Soutenance mémoire
Fish collection and experimental design
The experimentation has been do ne at the ISMER aquaculture station in Pointe-auPère, Québec (48°31 ‘N; 68°29’W). Fish used for this study were descendants from a stock of anadromous and resident-river lineages, both originating from Laval River and maintained at the Station aquicole de l’ISMER. Fish from both forms were reared in freshwater under similar natural temperature and photoperiod conditions, in six tanks of 500 liters each. Families were identified with different fins cut, mixed randomly in tanks and fed daily until fulfilment. In May, wh en fish were 17 months -old, both anadromous and river-resident were transferred to salt water (salinity being increasing by 2%0 per day until 28%0). These fish were maintained at 28 %0 until August. Samplings were done at day 0, before the transfer to saltwater, and after 3, 7, 14 and 60 days following transfer. Control fish from each family were maintained in fresh water during the whole experiment and sampled on the same days than those in salt water. Numbers of fish sampled for each group are shown in Table 1. For each sampling day, fish were anesthetized using MS222 (Ethyl 3- aminobenzoate methanesulfonate, 0.12g L- 1), blood sample were taken by caudal puncture, centrifuged and plasma was immediately frozen at -80°C for further analyses. The second and gill arch were sampled and immediately frozen at -80°C for further analyses of Na+/K +-ATPase activity measurement and isoforms expression.
Na + IK -A TPase isoform expression
Total RNA was extracted from approximately 10 mg of gill filaments using a RNeasy Plus Mini kit® (Qiagen Inc., Mississauga, ON, Canada) following manufacturer’s instructions. Isolated RNA was quantified using a spectrophotometer NanoVue (NanoDrop ND-I000 spectrophotometer v3 .3.0, NanoDrop Technologies Inc., DE, USA). RNA samples were run on an agarose gel (2%, with 500 f!g mLI Ethidum bromide marker) to verify both RNA integrity and quantity (Figure 2). First strand cDNA was obtained from 1 f!g RNA by using the QuantiTect Reverse Transcription Kit® (Qiagen Inc., Mississauga, ON, Canada) according to the manufacturer protocol, and the quantity obtained was evaluated using a Nano Vue. cDNA samples were stored at -20°C until quantitative realtime polymerase chain reaction analyses (RT-qPCR). The two target genes were the two isoformes of Na + /K+ -A TPase, al a and al b genes. Because the mRNA sequences for the al a and al b genes were not available for Salvelinus fontinalis on GenBank® database, non-specifie primers were designed from Oncorhynchus mykiss sequences (GenBank ID: A Y319391 and A Y319390 respectively). To verify that the primers designed for al a and al b genes amplified the target genes, brook charr gill cDNA was amplified by classic PCR with Amplitaq Gold Master Mix (Applied Biosystems, Foster city, CA) on a MyIQ Single Color Real-Time PCR Detection system
Na + Ir -A TPase activity and protein concentrations
Protein concentrations did not differ among days of sampling (F[4] = 0.399; p > 0.05), form (F[I] = 1.355; p > 0.05), or environment (F[I] = 0.002; p > 0.05), with a mean valor of 0.072 ± 0.016 mg protein mg tissu-I (Table 3). The absolute enzyme activity did not differ between anadromous and river-resident forms at any time of the experiment (F[l] = 0.807; p > 0.05, but there was a significant combined effect of environment and time of sampling (environment X time of sampling: F[4] = 12.965; P < 0.001 , Figure 4). On day 14, enzyme activity significantly raised both in saltwater (28%0) and freshwater groups. After 60 days, the Na+/K+-ATPase activity was significantly higher in salt- compared to fresh water. In fresh water, activity at day 60 was similar to the activity measured on day 0 (Figure 4). The same pattern as for absolute activity was observed for the specifie enzyme activity, with a combined significant effect of environment and time of sampling (environment X time of sampling: F[4] = 8.169, P < 0.001). In contrast with the results obtained with specifie activity, absolute activity was significantly higher at day 60 than at day 14 in salt water.
Discussion
The results presented in this study provide evidence that anadromous and riverresident charr from Laval Ri ver do not differ in their ability to regulate gill Na+/K+-ATPase activity (specifie and absolute) and to express a-subunit spec ifie isoforms of this enzyme with a graduai salinity increase to 28%0. Even if the two forms of Laval River charr are genetically distinct (Boula et al., 2002; Perry et al., 2005) and have shown differences in Na+/K+-ATPase activity on field study (Boula et al., 2002), no difference was observed either in Na+/K+-ATPase activity or in the relative expression of the two Na+/K+-ATPase isoforms between the two forms in response to changes in salinity. Similar gill Na + /K+- A TPase activity and isoform expression responses may express the persistence of anadromous behavior in brook charr, which would be the ancestral form according to Curry and colleagues (2010). Therefore, river-resident can have retained sorne osmoregulatory ability at the Na + /K+ -A TPase level in hyper-osmotic environment. In our study, when salinity reached 28%0, there was a sharp increase in gill Na + /K+ -A TPase activity in both forms. Plasma osmolality was significantly higher in river-resident th an in anadromous fish at 28%0 (Lemieux, personal comm., 2013) despite a similar Na+/K+-ATPase activity. This difference in plasma osmolality suggest difficulties acclimating to sea water with greater perturbations in plasma ion levels for the resident charr of Laval river in salt water.However, at the end of the experiment, plasma osmolality has retumed to control level for both forms (Lemieux, personal comm., 2013), suggesting an efficient ion excretion system. While gill Na+/K+-ATPase is an important enzyme in both salt and fresh water by creating the ionic gradient in chloride cell, this gradient is used by co-transporters and ions channels to excrete or secrete ions (Evans et al., 2005; McCormick, 2001). Nielsen and collaborators (2007) has shown an up regulation of Na+/K+I2Cr mRNA levels following seawater transfer of Atlantic salmon, a cotransporter implied in ion excretion in salt water, with landlocked salmon showing a time delay in up regulation of this cotransporter compared to anadromous Atlantic salmon. Increase in Na+/K+-ATPase activity In freshwater group after 14 days seems not related to plasma ions perturbations since this plasma osmolality stayed stable in this environment for the whole experiment (Lemieux, personal comm., 2013). After 60 days in saltwater, the enzyme activity was still high in the saltwater group, while a retum to control levelof Na+/K+-ATPase activity in fresh water after 60 days was observed. The increase in enzyme activity in fresh water after 14 days may be caused by a different factor, such as stress or immune response.
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Table des matières
REMERCIEMENTS
RÉSUMÉ
ABSTRACT
LISTE DES TABLEAUX
LISTE DES FIGURES
INTRODUCTION GÉNÉRALE
CHAPITRE 1 CARACTERISATION DE LA FONCTION NA+/K+- ATPASIQUE BRANCHIALE CHEZ DES OMBLES DE FONTAINE (SAL VELINUS FONTINALIS) ANADROMES ET RESIDENTS DE LA RIVIERE LAVAL
1.1 RESUME
1.2 ABSTRACT
1.3 INTRODUCTION
1.4 MATERIAL AND METHODS
1.5 RESULTS
1.6 DISCUSSION
CHAPITRE 2 DISCUSSION GENERALE
CONCLUSION ET PERSPECTIVES
RÉFÉRENCES BIBLIOGRAPHIQUES
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