High water temperature is among the main factors causing unwanted sexual maturation of male Atlantic salmon postsmolts in modern aquaculture facilities. The present study tested if a sudden drop in temperature from 15 degrees C to 8 degrees C, applied right before or after a 5-week LD12:12 winter signal regime (WS), would arrest or halt the maturation process previously stimulated by a period at high temperature (15 degrees C). Atlantic salmon parr (n = 900, initial mean weight = 51.8 + 7.3 g) were reared from 2 June to 3 November 2022 in a freshwater flow-through system under four temperature regimes: at 15.1 + 0.7 degrees C (constant 15 degrees C); at 8.1 + 0.7 degrees C (constant 8 degrees C); at 14.9 + 0.9 degrees C until 31 July, followed by 7.8 + 0.6 degrees C until the endpoint (15-8E, E for "Early"); and at 15.0 + 0.7 degrees C until 6 September, followed by 8.2 + 0.5 degrees C until the endpoint (15-8L, L for "Late"). The WS lasted from 31 July to 6 September in all treatments. Morphology (body weight, condition factor), maturation indicators that included gonadosomatic index (GSI), testis histology, and pituitary transcription of gonadotropin-releasing hormone receptor (gnrhr2bb alpha), follicle-stimulating hormone (fshb) and luteinizing hormone (lhb), as well as smoltification markers (gill Na+, K +-ATPase activity, plasma cortisol) were assessed periodically (9 samplings). At constant 15 degrees C, males displayed the highest growth and a synchronized onset of sexual maturation in response to the photoperiod cue, resulting in 100% of them maturing while displaying poor signs of hypo-osmoregulatory ability. In contrast, at constant 8 degrees C salmon grew the least, showed virtually no signs of maturation and the best signs of seawater tolerance. In the two treatments where temperature dropped from 15 degrees C to 8 degrees C, markers suggested that the maturation process also started in response to the increase in daylength, but in much lower proportion than at constant 15 degrees C. In these two treatments, the proportions of maturation and physiological changes were dependent on the duration of the early period at 15 degrees C (46 % maturing in 15-8L in November, and 24 % in 15-8E). The temperature drop also induced a reduction in growth and better signs of seawater tolerance in comparison to constant 15 degrees C. In addition, in a number of males that had initiated maturation at 15 degrees C "spontaneously" (only in response to internal triggers and not to increasing daylength), the drop in temperature appeared to induce a switch in testes from proliferative to meiotic and spermiogenic phases. All considered, our results suggest that a drop in temperature is unlikely to arrest the maturation process if the decision has been previously stimulated by a period at high temperature. The temperature drop can simply delay the rate at which the process develops if fish have not yet initiated spermatogenesis, but not removing its occurrence, or induce a switch towards late spermatogenesis phases if fish are already engaged in the process. This evidences the high risks that using high temperature represents for salmon producers at different stages during smolt production.

Long-term storage of extracted DNA, RNA, and samples for DNA and RNA extractions is usually done in ultra-low temperature freezers using the standard temperature of −80°C. While this standard was based on the maximum capacity of early generation ultra-low temperature freezers, this paradigm is challenged and initiatives support a switch to −70°C to save energy, reduce heat production, and increase the life expectancy of the freezers. The question arising from these initiatives regards the safety of the samples. Especially in complex biological samples, such as sediments, changes in long-term storage temperature have not been studied in detail. Here, we show that the concentration of extracted nucleic acids and nucleic acids in tissue or cells stored at both temperatures does not differ significantly from each other. The only significant differences found were explained by the variability within the samples over time but not between different temperatures or by dilution factor. In addition, we show that prokaryote community composition in sediment and DNA samples also remain stable at both temperatures. Only two treatments were significantly different in temperature, indicating that for RNA, storage at −70°C might be preferable. Consequently, we recommend storing samples for nucleic acid work at −70°C to reduce energy consumption and support more sustainable lab practices.

Maturation of Atlantic salmon male postsmolts is a concern in aquaculture due to its increasing occurrence under intensive rearing conditions and its negative impact on growth, welfare and seawater readiness. The effect of temperature and photoperiod on maturation was assessed in male postsmolts kept in freshwater. We used a 2 x 2 factorial design with two temperatures (12.5 and 15 degrees C) and two photoperiods (a group in continuous light LD24:0 or LL, and another receiving a 5-week LD12:12 winter signal or WS). Salmon in the four resulting treatments (1000 parr, initial mean weight 52.1 +/- 5.2 g) were reared in a flow-through system from 28 October 2019 to 30 May 2020. Morphology (body weight, condition factor), maturation indicators including gonadosomatic index (GSI), plasma 11-Ketotestosterone (11-KT), pituitary follicle-stimulating hormone beta-subunit (fshb) and luteinizing hormone beta-subunit (lhb) transcript levels, as well as smoltification markers (Na+, K+- ATPase activity) were assessed. Results revealed that rearing salmon at 15 degrees C was the most important factor promoting early maturation, leading to 100% of males maturing in late May in 15-WS, and 75% in 15-LL. However, the groups receiving a winter signal (WS) displayed a highly synchronized onset and progression of maturation specially at 15 degrees C, revealed by the low variability observed among individuals in GSI and fshb transcription after the WS. This evidences the role of the photoperiod switch from short to long day as zeitgeber for sexual maturation. On the contrary, under constant light (LL), entry into maturation was not synchronized among individuals, and onset of maturation occurred spontaneously in a proportion of males highly dependent upon temperature (75% in 15-LL, 25% in 12.5-LL). Signs of smoltification were poor at both temperatures, and the WS did not induce development of hypo-osmoregulatory abilities in any case. This suggests that a winter signal may not induce smoltification if introduced at high temperature or when fish have reached large size, and instead may increase the risk of a sexual maturation response. These findings are relevant for the aquaculture industry, since similar rearing conditions are currently used in the industry, including constant high water temperature and winter signal regimes. The use of such conditions can increase the risk of early maturation, as well as of poor hypo-osmoregulatory performance.

Early maturation of Atlantic salmon male postsmolts is undesirable in aquaculture due to its negative impact on growth, welfare and seawater adaptation, however it is an increasing problem under intensive rearing condi-tions. The effect of temperature and feeding ration on sexual maturation during the induction of smoltification was tested with a 3 x 2 factorial design including three temperatures (8, 12.5 and 18 degrees C) and two feeding rations (100% and 67%), from 28 October 2018 to 30 May 2019. Fish in the six resulting treatments (1800 parr, initial mean weight 23.1 +/- 7.2 g) were reared in flow-through under continuous light (LD24:0) except for a 5-week winter signal (LD12:12) introduced on 4 February 2019 to induce smoltification. A major effect of tempera-ture determining the life history pattern of each group was observed. At 18 degrees C, a majority of the males were maturing in May under both rations (100% males in 18-100% and 88.9% males in 18-67%). Fish at 18 degrees C experienced an early activation of the BPG (Brain-Pituitary-Gonad) axis and displayed very poor signs of smoltification. At 12.5 degrees C, early maturation was also present, although at lower proportion than at 18 degrees C and more dependent upon feed ration (40% of males maturing in 12.5-100%, 8.6% in 12.5-67%). Salmon at 12.5 degrees C displayed the best growth within each feed regime, some early signs of smolting but subsequent low Na+, K+- ATPase activity after the winter signal. At 8 degrees C, salmon showed best signs of smoltification and lowest growth, while no males matured. The feed restriction had a minor effect on maturation at 18 degrees C but a greater impact at 12.5 degrees C, evidenced by significant differences between 12.5-100% and 12.5-67% in gonadosomatic index, plasma 11-ketotestosterone, insulin-like growth factor-I, and pituitary fshb (follicle stimulating hormone beta-subunit gene) transcription. Onset of maturation was associated with the photoperiod change from short to long daylength, which probably also induced a slight activation of the BPG axis in non-maturing groups. Our results suggest that postsmolt producers must be cautious when using sustained high water temperatures, or the risk of early maturation and poor hypo-osmoregulatory performance will increase significantly. In addition, reducing the feeding ration will not help decrease incidence of maturation without significantly affecting growth.

In Atlantic salmon (Salmo salar) seasonal photoperiod is shown to regulate the onset of sexual maturation, yet which brain region(s) are involved and how light information impacts the neuroendocrine system are still not fully understood in teleosts. Detailed knowledge about the photoperiodic regulation of maturation in fish is still missing. In birds, it is shown that gonadotrophin releasing hormone (GNRH) is located in the same neurons as vertebrate ancient (VA) opsin suggesting a direct photoreceptive regulation for onset of sexual maturity. This study presents a comprehensive topographic mapping of gnrh2, gnrh3, kisspeptin 2 (kiss2) gonadotrophin inhibiting hormone (gnih) and VA opsin using in situ hybridization on mature Atlantic salmon brains. Neurons positive for gnrh3 are expressed in the olfactory bulb and ventral telencephalon while gnrh2 positive neurons are located dorsally in midbrain tegmentum. Gnih expressing cell bodies are present in the ventral thalamus and extend caudally to the hypothalamus with kiss2 expressing cells appearing in a lateral position. VA opsin positive cells are present in the telencephalon, the rostro-dorsal ring of left habenula, the ventral thalamus and the midbrain tegmentum. The results show no similar co-location as found in birds, hypothesizing that the photoreceptive modulation of Gnrh in salmon may interact through neuronal networks. The topography analyses of the essential neuroendocrine cells related to sexual maturation in Atlantic salmon brain show that diencephalic (thalamus, hypothalamus) and midbrain (tegmentum) regions seem central for controlling sexual maturation.

The effect of photoperiod manipulation on growth, smoltification and maturation was assessed in Atlantic salmon through an observational study performed in a commercial RAS facility from June (25.0 +/- 11.0 g) to October 2018 (151.1 +/- 25.5 g). Half of the commercial cohort 721 was raised in continuous light LD24:0 (LL) while the other received a 6-week LD12:12 winter signal (WS) for smoltification from 12 August to 21 September. Parameters related to growth (body weight, gene expression of pituitary gh1 and gh2, and liver ghr1, igf1 and igfbp1a), smoltification (condition factor, plasma sodium and cortisol, gill NKA activity and nka1a, nka1b and nkcc1a expression) and maturation (GSI and pituitary fshb and lhb expression) were analysed. Afterwards, a multivariate analysis was performed on production data from five commercial cohorts raised in the facility (including 721) to identify variables potentially linked to early maturation in RAS. Results from the observational study indicated weak compensatory growth and slightly better smoltification in WS, although signs of size-induced smoltification were present in LL. Smoltification indicators were poor in both treatments, suggesting that smolts may not be yet ready for seawater. No maturation was observed in any photoperiod treatment; however, the multivariate assessment suggested that such lack of maturation might be rather linked to the low mean temperature and SGR experienced by our cohort.

Seasonal timing is important for many critical life history events of vertebrates, and photoperiod is often used as a reliable seasonal cue. In mammals and birds, it has been established that a photoperiod-driven seasonal clock resides in the brain and pituitary, and is driven by increased levels of pituitary thyroid stimulating hormone (TSH) and brain type 2 iodothyronine deiodinase (DIO2), which leads to local increases in triiodothyronine (T3). In order to determine if a similar mechanism occurs in fish, we conducted photoperiod manipulations in anadromous (migratory) Atlantic salmon (Salmo salar) that use photoperiod to time the preparatory development of salinity tolerance which accompanies downstream migration in spring. Changing daylength from short days (light:dark (LD) 10:14) to long days (LD 16:8) for 20 days increased gill Na+/K+-ATPase (NKA) activity, gill NKAα1b abundance and plasma growth hormone (GH) levels that normally accompany increased salinity tolerance of salmon in spring. Long-day exposure resulted in five-fold increases in pituitary tshβb mRNA levels after 10 days and were sustained for at least 20 days. tshβb mRNA levels in the saccus vasculosus were low and not influenced by photoperiod. Increased daylength resulted in significant increases in dio2b mRNA levels in the hypothalamus and midbrain/optic tectum regions of the brain. The results are consistent with the presence of a photoperiod-driven seasonal clock in fish which involves pituitary TSH, brain DIO2 and the subsequent production of T3, supporting the hypothesis that this is a common feature of photoperiodic regulation of seasonality in vertebrates.

Smoltification is a metamorphic event in salmon life history, which initiates downstream migration and pre-adapts juvenile salmon for seawater entry. While a number of reports concern thyroid hormones and smoltification, few and inconclusive studies have addressed the potential role of thyrotropin (TSH). TSH is composed of a α-subunit common to gonadotropins, and a β-subunit conferring hormone specificity. We report the presence and functional divergence of duplicated TSH β-subunit paralogs ( tshβa and tshβb ) in Atlantic salmon. Phylogeny and synteny analyses allowed us to infer that they originated from teleost-specific whole genome duplication. Expression profiles of both paralogs in the pituitary were measured by qPCR throughout smoltification in Atlantic salmon from the endangered Loire-Allier population raised in a conservation hatchery. This revealed a striking peak of tshβb expression in April, concomitant with downstream migration initiation, while tshβa expression remained relatively constant. In situ hybridization showed two distinct pituitary cell populations, tshβa c ells in the anterior adenohypophysis, and tshβb cells near to the pituitary stalk, a location comparable to the pars tuberalis TSH cells involved in seasonal physiology and behaviour in birds and mammals. Functional divergence of tshβ paralogs in Atlantic salmon supports a specific role of tshβb in smoltification.

Suboptimal egg incubation temperature is a risk factor for the development of skeletal deformities in teleosts. Triplicate diploid and triploid Atlantic salmon, Salmo salar L., egg batches were incubated at 6, 8 and 10 °C up until first feeding, whereupon fish were reared on a natural temperature before examination for externally visible skeletal deformities (jaw and spine) and radiographed for vertebral deformities and morphology at the parr stage. Increasing incubation temperatures and triploidy increased the number of fish showing one or more deformed vertebrae. Triploids had significantly higher mean vertebrae cranio-caudal length (L) and dorsal-ventral height (H) ratio at 6 and 10 °C than diploids, but triploidy had no effect on mean vertebrae centra area. Triploids demonstrated an increase in lower jaw deformities with increased incubation temperature, whereas jaw deformities were rare in diploids. Fish incubated at 10 °C had a significantly lower mean vertebral number than fish incubated at 6 °C, and triploids had lower mean vertebral numbers than diploids. Diploid fish with 58 vertebrae had a significantly higher mean vertebral centra area than fish with 59 vertebrae, but vertebral number did not affect the mean vertebral L/H ratio. The results are discussed with respect to the welfare and production of farmed salmonids.

Heart deformities are a concern in aquaculture and are linked to egg incubation temperature. Diploid and triploid Atlantic salmon, Salmo salar L., were incubated at 6, 8 and 10 °C and analysed for aplasia of the septum transversum (n = 150 ploidy⁻¹ incubation temperature⁻¹). Heart morphology (size and shape) was assessed in fish incubated at 6 °C and in fish with and without aplasia of the septum transversum (n = 9 group⁻¹) incubated at 10 °C. Egg mortality was significantly higher in triploids than in diploids at all incubation temperatures, and increased egg incubation temperatures increased mortality in both ploidy. Triploids grew quicker than diploids after egg incubation at 10 °C, but not at 6 °C. Aplasia of the septum transversum occurred only in triploid fish after incubation at 6 °C and 8 °C (0.7% and 3.3%, respectively) and was significantly greater (P ≤ 0.05) in triploids after incubation at 10 °C compared with diploids (30% and 18%, respectively). Aplasia of the septum transversum significantly increased heart mass and resulted in a long flat ventricle compared with fish displaying a septum transversum. The results suggest triploid salmon should be incubated below 8 °C.

This study examined how triploidization and species hybridization affects skeletal related meristic characteristics in salmonids with the goal of understanding the maternal and paternal contribution. The study also investigated the hybrid sea louse infection rate in hybrid salmonids. In order to do so, a number of vertebrae, scales along the lateral line, and dorsal fin rays were measured in diploid and triploid Atlantic salmon, Arctic char, and Atlantic salmon (female) X Arctic char (male) hybrids. The success of triploidization was 100%, and some spontaneous triploid individuals (similar to 15%) were found among the hybrids. In general, hybrids displayed intermediate counts for all three characteristics with salmon displaying the highest number of dorsal fin rays, and char displaying the highest number of scales and vertebrae. The effect of triploidization was always strongest among the hybrids. However, the direction of the effect differed between structures in the hybrid; triploidization increased the number of vertebrae and reduced the number of fin rays towards the level observed in diploid char, and reduced the number of scales towards the level observed in diploid salmon. We conducted a second experiment whereby diploid Atlantic salmon and Atlantic salmon (female) X Arctic char (male) hybrids were exposed to an experimental challenge with the parasitic salmon louse (Lepeophtheirus salmonis). There was no difference in the sea-lice infection level between Atlantic salmon and the hybrids. This study shows that the present model with diploid and triploid Atlantic salmon (female) X Arctic char (male) hybrids may be a useful tool for the study of which traits are maternally and paternally inherited in salmonids. The understanding of morphological development with regard to meristic traits could be advantageous for both hybrid and triploid fish domestication.