Understanding postsmolt maturation in Atlantic salmon in the context of new, closed production systems

During the recent years production of farmed Atlantic salmon has changed. While the production still is divided in two phases, first in tanks on land followed by cages in seawater, the production time in tanks and fish size at transfer from tanks to cages have increased. This is mainly due to the negative impacts imposed by the parasitic salmon louse in sea-cage aquaculture. With the increase in production volume and fish size on land, the amount of fish produced in recirculated heated water has also increased. This has created new challenges in form of more un-wanted sexual maturation, especially in males, where fish typically mature after reaching only a few hundred grams in body size. This type of maturity reduces survival, growth and welfare. Earlier studies have shown that the vgll3 locus is important for the determination of age at sexual maturity in adult Atlantic salmon. There are two variants of vgll3, one for low age at maturation (early) and one for high age at maturation (late). How vgll3 impact of early maturity when salmon are farmed in heated water in tanks is, however, unknown. The pituitary gland is a key endocrine organ in the regulation of sexual maturation in salmon. Follicle stimulation hormone (fsh) is produced and released by the pituitary. The role of this key puberty hormone in the regulation of early maturity is unknown. To gain research on early maturity in salmon and the role of vgll3 and the pituitary, this project is tailored to develop methods to: (i) induce early maturity in salmon males; (ii) produce all-male salmon with different vgll3 genotypes; (iii) keep pituitaries in tissue culture; (vi) measure fsh protein level; (v) gene-edit salmon to knock out the fsh receptor. In salmon, males have two different sex chromosomes (X,Y), while females have two equal chromosomes (X,X), and the sex ratio is normally 50/50. To effectively study sexual maturity in male salmon we developed, for the first time, a method to produce all-male populations with different vgll3 genotypes. These were produced by fertilizing eggs with sperm from supermales. Supermales have 2 Y chromosomes instead of the normal XY, and therefore produces all-male offspring (XX x YY = XY). In parallel with this activity, we developed a method to induce early maturity in salmon males. In this model were the fish are reared at 16C under continuous light (LL), we often observe >90% male maturity. We tested the all-male populations in this model and found that males with the early vgll3 genotype developed 100% maturation, while those with late developed 25% maturation. We produced new all-male populations the following year and found 90% maturation in the early vgll3 genotype and 41% maturation in the late at 16C/LL. This shows that the early vgll3 genotype is a risk factor for early un-wanted maturity in farmed salmon. We have developed a method (ELISA) to measure fsh level. The sensitivity of the method is 0.8 ng/ml, and the standard curve is linear between 1 and 50 ng/ml. Plasma values in salmon vary between <10 ng/ml (juveniles) and 80 ng/ml (sexual mature). We have also developed a method to keep salmon pituitaries alive for up to 9 days in tissue culture. Salmon were reared at 16C/LL and pituitaries from both mature and immature males were dissected out and put into culture. When potential regulators of fsh were added, we found the strongest response on fsh gene expression in pituitaries from immature males. This indicates that fsh was already activated in the pituitaries from mature males. The fsh ELISA was used to determine how much fsh the pituitaries released into the tissue culture medium, and the analysis showed that fsh was only released in cultures with pituitaries from immature males. I addition to the tissue culture experiments, we did RNA sequencing of pituitaries from male reared at 16C/LL. 152 genes were differentially expressed in immature vs. maturing individuals. ~2/3 of the genes were up-regulated, e.g. fsh and other pituitary hormones, and other known steroid and puberty related genes. We established a fshr (fsh receptor) knock out (KO) salmon mutant using CRISPR-Cas9 technology. To examine sexual maturity in males, fshr KO and normal (NO) salmon were reared in common garden at 16C/LL for 3 months followed by natural light and temperature for a further 6 months. We sampled fish 1, 2, 5 and 9 months after commencing exposure to 16C/LL. In months 5 and 9, we observed slightly (5 months) and clearly (9 months) smaller gonads and lower plasma steroid level in KO compared NO males. The results indicate that the maturation cycle was shortened in fshr KO mutants. One year later, a F1 generation was created by crossing fshr KO fish (2 KO males crossed with 3 KO females). In the spring 2020, the F1 generation was subjected 16C/LL to stimulate maturity. Sampling is ongoing. Preliminary results show that KO males do not mature while NO males mature.

Det har vært store endringer i hvordan en oppdretter laks de siste årene. Mens produksjon fremdeles er todelt med en fase på land i kar etterfulgt og en fase i merd i sjøvann, har størrelsen på fisken ved flytting fra kar til merd økt. I tillegg blir mer fisk produsert i resirkulert oppvarmet vann i karfasen. Begge disse endringene har gitt mer tidlig uønsket kjønnsmodning, spesielt hos hannfisk. Tidlig kjønnsmodning gir redusert vekst, overlevelse og velferd. I dette prosjektet har vi vist at hannlaks kan begynne å kjønnsmodne samtidig som den smoltifiserer dersom den oppdrettes ved kontinuerlig belysning i oppvarmet vann. Smoltifisering er prosessen der fisken forbereder seg til overgangen fra fersk- til saltvann. Siden smoltmodning vil kunne gi negative konsekvenser har vi i dette prosjektet også utviklet flere viktige forskningsverktøy for videre studier innen kunnskapsfeltet. Dette vil kunne sikre gode produksjonsresultater samtidig som fiskevelferd blir ivaretatt.

With the increasing incidence of unwanted pubertal development in male Atlantic salmon post-smolts raised in new production systems, it is crucial to understand the physiological mechanisms triggering this development, in order to gain knowledge on how to avoid entrance into precocious puberty. This knowledge can serve as a resource for sustainable salmon farming, aiding in the development of best practice knowledge for preventing early puberty in new production systems. Previous work in fish identified the pituitary hormone, follicle-stimulating hormone (Fsh), as the major factor triggering entry into and completion of puberty. Detailed knowledge about the regulation of Fsh synthesis and secretion is therefore of decisive relevance for understanding the regulation of puberty, but is rather fragmentary in fish. The proposed project combines recent methodological developments (e.g. availability of clonal salmon lines; deep sequencing of coding and non-coding [pituitary] RNAs in the context of the recently published, annotated salmon genome) to make significant progress in studies on the epigenetic, transcriptional and post-transcriptional control of Fsh production and release. The data obtained will be highly relevant for salmon production biology and of high scientific quality, by providing both, practically relevant and scientifically original information on the biological basis of the plasticity of Atlantic salmon reproductive physiology.