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- Modulation of intestinal growth and differentiation by photoperiod and dietary treatment during smoltification in Atlantic salmon (Salmo salar, L.)Publication . Fernandes Duarte, Vilma Vanessa; Gaetano, Pasqualina; Striberny, Anja; Hazlerigg, David; Jørgensen, Even H.; Fuentes, Juan; Campinho, Marco AntónioAtlantic salmon undergo smoltification, a process that prepares the fish to enter and thrive in seawater (SW). Several physiological changes occur during smolting, especially in osmoregulatory tissues, the gill, the kidney, and the intestine. Here we characterized the effects on intestinal morphogenesis of two different, commonly used smoltification regimes during the end of the freshwater phase, photoperiod and/or the addition of salt and amino acid supplements in the diet. We focused on intestinal morphological differentiation, i.e., external perimeter, absorptive perimeter, tissue thickness, and villi density. In addition, we quantified cell proliferation (PCNA positive) and Na+, K+-ATPase (NKA) and Na+, K+,2Cl- (NKCCs) co-transporters expression and enterocyte apicobasal distribution by immunohistochemistry. These analyses show that the anterior and posterior intestines have different developmental dynamics during smoltification. In both intestinal regions, photoperiod and dietary treatment increased the absorptive perimeter. In addition, diet and photoperiod treatments differentially stimulated NKA protein expression in the anterior intestine. NKCC apical-basolateral expression in the enterocytes increased after SW entry in the anterior and posterior intestines. In conclusion, our results show that, as smoltification progresses, the anterior intestine responds more readily to experimental conditions than the posterior intestine. In our study, photoperiod and dietary treatment seem to enhance the development of the capacity to tolerate SW.
- Photoperiod and dietary treatment in freshwater modulate the short-term intestinal response to seawater in atlantic salmon (salmo salar)Publication . Gaetano, Pasqualina; Fernandes Duarte, Vilma Vanessa; Striberny, Anja; Hazlerigg, David; Jørgensen, Even H.; Campinho, Marco António; Fuentes, JuanStimulation and timing of smoltification are essential for successful Atlantic salmon (Salmo salar) aquaculture. This study investigated intestinal responses during dietary and photoperiod manipulation in freshwater (FW) and after a subsequent seven days residence in seawater (SW). "Small" and "large" Atlantic salmon parr (-40 g and -130 g respectively) were treated in FW for 12 weeks and thereafter transferred to SW for seven days. During the FW phase, fish underwent two different light conditions, 24 L:0D - 24 L ("LL-LL" groups) and 7 L:17D - 24 L ("SP-LL" groups) or fed with either regular feed ("LL-LL C" and "SP-LL C" groups) or feed enriched with a salt mix plus free tryptophan ("LL-LL + diet" and "SP-LL + diet" groups). We analyzed Na+/K+-ATPase (NKA) activity, tissue bioelectrical properties in Ussing chambers, and intestinal fluid composition. The NKA activity showed minor variations in relation to fish size, treatments, or intestinal region (anterior or posterior). Photoperiod modulated epithelial bioelectrical properties (Isc and Rt) of the anterior and posterior intestine, particularly transepithelial resistance (Rt). Pharmacological experiments, targeting apical Na+/K+/2Cl- (NKCC2) and Na+/ Cl (NCC) co-transporters revealed intestinal region-and water salinity-dependent effects. In addition, stimu-lation of the intracellular cAMP with forskolin and IBMX showed intestinal region-, water salinity, and treatment-dependence responses with clear functional specialization of the anterior and posterior intestine. The intestinal fluid composition reflected the ability to process ingested SW and showed little variation in large fish. In sum-mary, our data suggest a better pre-adaptation of the intestine during light-stimulated smoltification (SP-LL groups), and the combination of light and diet might give, in an industrial aquaculture setting, an advantage to smaller, but not larger smolts. Intestinal fluid composition in small fish can be used as an index of intestinal function and may act as a long-term performance proxy in SW Atlantic salmon.
- More than one way to smoltify a salmon? Effects of dietary and light treatment on smolt development and seawater growth performance in Atlantic salmonPublication . Striberny, Anja; Lauritzen, Daniel E.; Fuentes, Juan; Campinho, Marco António; Gaetano, Pasqualina; Duarte, Vilma; Hazlerigg, David G.; Jorgensen, Even H.Post-smolt mortality and stunted growth is a problem in Norwegian salmon farming that can be partly traced back to sub-optimal smolt quality in connection with ongoing changes in smolt production practices. In the present study, we compared smolt development and post-smolt performance in seawater (SW) of Atlantic salmon subjected to 1) traditional light treatment (short photoperiod (SP) long photoperiod (LL)), 2) a dietary treatment alone (LL-LL + diet), 3) a combination of light and diet (SP-LL + diet) treatment and 4) no stimulation (LL-LL control) during the freshwater (FW) phase. The transition diet consisted of feed supplemented with a salt mixture and the amino acid tryptophan. Effects of light and dietary treatment were tested on two different size classes (40 and 130 g) of parr. Response patterns to treatments were independent of initial size and findings discussed below apply to both. Fish in the continuous light groups (LL-LL and LL-LL + diet) had the highest mass gain during the FW phase while light treated fish had a reduction in condition factor indicative of a true smolting. Hypoosmoregulatory ability in May was increased by diet, but not by light. However, a strong and treatment independent decrease in gill NKA alpha 1a gene expression indicated a complete shift from a FWto a SW type gill Na+, K+-ATPase. Moreover, plasma Cl- concentration and osmolality after 7 days in SW in May were well within the range expected for fully SW adapted fish in all treatment groups. Despite good hypoosmoregulatory abilities in all fish, there were strong treatment dependent effects on their growth during the two month SW residence. SW growth was negatively correlated with growth and change in K during the last part of the FW phase. A markedly higher specific growth rate and appetite in the SP-LL group than in the LL-LL group was likely a result of the light stimulated endocrine changes that occurred during smolting. Dietary treatment seemed to compensate for the lack of light treatment since feed intake and growth in LL-LL + diet fish was comparable to that in the SP-LL treated smolts. Feed intake and growth in the SP-LL + diet groups was highest, giving support for a notion that light and diet act through separate and additive mechanisms.
- First molecular characterization of grapevine virus B (GVB) in portuguese grapevine cultivars and improvement of the RT-PCR detection assayPublication . Fonseca, Filomena; Fernandes Duarte, Vilma Vanessa; Santos, Margarida Teixeira; Brazão, João; Eiras-Dias, EduardoThis work describes the first molecular characterization of grapevine virus B (GVB) in Portuguese grapevine cultivars. During a routine screening of 44 accessions in the National Collection of Grapevine Varieties (CAN PRT051), 17 were found infected with GVB in DAS-ELISA assays with commercial antibodies. However, only six of the corresponding isolates were successfully amplified using primer pairs described in the literature. The sequence variants (ORF4-3'UTR, 1147 nt) retrieved from these isolates segregated into two phylogenetic groups, which included sequences from complete genomes available in GenBank. The highly discrepant results obtained using serological and RT-PCR-based diagnostic tools led to the design of a primer pair for detection of GVB, which allowed the amplification of a 606-bp GVB-specific fragment from all DAS-ELISA-positive isolates and also revealed the existence of false negatives in the serological testing.
- Molecular responses in the intestine of Atlantic salmon (Salmo salar) following light and diet stimulation of smoltification: Potential molecular markers for a seawater-ready smoltPublication . Gaetano, Pasqualina; Fernandes Duarte, Vilma Vanessa; Striberny, Anja; Hazlerigg, David G.; Jørgensen, Even H.; Campinho, Marco António; Fuentes, JuanThe transfer to seawater (SW) represents a critical stage in the production of Atlantic salmon. The success of the transfer links with the optimal development of hypo-osmoregulatory capacities during smoltification. While various strategies are adopted in aquaculture to stimulate smoltification, considerable fish loss still occurs after transfer to sea cages. Therefore, we investigated the molecular responses in the anterior and posterior intestine of Atlantic salmon, following 1) a photoperiod treatment (24 h light (L):0 h dark (D) → 24 L:0D vs. 7 L:17D → 24 L:0D) and 2) dietary treatment (regular feed or feed enriched with a salt mix/tryptophan), combined with, or without a photoperiodic treatment in freshwater (FW), to evaluate how intestinal osmoregulatory mechanisms are modulated by these treatments, and to identify potential intestinal markers indicative of a SW-ready smolt. Using quantitative real-time PCR (qPCR), we investigated transcript levels of transporters and channels involved in ion movements through the enterocytes, tight junction components, and receptors (i.e., calcium-sensing re ceptor and prolactin receptor). The two intestinal regions showed different gene profiles and responsiveness towards the experimental treatments. In the anterior intestine, the exposure to short photoperiod (7 L:17D) upregulated Na+/K+ − ATPase subunit alpha 1c (nkaα1c), Na+/K+/2Cl− cotransporter 1 (nkcc1), Na+/K+/2Cl− cotransporter 2 (nkcc2), Cl− /HCO− 3 exchanger Slc26a6 (slc26a6), and cystic fibrosis transmembrane conductance regulator I (cftrI), in FW and SW. Also, Na+/K+ − ATPase subunit alpha 1b (nkaα1b), occludin (ocln), and prolactin receptor (prlr) were upregulated in FW and claudin 15 (cldn15) in SW groups exposed to this photo period. The posterior intestine was less responsive to the experimental treatments, although upregulation of nkcc1, nkcc2, slc26a6, and cftrI was observed in FW in the short photoperiod groups. Hence, our findings show that exposure to a winter signal in FW more effectively activates hypo-osmoregulatory mechanisms in the in testine of Atlantic salmon, where a coordinated and complementary role of the anterior and posterior intestine ensures optimal SW processing. Dietary treatment had a positive but more marginal effect on the regulation of the genes investigated, mainly enhancing the impact of short photoperiod when the two treatments were combined. Overall, we propose the apical Na+/K+/2Cl− cotransporter, nkcc2, and the apical Cl− / HCO− 3 exchanger, slc26a6, as potential FW molecular markers in the anterior intestine to assess “SW-readiness” in Atlantic salmon smolts.
- Molecular responses in the intestine of Atlantic Salmon (Salmo salar) following light and diet stimulation of smoltification: Potential molecular markers for a seawater-ready smoltPublication . Gaetano, Pasqualina; Fernandes Duarte, Vilma Vanessa; Striberny, Anja; Hazlerigg, David G.; Jørgensen, Even H.; Campinho, Marco António; Fuentes, JuanThe transfer to seawater (SW) represents a critical stage in the production of Atlantic salmon. The success of the transfer links with the optimal development of hypo-osmoregulatory capacities during smoltification. While various strategies are adopted in aquaculture to stimulate smoltification, considerable fish loss still occurs after transfer to sea cages. Therefore, we investigated the molecular responses in the anterior and posterior intestine of Atlantic salmon, following 1) a photoperiod treatment (24 h light (L):0 h dark (D) -* 24 L:0D vs. 7 L:17D -* 24 L:0D) and 2) dietary treatment (regular feed or feed enriched with a salt mix/tryptophan), combined with, or without a photoperiodic treatment in freshwater (FW), to evaluate how intestinal osmoregulatory mechanisms are modulated by these treatments, and to identify potential intestinal markers indicative of a SW-ready smolt. Using quantitative real-time PCR (qPCR), we investigated transcript levels of transporters and channels involved in ion movements through the enterocytes, tight junction components, and receptors (i.e., calcium-sensing receptor and prolactin receptor). The two intestinal regions showed different gene profiles and responsiveness towards the experimental treatments. In the anterior intestine, the exposure to short photoperiod (7 L:17D) upregulated Na+/K+ - ATPase subunit alpha 1c (nka alpha 1c), Na+/K+/2Cl- cotransporter 1 (nkcc1), Na+/K+/2Clcotransporter 2 (nkcc2), Cl- /HCO-3 exchanger Slc26a6 (slc26a6), and cystic fibrosis transmembrane conductance regulator I (cftrI), in FW and SW. Also, Na+/K+ - ATPase subunit alpha 1b (nka alpha 1b), occludin (ocln), and prolactin receptor (prlr) were upregulated in FW and claudin 15 (cldn15) in SW groups exposed to this photoperiod. The posterior intestine was less responsive to the experimental treatments, although upregulation of nkcc1, nkcc2, slc26a6, and cftrI was observed in FW in the short photoperiod groups. Hence, our findings show that exposure to a winter signal in FW more effectively activates hypo-osmoregulatory mechanisms in the intestine of Atlantic salmon, where a coordinated and complementary role of the anterior and posterior intestine ensures optimal SW processing. Dietary treatment had a positive but more marginal effect on the regulation of the genes investigated, mainly enhancing the impact of short photoperiod when the two treatments were combined. Overall, we propose the apical Na+/K+/2Cl- cotransporter, nkcc2, and the apical Cl- / HCO-3 exchanger, slc26a6, as potential FW molecular markers in the anterior intestine to assess "SW-readiness" in Atlantic salmon smolts.