizes during a simulated seawater migration and thereafter a premature return to freshwater after 4 weeks in seawater (pre-adult louse). Un-infected trout served as control. Male trout used longer time to develop full hypo-osmoregulatory ability in seawater and showed a stronger response in plasma glucose and lactate following simulated premature return to freshwater, compared to female trout. Response to salmon louse was stronger in female trout, shown by stronger osmotic stress by chalimus (plasma Cl-) and pre-adult louse (plasma osmolality) and elevated relative liver size (hepatosomatic index) by pre-adult louse in female compared to male trout. Moreover, high plasma cortisol in infected female and low plasma cortisol in infected male trout produced a significant treatment-sex interaction on plasma cortisol. Lice infection intensity was initially higher in male (0.18 lice g-1) compared to female trout (0.11 lice g-1) at the chalimus stage, but equal between sexes at the pre-adult stage (male 0.15 and female 0.17 lice g-1). This study showed that female trout were better adapted for changes in water salinity, while male trout were more robust against salmon louse infection. These results suggests that the elevated salmon louse infection pressure generated by salmon farming have strong and unexplored negative effects on wild sea trout populations. Further research on this topic is vital for the conservation of wild sea trout populations.

Pituitary Gonadotropin Gene Expression During Induced Onset of Postsmolt Maturation in Male Atlantic Salmon: In Vivo and Tissue Culture Studies.

Precocious male maturation causes reduced welfare and increased production costs in Atlantic salmon (Salmo salar) aquaculture. The pituitary produces and releases follicle-stimulating hormone (Fsh), the gonadotropin triggering puberty in male salmonids. However, little is known about how Fsh production is regulated in Atlantic salmon. We examined, in vivo and ex vivo, transcriptional changes of gonadotropin-related genes accompanying the initial steps of testis maturation, in pituitaries of males exposed to photoperiod and temperature conditions promoting maturation (constant light and 16°C). Pituitary fshb, lhb and gnrhr2bba transcripts increased in vivo in maturing males (gonado-somatic index > 0.1%). RNA sequencing (RNAseq) analysis using pituitaries from genetically similar males carrying the same genetic predisposition to mature, but differing by responding or not responding to stimulatory environmental conditions, revealed 144 differentially expressed genes, ~2/3rds being up-regulated in responders, including fshb and other pituitary hormones, steroid-related and other puberty-associated transcripts. Functional enrichment analyses confirmed gene involvement in hormone/steroid production and gonad development. In ex vivo studies, whole pituitaries were exposed to a selection of hormones and growth factors. Gonadotropin-releasing hormone (Gnrh), 17ß-estradiol (E2) and 11-ketotestosterone (11-KT) up-regulated gnrhr2bba and lhb, while fshb was up-regulated by Gnrh but down-regulated by 11-KT in pituitaries from immature males. Also pituitaries from maturing males responded to Gnrh and sex steroids by increased gnrhr2bba and lhb transcript levels, but fshb expression remained unchanged. Growth factors (inhibin A, activin A and insulin-like growth factor 1) did not change gnrhr2bba, lhb or fshb transcript levels in pituitaries either from immature or maturing males. Additional pituitary ex vivo studies on candidates identified by RNAseq showed that these transcripts were preferentially regulated by Gnrh and sex steroids, but not by growth factors, and that Gnrh/sex steroids were less effective when incubating pituitaries from maturing males. Our results suggest that a yet to be characterized mechanism up-regulating fshb expression in the salmon pituitary is activated in response to stimulatory environmental conditions prior to morphological signs of testis maturation, and that the transcriptional program associated with this mechanism becomes unresponsive or less responsive to most stimulators ex vivo once males had entered pubertal developmental in vivo.

Loss of stra8 Increases Germ Cell Apoptosis but Is Still Compatible With Sperm Production in Atlantic Salmon (Salmo salar).

Entering meiosis strictly depends on stimulated by retinoic acid 8 (Stra8) gene function in mammals. This gene is missing in a number of fish species, including medaka and zebrafish, but is present in the majority of fishes, including Atlantic salmon. Here, we have examined the effects of removing stra8 on male fertility in Atlantic salmon. As in mammals, stra8 expression was restricted to germ cells in the testis, transcript levels increased during the start of puberty, and decreased when blocking the production of retinoic acid. We targeted the salmon stra8 gene with two gRNAs one of these were highly effective and produced numerous mutations in stra8, which led to a loss of wild-type (WT) stra8 expression in F0 salmon testis. In maturing stra8 crispants, the spermatogenetic tubuli were partially disorganized and displayed a sevenfold increase in germ cell apoptosis, in particular among type B spermatogonia and spermatocytes. The production of spermatogenic cysts, on the other hand, increased in maturing stra8 crispants. Gene expression analysis revealed unchanged (lin28a, ret) or reduced levels (egr1, dusp4) of transcripts associated with undifferentiated spermatogonia. Decreased expression was recorded for some genes expressed in differentiating spermatogonia including dmrt1 and ccnd2 or in spermatocytes, such as ccna1. Different from Stra8-deficient mammals, a large number of germ cells completed spermatogenesis, sperm was produced and fertilization rates were similar in WT and crispant males. While loss of stra8 increased germ cell apoptosis during salmon spermatogenesis, crispants compensated this cell loss by an elevated production of spermatogenic cysts, and were able to produce functional sperm. It appears that also in a fish species with a stra8 gene in the genome, the critical relevance this gene has attained for mammalian spermatogenesis is not yet given, although detrimental effects of the loss of stra8 were clearly visible during maturation.

Vertebral deformities in interspecific diploid and triploid salmonid hybrids.

Vertebral deformities in salmonid interspecific hybrids, some of which were triploidised, were assessed across three separate year classes during the freshwater life stage. Initially, eggs from a farmed Atlantic salmon Salmo salar were crossed with the sperm from a S. salar, arctic char Salvelinus alpinus or brown trout Salmo trutta. For S. salar × S. trutta, half the eggs were triploidised. In a second- and third-year class, the eggs from a farmed S. salar were crossed with the sperm from either a S. salar or a S. trutta, and half of each group was triploidised. In the two initial-year classes, all hybrids were larger than the S. salar controls, and triploid S. salar × S. trutta were larger than diploid counterparts. In the third-year class, the S. salar × S. trutta were smaller than the S. salar, in contrast to the initial 2 year classes, although the triploid hybrids were still larger than the diploids. In the third-year class, a high degree of spontaneous triploidy was also observed in the putative diploid groups (between 16 and 39%). Vertebral deformities were consistently higher in pressure-shocked triploids than diploids, irrespective of hybridisation, but there was no consistent effect of hybridisation among experiments. Although this study was not able to explain the contrasting results for vertebral deformities between year classes, triploid S. salar × S. trutta can demonstrate impressive freshwater growth that could be of interest for future farming programmes.

Development of supermale and all-male Atlantic salmon to research the vgll3 allele - puberty link.

BACKGROUND: Farmed Atlantic salmon are one of the most economically significant global aquaculture products. Early sexual maturation of farmed males represents a significant challenge to this industry and has been linked with the vgll3 genotype. However, tools to aid research of this topic, such as all-male and clonal fish, are still lacking. The present 6-year study examined if all-male production is possible in Atlantic salmon, a species with heteromorphic sex chromosomes (males being XY, females XX), and if all-male fish can be applied to further explore the vgll3 contribution on the likelihood of early maturation. RESULTS: Estrogen treatment of mixed sex yolk sac larvae gave rise to one sexually mature hermaphrodite with a male genotype (XY) that was used to produce both self-fertilized offspring and androgenetic double haploid (dh) offspring following egg activation with UV treated sperm and pressure shock to block the first mitotic division. There were YY supermales among both offspring types, which were crossed with dh females. Between 1 and 8% of the putative all-male offspring from the eight crosses with self-fertilized supermales were found to have ovaries, and 95% of these phenotypic females were also genetically female. None of the offspring from the one dh supermale cross had ovaries. When assessing the general contribution of the vgll3 locus on the likelihood of early post-smolt sexual maturation (jacking) in the all-male populations we found individuals that were homozygous for the early maturing genotype (97%) were more likely to enter puberty than individuals that were homozygous for the late maturing genotype (26%). However, the likelihood of jacking within individuals with an early/late heterozygous genotype was higher when the early allele came from the dam (94%) compared to the sire (45%). CONCLUSIONS: The present results show that supermale Atlantic salmon are viable and fertile and can be used as a research tool to study important aspects of sexual maturation, such as to further explore the sex dependent parental genetic contribution to age at puberty in Atlantic salmon. In addition, we report the production of viable double haploid supermale fish.

Effects of laboratory salmon louse infection on osmoregulation, growth and survival in Atlantic salmon.

Anadromous Atlantic salmon (Salmo salar) rely on long ocean migrations to build energy stores for maturation and spawning. In seawater, wild Atlantic salmon are threatened by high salmon lice (Lepeophtheirus salmonis) infestation levels resulting from intensive salmonid sea-cage aquaculture. Salmon lice infection can cause a stress response and an osmotic imbalance in the host. The lice infection intensity threshold values for these responses, however, remain to be identified in Atlantic salmon. In order to define this under laboratory conditions, individually tagged F1 wild origin Atlantic post-smolts (40 g) were infected with salmon lice copepodids or left as uninfected controls. Twenty-eight days post infection, infected post-smolts had a mean of 0.38 (range of 0.07-0.9) mobile lice g-1 fish weight. During this period, specific growth rates (SGRs) were lower in infected than control fish (0.4 vs 1.0% day-1). Higher plasma Na+, Cl- and osmolality in infected fish also indicate osmoregulatory impairment. SGR correlated negatively with plasma Na+, Cl-, osmolality and cortisol in the infected, but not in the control group. Infection intensity (lice g-1 fish) correlated positively with mortality rate and plasma Na+, Cl-, osmolality and cortisol and correlated negatively with SGR and condition factor. Calculated lice intensity threshold values for changes in plasma ions were 0.18 lice g-1 for plasma Cl-, and 0.22 lice g-1 for plasma Na+. Moribund infected fish occurred at infection intensities above 0.2 lice g-1, and these fish had extreme plasma Cl-, Na+, osmolality and cortisol levels. There was a positive correlation between plasma cortisol and plasma Na+, Cl- and osmolality in infected fish. This study provides vital information that can be used to define thresholds in the monitoring and conservation of wild Atlantic salmon populations affected by aquaculture-driven salmon lice infestations.

Termination of puberty in out-of-season male Atlantic salmon smolts.

Environmental conditions are known to contribute to the phenotypic plasticity in the age of sexual maturation of Atlantic salmon (Salmo salar). Here, we report on an observation of out-of-season male Atlantic salmon initiating puberty as pre-smolts (jacks) but failing to complete maturation as post-smolts. Jacks were identified based on elevated plasma 11-ketotestosterone (range, 3-12 ng/ml) and the occurrence of type B spermatogonia in January 2017. However, these males failed to show running milt as post-smolts at the expected time in May 2017. Subsequently, 6 out of the 21 (32%) suspected "terminated jacks" went on to become grilse, whereas only 1 of the 22 (5%) males that showed no signs of initiating puberty in January became grilse in December 2017. Therefore, "terminated" jacks were more likely to mature as grilse than the males that remained immature. Why these pubertal pre-smolt males did not complete maturation is unclear but could be related to the transfer of fish from conditions of warm water and long days, risk factors for early maturation, to conditions of cold water and short days, which are expected to delay the age of maturation. We provide a description of the conditions under which male Atlantic salmon appear to have terminated the process of sexual maturation.

Vertebral column deformity in adult wild haddock (Melanogrammus aeglefinus).

This study describes radiological malformations in the vertebral column of adult sexually mature wild haddock, Melanogrammus aeglefinus collected from Masfjorden, Western Norway, in 2014 and 2015. There were seven deformed fish (33% of sampled fish), five with mild deformities and two with severe deformities. The deformity types discovered were lordosis, vertebra body asymmetry, compression, fusion (ankylosis), remodelling, and haemal spine hyperostosis. The two severely deformed fish were as follows: (a) one specimen with 29 deformed vertebrae within the region vertebrae nos. 1-30, including two sites of lordosis; (b) one specimen with 15 deformed vertebrae within the region vertebrae nos. 31-52 (most caudal vertebrae). The results of this study show that wild haddock can survive severe vertebra deformities, both in the trunk and tail region, and reach adulthood and sexual maturity. Whether normal nondeformed wild conspecifics would mate and spawn with these individuals and thereby complete the life cycle of the deformed individuals is, however, unknown. A possible link between bone fracture and fish hyperostosis is discussed.

The frequency of spontaneous triploidy in farmed Atlantic salmon produced in Norway during the period 2007-2014.

BACKGROUND: Spontaneous triploidy has been reported in a number of fish species, and is often linked with in vivo or in vitro ageing of eggs post ovulation. Here, we provide the first investigation into the frequency of spontaneous triploidy in farmed Atlantic salmon by analysing more than 4000 fish from 55 farms, and approximately 1000 recaptured escapees, all sampled in the period 2007-2014. In addition, we compare microsatellite genotyping against flow cytometry and red blood cell diameter in a set of 45 putatively diploid and 45 putatively triploid Atlantic salmon. RESULTS: The three methods implemented for ploidy determination gave consistent results, thus validating the methods used here. Overall, 2.0% spontaneous triploids were observed in salmon sampled on farms. The frequency of spontaneous triploids varied greatly among sea cages (0-28%), but they were observed in similar frequencies among the three primary breeding companies (1.8-2.4%). Spontaneous triploids were observed in all farming regions in Norway, and in all years sampled. Spontaneous triploids were also observed among the escapees recaptured in both the marine environment and in rivers. CONCLUSIONS: Spontaneous triploidy in commercially produced Atlantic salmon is likely to be a result of the practices employed by the industry. For logistical reasons, there is sometimes a pause of hours, and in some cases overnight, between killing the female broodfish, removal of her eggs, and fertilization. This gives the eggs time to age post ovulation, and increases the probability of duplication of the maternal chromosome set by inhibition of the second polar body release after normal meiosis II in the oocyte.

Aplasia of the septum transversum has no effect on plasma biochemistry following an acute hypoxic event in Atlantic salmon.

Aplasia of the septum transversum (AST) is a malformation that results in alterations in ventricle morphology. The condition has been linked to increased mortality during periods of increased cardiac demand in Atlantic salmon Salmo salar. The blood plasma biochemical response to an acute hypoxic event (1 h at 31-39% O2saturation) was investigated in fish with and without a septum transversum to assess levels of anaerobic respiration (lactate) and the stress response (cortisol, glucose, osmolality, Na⁺, Cl⁻ and K⁺). AST had no effect on body size parameters or relative ventricular mass. The hypoxic event increased the levels of anaerobic respiration and induced a typical stress response. Contrary to our expectations, AST had no effect on any plasma parameter in normoxia or following severe acute hypoxia. We conclude that in the current scenario, AST does not affect the levels of anaerobic respiration or the plasma stress response in Atlantic salmon.