Role of the kidneys in acid-base regulation and ammonia excretion in freshwater and seawater fish: implications for nephrocalcinosis

Maintaining normal pH levels in the body fluids is essential for homeostasis and represents one of the most tightly regulated physiological processes among vertebrates. Fish are generally ammoniotelic and inhabit diverse aquatic environments that present many respiratory, acidifying, alkalinizing, ionic and osmotic stressors to which they are able to adapt. They have evolved flexible strategies for the regulation of acid-base equivalents (H+, NH4+, OH− and HCO3−), ammonia and phosphate to cope with these stressors. The gills are the main regulatory organ, while the kidneys play an important, often overlooked accessory role in acid-base regulation. Here we outline the kidneys role in regulation of acid-base equivalents and two of the key ‘urinary buffers’, ammonia and phosphate, by integrating known aspects of renal physiology with recent advances in the molecular and cellular physiology of membrane transport systems in the teleost kidneys. The renal transporters (NHE3, NBC1, AE1, SLC26A6) and enzymes (V-type H+ATPase, CAc, CA IV, ammoniagenic enzymes) involved in H+ secretion, bicarbonate reabsorption, and the net excretion of acidic and basic equivalents, ammonia, and inorganic phosphate are addressed. The role of sodium-phosphate cotransporter (Slc34a2b) and rhesus (Rh) glycoproteins (ammonia channels) in conjunction with apical V-type H+ ATPase and NHE3 exchangers in these processes are also explored. Nephrocalcinosis is an inflammation-like disorder due to the precipitation of calcareous material in the kidneys, and is listed as one of the most prevalent pathologies in land-based production of salmonids in recirculating aquaculture systems. The causative links underlying the pathogenesis and etiology of nephrocalcinosis in teleosts is speculative at best, but acid-base perturbation is probably a central pathophysiological cause. Relevant risk factors associated with nephrocalcinosis are hypercapnia and hyperoxia in the culture water. These raise internal CO2 levels in the fish, triggering complex branchial and renal acid-base compensations which may promote formation of kidney stones. However, increased salt loads through the rearing water and the feed may increase the prevalence of nephrocalcinosis. An increased understanding of the kidneys role in acid-base and ion regulation and how this relates to renal diseases such as nephrocalcinosis will have applied relevance for the biologist and aquaculturist alike.publishedVersio

Atlantic salmon male post-smolt maturation can be reduced by using a 3-hour scotophase when inducing smoltification

Photoperiod regulates the occurrence of unwanted male post-smolt maturation during the production of large (>100 g) Atlantic salmon (Salmo salar) smolts. However, the optimal daylength for triggering smoltification, but not male puberty, has yet to be established. We used either continuous light (24:0 light/dark) or long days (18:6 and 21:3) after a six week “winter” zeitgeber (12:12) to induce smoltification in fish of around 120 g reared at 16 °C. The fish were sampled 1, 2, 3, and 6 weeks after the initiation of the three different photoperiod treatments (n = 153 males in total with 9–18 males/photoperiod/time point). As expected, the smoltification indicator gill Na+/K+-ATPase (NKA) was significantly (p < 0.05) elevated and peaked 2 to 3 weeks after the initiation of the different photoperiods. Pubertal males were identified in all treatments via the combined use of relative testis size and histology, plasma 11-ketotestosterone, changes in body condition, and growth rate. The total incidence of puberty was significantly higher among males on continuous light at 33% (n = 16/49) compared to 10% (6/61) and 12% (5/43) in 21:3 and 18:6, respectively. The incidence of puberty increased over time in all photoperiods, with 62% (8/13), 19% (3/16), and 38% (3/8) of the males from 24:0, 21:3, and 18:6 pubertal at week 6, respectively. The mean weight of males that went on to initiate puberty was significantly higher (13%) at the beginning of the trial compared to those that remained immature (mean weight, 127 vs 112 g, respectively), but there was no initial difference in body condition. Puberty significantly reduced gill NKA by 35% compared to immature males at week six but had no effect at earlier time-points. Photoperiod had no effect on the female GSI, and they were all considered immature. In conclusion, the incidence of male puberty during smoltification is regulated by photoperiod and leads to an earlier decline in a key indicator of seawater readiness. As such, photoperiods with a short scotophase (21:3 or 18:6) following the winter zeitgeber in a square-wave (long-short-long day) smolt regime are recommended to limit the incidence of male puberty.publishedVersio

Increased Thermal Challenges Differentially Modulate Neural Plasticity and Stress Responses in Post-Smolt Atlantic Salmon (Salmo salar)

The successful transfer of farmed post-smolt Atlantic salmon (Salmo salar) depends on proper stress responses and cognitive functions during the early seawater (SW) phase. However, with increasing summer oceanic temperatures, these processes may become a challenge, implicating allostasis and welfare. Therefore, we examined the effect of post-smolt transfer from 10°C SW to elevated temperatures (13°C, 16°C, and 18°C) on plasma cortisol and telencephalic genes modulating cognition (neurod, bdnf, pcna, and c-fos) and stress-axis regulation (crf, crfbp, mr, gr1, gr2, and hsd11b2). Fish were sampled at i) 1 day following transfer, ii) 45 days of acclimation, and iii) 45 days and 1 h after an acute challenge test (ACT) using confinement stress. Fish transferred to 13°C retained stress responses, elevating levels of cortisol, crf, mr, gr2, c-fos, and bdnf and maintaining levels of neurod and pcna. Contrastingly, although cortisol increased at 16°C, telencephalic genes reverted to an inhibition of stress responses, increasing crfbp and gr1 complemented with dampened bdnf, neurod, and c-fos responses. However, transferring post-smolts to 18°C showed the most adverse effects, having absent stress responses (cortisol and c-fos), elevated crfbp, and a suppression of hsd11b2 and neurod. The hsd11b2 downregulation implies low cortisol inhibition in line with absent modulations in corticosteroid receptors and stress responses. These results suggest that the transfer to 16°C and 18°C inhibits the normal reactive response of post-smolts. Following acclimation (45 days), cortisol levels were basal for all groups; however, post-smolts at 16°C and 18°C maintained a telencephalic inhibition of key regulatory genes (crf, mr, gr2, and hsd11b2), alongside a lower mr/gr1 ratio, an indicator of chronic allostatic load. Moreover, neural plasticity (neurod and pcna) was suppressed at 16°C and 18°C, suggesting impacts of elevated allostatic loads with potentially inferior cognitive capacities. Despite maintaining similar plasma cortisol responses to ACTs, post-smolts at 16°C and 18°C elevated neural activation (c-fos) to stress, implying greater challenges, with the 18°C group also elevating the level of bdnf. In summary, the telencephalon shows that post-smolts transferred to 16°C and 18°C continue to struggle with the thermal allostatic loads even after acclimation, which is not revealed by plasma cortisol levels, grounding the importance of telencephalic measures in identifying environmental thresholds and hidden challenges.publishedVersio

Triploid Atlantic salmon and triploid Atlantic salmon × brown trout hybrids have better freshwater and early seawater growth than diploid counterparts

The use of reproductively sterile triploid salmonids would enhance the environmental sustainability of the aquaculture industry by preventing genetic exchange between escapees and wild conspecifics. To this end, we assessed smoltification and early seawater performance (241 days) following a yearling production cycle (i.e. spring smolts) in diploid and triploid female Atlantic salmon (Salmo salar) × male brown trout (Salmo trutta) hybrids compared to purebred diploid and triploid salmon. During freshwater rearing (n = 180/group), hybrids demonstrated a degree of bimodality in body size, significantly (p < 0.05) more so in diploid than triploid hybrids (11 and 37% in the lower mode, respectively) that was not seen in purebred salmon of either ploidy. This resulted in diploid hybrids being 66% smaller on average at sea transfer, whereas no hybridisation effect was seen in triploids, and both triploid groups were significantly heavier (16–43%) than diploid salmon. Irrespective of ploidy, lower mode hybrids grew poorly and showed low survival in seawater, suggesting they had failed to undergo smoltification. However, the upper mode diploid hybrids showed a similar Na+/K+-ATPase (NKA) enzyme activity surge during the spring as in diploid and triploid salmon, despite a higher ratio of the freshwater to seawater mRNA abundance of the NKA subunits (nkaα1a and nkaα1b) and a reduced plasma cortisol surge. At the end of the experimental period, both hybrids weighed significantly less than their salmon counterparts although the hybrid effect was again greater in diploids (71% smaller) than triploids (6% smaller). In addition, both triploid groups were on average heavier (15–22%) than diploid salmon. As such, both triploid Atlantic salmon and triploid hybrids can show enhanced growth performance from juveniles up to post-smolts compared to diploid salmon in an aquaculture setting.publishedVersio

Triploid atlantic salmon (Salmo salar L.) postsmolts accumulate prevalence more slowly than diploid salmon following bath challenge with salmonid alphavirus subtype 3

Triploid Atlantic salmon (Salmo salar L.) may play an important role in the sustainable expansion of the Norwegian aquaculture industry. Therefore, the susceptibility of triploid salmon to common infections such as salmonid alphavirus (SAV), the causative agent of pancreas disease (PD), requires investigation. In this study, shortly after seawater transfer, diploid and triploid post-smolts were exposed to SAV type 3 (SAV3) using a bath challenge model where the infectious dose was 48 TCID50 ml-1 of tank water. Copy number analysis of SAV3 RNA in heart tissue showed that there was no difference in viral loads between the diploids and triploids. Prevalence reached 100% by the end of the 35-day experimental period in both infected groups. However, prevalence accumulated more slowly in the triploid group reaching 19% and 56% at 14 and 21 days post exposure (dpe) respectively. Whereas prevalence in the diploid group was 82% and 100% at the same time points indicating some differences between diploid and triploid fish. Both heart and pancreas from infected groups at 14 dpe showed typical histopathological changes associated with pancreas disease. Observation of this slower accumulation of prevalence following a natural infection route was possible due to the early sampling points and the exposure to a relatively low dose of virus. The triploid salmon in this study were not more susceptible to SAV3 than diploid salmon indicating that they could be used commercially to reduce the environmental impact of escaped farmed fish interbreeding with wild salmon. This is important information regarding the future use of triploid fish in large scale aquaculture where SAV3 is a financial threat to increased production.publishedVersio

Triploidy leads to a mismatch of smoltification biomarkers in the gill and differences in the optimal salinity for post-smolt growth in Atlantic salmon

Sterile triploid Atlantic salmon (Salmo salar) show inconsistent seawater grow-out, but the reason why remains unclear. The purpose of this study was to determine the salinity optima of triploid post-smolts. Diploids and triploids were assessed for smoltification status during an underyearling smolt regime before being transferred to one of four different salinities, 0, 11, 23 and 35 ppt at 12 °C and under 24 h continuous light for 83 days. During this period, fish growth, plasma biochemistry, and production traits (vertebral deformities, ocular cataracts, sexual maturation) were monitored. Molecular biomarkers in the gill (nkaα1a, nkaα1b, nkcc1a) suggested triploids reached peak smolt earlier than diploids and began the desmoltification process before the start of the salinity treatments, however this was not reflected in gill Na+/K+-ATPase enzyme activity. At the initiation of the salinity treatments triploids were significantly larger than diploids (mean weight g ± SE: 71 ± 0.7 and 87.2 ± 0.8 for diploids and triploids, respectively) and there was a ploidy effect on post-smolt growth, with body weight showing a clearer positive trend with salinity in diploids (0 < 11 = 23 = 35 ppt) than in triploids (0 < 11 < 35 = 23 ppt) (final mean weight g ± SE: 255.2 ± 7.4, 303.9 ± 9, 313.9 ± 9 and 342.4 ± 12 for diploids and 322.9 ± 9.7, 361.7 ± 10.7, 425.9 ± 12.1, 415.2 ± 12.2 for triploids at 0, 11, 23, and 35 ppt, respectively). Plasma Na+ and Cl− increased, but plasma pH decreased, with increasing salinity in both ploidy. However, ploidy only had transient effects on plasma biochemistry depending on the salinity treatment. There was no ploidy effect on vertebral deformities (21% of both ploidy had one or more deformed vertebra). In contrast, triploids had a significantly higher prevalence of ocular cataracts (84 vs 98% in diploids and triploids, respectively) with a higher mean cataract score (mean ± SE: 1.93 ± 0.1 and 2.78 ± 0.1 for diploids and triploids, respectively), but a significantly lower prevalence of pubertal male post-smolts (15 vs 2% in diploids and triploids, respectively). Salinity treatment had no effect on vertebral deformities, cataracts, or post-smolt sexual maturation. In summary, there was a ploidy mismatch for smoltification biomarkers in the gill and salinity had a strong effect on post-smolt growth, but the effects were ploidy dependent.publishedVersio

Validating timing of salmon smolt runs obtained by telemetry studies

Telemetry tags are increasingly used in management to monitor the migration timing of Atlantic salmon smolts (Salmo salar L.). It has been postulated that (1) effects from tagging and handling may alter migration behaviour, and (2) that the selection of fish during sampling is not representative of all migrating smolts, yielding bias in migration timing estimates. In the River Dale (Vestland, Norway), five groups of wild Atlantic salmon smolts (N = 385) were tagged in April–May and recaptured in a wolf trap. Migration timing was then compared to the untagged population. Migration timing differed between tagged (12 mm PIT) and untagged fish for 4/5 groups. Only fish tagged at the first time point did not have significantly different timing of migration from untagged counterparts. The relationship between length and the timing of migration was different for initial length and length at recapture; initial length suggested earlier migration of longer fish, but the extra time spent in the river prior to migrating for smolts that were initially smaller compensated for the size difference. The tagging protocol is crucial to obtaining representative migration timing results. Smolts should be tagged as early as possible and include the entire size distribution.publishedVersio

Exposure to cold temperatures differentially modulates neural plasticity and stress responses in post-smolt Atlantic salmon (Salmo salar)

The transfer success of farmed post-smolt Atlantic salmon (Salmo salar) to sea-cages rely on neural adaptions to promote stress resilience. As low temperatures impact physiology, this suggests that off-season transfer to cold waters may be challenging. To address this, post-smolts reared at 13 °C seawater were abruptly transferred to 10 °C, 7 °C, and 4 °C, then acclimated to these respective temperatures for 58-days followed by an acute challenge test (ACT) using confinement stress. Plasma and brain samples were collected after i) the abrupt temperature transfer at 1-h and 1-day, ii) 58-days of acclimation, and iii) 1-h post ACT. In tandem to measuring plasma cortisol levels, the expression of key genes involved in telencephalic regulation (crf, crfbp, mr, gr1, gr2 and hsd11b2) and neural plasticity (neurod, bdnf, pcna, and cfos) were analyzed. Post-smolts exposed to the 7 °C and 4 °C displayed the largest alteration in telencephalic functions, differentially regulating mr and gr1, to elevate the mr/g1 ratio for downregulating Gr1, proposing an elevated stress loads. After acclimation, these coincided with blunted stress responses capacities to ACTs for both cortisol and telencephalic neural activity (cfos), suggesting a continuation of challenges and reduced the capacity to mount a stress response. Concomitantly, these telencephalic alterations in CRs coincided with a differential modulation in neural plasticity, measured as elevated bdnf and neurod during the abrupt transfer period (acute) and after acclimation (prolonged), respectively, revealing neural responses are still robustly maintained to retain a degree of stress resilience. However, exposure of post-smolts to 4 °C clearly induced the most adverse and suppressive effects in telencephalic functions, cued by a suppression in pcna and stress response capacities, downregulation in the CRF system, and largest elevation in the mr/g1 ratio. Conversely, acclimating post-smolts to 7 °C elevated 11hsdb2 proposing a greater inhibition of cortisol action that may point to still adequate maintenance of CR and neural processes. Taken together, these findings show that cold temperatures alter key neural processes required for maintaining proper stress management, providing an alternative explanation for reductions in fish stress reactivity commonly observed with declining temperature. Therefore, exposing post-smolts at 13 °C to temperature reductions of 6 °C or greater should be avoided in aquaculture.publishedVersio

Expression and localization of the aryl hydrocarbon receptors and cytochrome P450 1A during early development of Atlantic cod (Gadus morhua)

The aryl hydrocarbon receptor (Ahr) is a ligand-activated transcription factor that mediates the toxicity of dioxins and dioxin-like compounds (DLCs) in vertebrates. Two clades of the Ahr family exist in teleosts (Ahr1 and Ahr2), and it has been demonstrated that Ahr2 is the main protein involved in mediating the toxicity of dioxins and DLCs in most teleost species. Recently, we characterized the Atlantic cod (Gadus morhua) Ahr1a and Ahr2a receptors. To further explore a possible subfunction partitioning of Ahr1a and Ahr2a in Atlantic cod we have mapped the expression and localization of ahr1a and ahr2a in early developmental stages. Atlantic cod embryos were continuously exposed in a passive-dosing exposure system to the Ahr agonist, benzo[a]pyrene (B[a]P), from five days post fertilization (dpf) until three days post hatching (dph). Expression of ahr1a, ahr2a, and the Ahr-target genes, cyp1a and ahrrb, was assessed in embryos (8 dpf and 10 dpf) and larvae (3 dph) with quantitative real-time PCR analyses (qPCR), while in situ hybridization was used to assess the localization of expression of ahr1a, ahr2a and cyp1a. Quantitative measurements showed an increased cyp1a expression in B[a]P-exposed samples at all sampling points, and for ahr2a at 10 dpf, confirming the activation of the Ahr-signalling pathway. Furthermore, B[a]P strongly induced ahr2a and cyp1a expression in the cardiovascular system and skin, respectively, of embryos and larvae. Induced expression of both ahr2a and cyp1a was also revealed in the liver of B[a]P-exposed larvae. Our results suggest that Ahr2a is the major subtype involved in mediating responses to B[a]P in early developmental stages of Atlantic cod, which involves transcriptional regulation of biotransformation genes, such as cyp1a. The focused expression of ahr1a in the eye of embryos and larvae, and the presence of ahr2a transcripts in the jaws and fin nodes, further indicate evolved specialized roles of the two Ahrs in ontogenesis.publishedVersio

Comparison of anadromous and landlocked Atlantic salmon genomes reveals signatures of parallel and relaxed selection across the Northern Hemisphere

Most Atlantic salmon (Salmo salarL.) populations follow an anadromous life cycle, spending early life in freshwater, migrating to the sea for feeding, and returning to rivers to spawn. At the end of the last ice age similar to 10,000 years ago, several populations of Atlantic salmon became landlocked. Comparing their genomes to their anadromous counterparts can help identify genetic variation related to either freshwater residency or anadromy. The objective of this study was to identify consistently divergent loci between anadromous and landlocked Atlantic salmon strains throughout their geographical distribution, with the long-term aim of identifying traits relevant for salmon aquaculture, including fresh and seawater growth, omega-3 metabolism, smoltification, and disease resistance. We used a Pool-seq approach (n = 10-40 individuals per population) to sequence the genomes of twelve anadromous and six landlocked Atlantic salmon populations covering a large part of the Northern Hemisphere and conducted a genomewide association study to identify genomic regions having been under different selection pressure in landlocked and anadromous strains. A total of 28 genomic regions were identified and includedcadm1on Chr 13 andppargc1aon Chr 18. Seven of the regions additionally displayed consistently reduced heterozygosity in fish obtained from landlocked populations, including the genes gpr132, cdca4, and sertad2 on Chr 15. We also found 16 regions, includingigf1on Chr 17, which consistently display reduced heterozygosity in the anadromous populations compared to the freshwater populations, indicating relaxed selection on traits associated with anadromy in landlocked salmon. In conclusion, we have identified 37 regions which may harbor genetic variation relevant for improving fish welfare and quality in the salmon farming industry and for understanding life-history traits in fish.Peer reviewe