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Fitness difference between cryptic salinity-related phenotypes of sea bass (Dicentrarchus labrax)

Bruno Guinand 1,2, Nolwenn Quéré 1,2, Frédérique Cerqueira 1,3, Erick Desmarais 1,3,
François Bonhomme 1,2

1 Institut des Sciences de l’Evolution de Montpellier, CNRS-UMR 5554 (Université Montpellier 2), cc65, 34095 Montpellier cedex 5, France. E-mail: bruno.guinand@um2.fr
2 Station Méditerranéenne de l’Environnement Littoral, 2 Avenue des Chantiers, 34200 Sète, France.
3 LabEx CeMEB (Centre Méditerranéen Environnement Biodiversité), Université Montpellier II, place E. Bataillon, cc63, 34095 Montpellier Cedex 5, France.

Summary: The existence of cryptic salinity-related phenotypes has been hypothesized in the “euryhaline” sea bass (Dicentrarchus labrax). How differential osmoregulation costs between freshwater and saltwater environments affect fitness and phenotypic variation is misunderstood in this species. During an experiment lasting around five months, we investigated changes in the whole body mass and in the expression of growth-related genes (insulin-like growth factor 1 [IGF-1]; growth hormone receptor [GHR]) in the intestine and the liver of sea bass thriving in sea water (SSW), successfully acclimated to freshwater (SFW), and unsuccessfully acclimated to freshwater (UFW). Albeit non-significant, a trend toward change in body mass was demonstrated among SSW, UFW and SFW fish, suggesting that SSW fish were a mixture of the other phenotypes. Several mortality peaks were observed during the experiment, with batches of UFW fish showing higher expression in the osmoregulatory intestine due to down-regulation of genes in the liver and significant up-regulation of GHR in the intestine compared with SFW fish. Energy investment toward growth or ion homeostasis hence partly mediates the fitness difference between cryptic SFW and UFW phenotypes. The use of a genetic marker located within the IGF-1 gene showed no genotype-phenotype relationship with levels of gene expression.

Keywords: phenotype; gene expression; growth hormone receptor; insulin-like growth factor 1; sea bass.

Diferencia de eficacia biológica entre fenotipos crípticos relacionados con salinidad en la lubina (Dicentrarchus labrax)

Resumen: En la especie “eurihalina” de la lubina (Dicentrarchus labrax) se ha planteado la existencia de fenotipos crípticos relacionados con la salinidad. En esta especie los costes diferenciales de osmoregulación a la adaptación en de agua dulce y salada son aún desconocidos. Durante un experimento de aproximadamente 5 meses, se investigó los cambios en la masa corporal y en la expresión de genes relacionados con el crecimiento (factor de crecimiento similar a la insulina 1 [IGF-1]; receptor de la hormona del crecimiento [GHR]) en el intestino y el hígado de lubina en individuos que prosperan en agua de mar (SSW), individuos aclimatados con éxito con el agua dulce (SFW), e individuos no aclimatados al agua dulce (UFW). Aunque no es significativa, se observa una tendencia de cambio en la masa corporal entre individuos SSW, UFW y SFW. Estos resultados sugieren que los individuos SSW son una mezcla de los otros fenotipos. Se observaron varios picos de mortalidad durante el experimento, con lotes de peces UFW que presentan una expresión génica más elevada en el intestino osmoregulador, debido a la regulación a la baja de genes en el hígado y regulación hacia arriba en la GHR del intestino cunado se compara con los peces SFW. Por lo tanto, la inversión de energía hacia el crecimiento o la homeostasis iónica explica en parte la diferencia de adaptación entre los crípticos fenotipos SFW y UFW. El uso de un marcador genético localizado dentro del gen de IGF-1 no demuestra relación genotipo-fenotipo con los niveles de expresión génica.

Palabras clave: fenotipo; expression de genes; receptor de la hormona del crecimiento; factor de crecimiento similar a la insulina 1; lubina.

Citation/Como citar este artículo: Guinand B., Quéré N., Cerqueira F., Desmarais E., Bonhomme F. 2014. Fitness difference between cryptic salinity-related phenotypes of sea bass (Dicentrarchus labrax). Sci. Mar. 78(4): 493-503. doi: http://dx.doi.org/10.3989/scimar.03992.02C

Editor: J. Viñas.

Received: December 9, 2013. Accepted: July 29, 2014. Published: October 3, 2014.

Copyright: © 2014 CSIC. This is an open-access article distributed under the Creative Commons Attribution-Non Commercial Lisence (by-nc) Spain 3.0.

Contents

Summary
Resumen
Introduction
Materials and methods
Results
Discussion
Acknowledgements
References

IntroductionTop

Marine species often display phenotypic diversity to acclimate or to adapt to their local environment. Phenotypic variation within species can especially drive patterns in the distribution, abundance, and ecological roles of organisms (Pfennig et al. 2010Pfennig D.W., Wund M.A., Snell-Rodd E.C., et al. 2010. Phenotypic plasticity’s impact on diversification and speciation. Trends Ecol. Evol. 25: 459-467., Sotka 2012Sotka E.E. 2012. Natural selection, larval dispersal, and the geography of phenotypes in the sea. Integr. Comp. Biol. 52: 538-545.). At the intraspecific level, phenotypic variation as illustrated by the existence of different morphs, developmental pathways, life-history strategies or behaviours is thought to be adaptive and to generally translate into better phenotype-environment matching in response to fluctuating environmental conditions (e.g. Van Valen 1965Van Valen L. 1965. Morphological variation and width of ecological niche. Am. Nat. 99: 377-390., Ghalambor et al. 2007Ghalambor C.K., McKay J.K., Carroll S.P., et al. 2007. Adaptive versus non-adaptive phenotypic plasticity and the potential for contemporary adaptation in new environments. Funct. Ecol. 21: 394-407., but see Marshall et al. 2010Marshall D.J., Monro K., Bode M., et al. 2010. Phenotype-environment mismatches reduce connectivity in the sea. Ecol. Lett. 13: 128-140.). However, the assessment of phenotypic diversity relies on situations in which different and discrete phenotypes are easily observed or situations in which phenotypes are cryptic. In the latter case, the array of phenotypes is obviously underestimated and can obscure interpretation of performance and fitness of individuals in distinct habitats. When an environmental or stress-induced stimulus is applied, one formerly cryptic phenotype may react to the stimulus and become defined by other attributes, increasing its fitness. This phenotypic switch may occur only after stress has accumulated over individuals until reaching the level inducing the stress response, i.e. the time at which previously cryptic phenotypes are uncovered (Hoffmann and Parsons 1991Hoffmann A.A., Parsons P.A. 1991. Evolutionary genetics and environmental stress. Oxford University Press, Oxford, 296 pp., Gabriel et al. 2005Gabriel W., Luttbeg B., Sih A., et al. 2005. Environmental tolerance, heterogeneity, and the evolution of reversible plastic responses. Am. Nat. 166: 339-353.). Delayed response depends on environmental tolerance, on stress intensity, and for how long cryptic phenotypes are submitted to the stressor environment to unravel performance/fitness differences in each phenotype, rather than simple variation reflecting short-term acclimation response (Palaima 2007Palaima A. 2007. The fitness cost of generalization: present limitations and future possible solutions. Biol. J. Linn. Soc. 90: 583-590.).

The distributional range of euryhaline European sea bass (Dicentrarchus labrax) extends from Mauritania to Norway and the Mediterranean Sea. It is an economically important fish that naturally inhabits marine, lagoon and estuarine environments (e.g. Dufour et al. 2009Dufour V., Cantou M., Lecomte F. 2009. Identification of sea bass (Dicentrarchus labrax) nursery areas in the north-western Mediterranean Sea. J. Mar. Biol. Ass. UK 89: 1367-1374., Vasconcelos et al. 2010Vasconcelos R.P., Reis-Santos P., Maia A., et al. 2010. Nursery use patterns of commercially important marine fish species in estuarine systems along the Portuguese coast. Estuar. Coast. Shelf Sci. 86: 613-624.). Only minor meristic and morphological phenotypic differences necessitating detailed studies to be identified exist in sea bass, and their interaction with fitness is unknown (Barnabé 1973Barnabé G. 1973. Étude morphologique du loup, Dicentrarchus labrax (L.) de la région de Sète. Rev. Trav. Instit. Pêches Marit. 37: 397-410., Corti et al. 1996Corti M., Loy A., Cataudella S. 1996. Form changes in the sea bass, Dicentrarchus labrax (Moronidae: Teleostei), after acclimation to freshwater: an analysis using shape coordinates. Env. Biol. Fishes 47: 165-175., Loy et al. 1999Loy A, Corti M., Cataudella S. 1999. Variation in gill rakers number during growth of the sea bass, Dicentrarchus labrax (Perciformes: Moronidae), reared at different salinities. Env. Biol. Fishes 55: 391-398., Bahri-Sfar and Ben Hassine 2009Bahri-Sfar L., Ben Hassine O.K. 2009. Clinal variations of discriminative meristic characters of sea bass, Dicentrarchus labrax (Moronidae, Perciformes) populations on Tunisian coasts. Cybium 33: 211-218., Costa et al. 2010Costa C., Vandeputte M., Antonucci F., et al. 2010. Genetic and environmental influences on shape variation in the European sea bass (Dicentrarchus labrax). Biol. J. Linn. Soc. 101: 427-436.). Concurrently, sea bass has repeatedly demonstrated different capabilities to acclimate freshwater (FW) in both experimental and natural conditions (Chervinski 1974Chervinski J. 1974. Sea bass, Dicentrarchus labrax L. (Pisces, Serranidae) a “police fish” in freshwater ponds and its adaptability to various saline condition. Isr. J. Aquacult. - Bamidgeh 26: 110-113., Dendrinos and Thorpe 1985Dendrinos P., Thorpe J.P. 1985. Effects of reduced salinity on growth and body composition in the European bass Dicentrarchus labrax (L.). Aquaculture 49: 333-358., Cataudella et al. 1991Cataudella S., Allegrucci G., Bronzi P., et al. 1991. Multidisciplinary approach to the optimisation of sea bass (Dicentrarchus labrax) rearing in freshwater – Basic morpho-physiology and osmoregulation. In: De Pauw N. and Joyce J. (eds), Aquaculture and the environment. European Aquaculture Society - Special Publication n° 14. Bredene, Belgium, pp. 55-57., Venturini et al. 1992Venturini G., Cataldi E., Marino G., et al. 1992. Serum ions concentration and ATPase activity in gills, kidney and oesophagus of European sea bass (Dicentrarchus labrax, Pisces, Perciformes) during acclimation trials to fresh water. Comp. Biochem. Physiol. A103: 451-454., Allegrucci et al. 1994Allegrucci G., Fortunato C., Cataudella S., et al. 1994. Acclimation to fresh water of the sea bass: evidence of selective mortality and allozyme genotypes. In: Beaumont A.R. (ed.), Genetics and evolution of marine organisms. Chapman and Hall, London, pp. 486-502., Marino et al. 1994Marino G., Cataldi E., Pucci P., et al. 1994. Acclimation trials of wild and hatchery sea bass (Dicentrarchus labrax) fry at different salinities. J. Appl. Ichthyol. 10: 57-63., Jensen et al. 1998Jensen K., Madsen S.S., Kristiansen K. 1998. Osmoregulation and salinity effects on the expression and activity of Na+,K+-ATPase in the gills of European sea bass, Dicentrarchus labrax (L.). J. Exp. Zool. 282: 290-300., Eroldogan and Kumlu 2002Eroldogan O.T., Kumlu M. 2002. Growth performance, body traits and fillet composition of the European sea bass (Dicentrarchus labrax) reared in various salinities and freshwater. Turk. J. Vet. Anim. Sci. 26: 993-1001., Varsamos et al. 2002Varsamos S., Diaz J.P., Charmantier G., et al. 2002. Branchial chloride cells in sea bass (Dicentrarchus labrax) adapted to fresh water, seawater, and doubly concentrated seawater. J. Exp. Zool. 293: 12-26., Nebel et al. 2005Nebel C., Romestand B., Nègre-Sadargues G., et al. 2005. Differential freshwater adaptation in juvenile sea-bass Dicentrarchus labrax: involvement of gills and urinary system. J. Exp. Biol. 208: 3859-3871., Boutet et al. 2007Boutet I., Lorin-Nebel C., De Lorgeril J., et al. 2007. Molecular characterisation of prolactin and analysis of extrapituitary expression in the European sea bass Dicentrarchus labrax under various salinity conditions. Comp. Biochem. Physiol. D2: 74-83., Giffard-Mena et al. 2008Giffard-Mena I., Lorin-Nebel C., Charmantier G., et al. 2008. Adaptation of the sea-bass (Dicentrarchus labrax) to fresh water: Role of aquaporins and Na+/K+-ATPases. Comp. Biochem. Physiol. A150: 332-338., Dufour et al. 2009Dufour V., Cantou M., Lecomte F. 2009. Identification of sea bass (Dicentrarchus labrax) nursery areas in the north-western Mediterranean Sea. J. Mar. Biol. Ass. UK 89: 1367-1374.). Sea bass could then be a mixture of cryptic phenotypes with distinct environmental tolerance and fitness regarding salinity, rather than a single unconditional, plastic, euryhaline phenotype as traditionally reported in textbooks (Pickett and Pawson 1994Pickett G.D., Pawson M.G. 1994. Sea bass biology, exploitation and conservation. Fish and fisheries series, Chapman and Hall, London, 337 pp., Sánchez Vázquez and Muñoz-Cueto 2014Sánchez Vázquez F.J., Muñoz-Cueto J.A. 2014. Biology of European sea bass. CRC Press, Cambridge, 436 pp.). Despite numerous reports of differential sea bass mortality when facing FW, observations were often a posteriori interpretations of experiments with very diverse objectives, and not studies dedicated to understanding fitness differences among individuals or phenotypes. The dynamics and the root of fitness difference have been very poorly assessed in sea bass, despite recent studies reporting histological observations (Nebel et al. 2005Nebel C., Romestand B., Nègre-Sadargues G., et al. 2005. Differential freshwater adaptation in juvenile sea-bass Dicentrarchus labrax: involvement of gills and urinary system. J. Exp. Biol. 208: 3859-3871.), or variation in patterns of gene expression that differ among juvenile sea bass successfully or unsuccessfully adapted to FW (Boutet et al. 2007Boutet I., Lorin-Nebel C., De Lorgeril J., et al. 2007. Molecular characterisation of prolactin and analysis of extrapituitary expression in the European sea bass Dicentrarchus labrax under various salinity conditions. Comp. Biochem. Physiol. D2: 74-83., Giffard-Mena et al. 2008Giffard-Mena I., Lorin-Nebel C., Charmantier G., et al. 2008. Adaptation of the sea-bass (Dicentrarchus labrax) to fresh water: Role of aquaporins and Na+/K+-ATPases. Comp. Biochem. Physiol. A150: 332-338.).

Indeed, fitness differences may have roots in the differential expression of the genes and variation in patterns of gene expression represents itself an expression of phenotypic variation (Larsen et al. 2011Larsen P.F., Schulte P.M., Nielsen E.E. 2011. Gene expression analysis for the identification of selection and local adaptation in fishes. J. Fish Biol. 78:1-22.). Fitness differences also emerge from competing demands that forces organisms like fish to adjust their metabolism to environmental conditions without compromising homeostasis and energetic budgets (Guderley and Pörtner 2010Guderley H., Pörtner H.O. 2010. Metabolic power budgeting and adaptive strategies in zoology: examples from scallops and fish. Can. J. Zool. 88: 753-763.). Growth and body mass are major fitness-related traits (Roff 1992Roff D.A. 1992. The evolution of life histories. Chapman and Hall, New York, 595 pp.). The control of growth involves a multifaceted system of regulation, using cellular controls that are modulated by the various endocrine signals of the growth hormone-insulin-like growth factor 1 (GH–IGF-I) axis (Reinecke 2010Reinecke M. 2010. Influences of the environment on the endocrine and paracrine fish growth hormone–insulin-like growth factor-I system. J. Fish Biol. 76:1233-1254., Reindl and Sheridan 2012Reindl K.L., Sheridan M.A. 2012. Peripheral regulation of the growth hormone-insulin-like growth factor system in fish and other vertebrates. Gene Comp. Endocrinol. 163: 231-245.). Indeed, though they are not the only hormones involved in the control of growth, GH and IGF-1 interact together in a complex manner, likely mediated through binding of GH to the GH receptor (GHR) (Wood et al. 2005Wood A.W., Duan C., Bern H.A. 2005. Insulin-like growth factor signaling in fish. Internat. Rev. Cytol. 243: 215-285.). In the liver, this association induces the expression of target genes, including IGF-I, which is responsible for most of the growth effects of GH (Wood et al. 2005Wood A.W., Duan C., Bern H.A. 2005. Insulin-like growth factor signaling in fish. Internat. Rev. Cytol. 243: 215-285., Reinecke 2010Reinecke M. 2010. Influences of the environment on the endocrine and paracrine fish growth hormone–insulin-like growth factor-I system. J. Fish Biol. 76:1233-1254.). Aside from its role in somatic growth, the GH–IGF-I axis also plays a role in osmoregulation, mediating a wide range of cellular, tissue and physiological adjustments in fish (Duan 1997Duan C. 1997. The insulin-like growth factor system and its biological actions in fish. Am. Zool. 37: 491-503., Reinecke 2010Reinecke M. 2010. Influences of the environment on the endocrine and paracrine fish growth hormone–insulin-like growth factor-I system. J. Fish Biol. 76:1233-1254.). As osmoregulation is an energy-demanding process, it naturally competes with growth (Bœuf and Payan 2001Bœuf G., Payan P. 2001. How should salinity influence fish growth? Comp. Biochem. Physiol. C130: 411-423.), and especially with the roles of GHR and mostly IGF-I in growth (Duan 1997Duan C. 1997. The insulin-like growth factor system and its biological actions in fish. Am. Zool. 37: 491-503., Moriyama et al. 2000Moriyama S., Ayson F.G., Kawauchi H. 2000. Growth regulation by insulin-like growth factor-I in fish. Biosci. Biotech. Biochem. 64: 1553-1562., Calduch-Giner et al. 2003Calduch-Giner J.A., Mingarro M., de Celis S.V.R., et al. 2003. Molecular cloning and characterization of gilthead sea bream, (Sparus aurata) growth hormone receptor (GHR). Assessment of alternative splicing. Comp. Biochem. Physiol. B136: 1-13., Côté et al. 2007Côté G., Perry G., Blier P., et al. 2007. The influence of gene-environment interactions on GHR and IGF-1 expression and their association with growth in brook charr, Salvelinus fontinalis (Mitchill). BMC Genet. 8: 87.). How IGF-I simultaneously acts on osmoregulatory potential and growth is poorly understood in fish because studies have primarily concentrated on extrahepatic IGF-I expression in tissues sensitive to nutritional status (muscle; e.g. Montserrat et al. 2007Montserrat N., Gabillard J.C., Capilla E., et al. 2007. Role of insulin, insulin-like growth factors, and muscle regulatory factors in the compensatory growth of the trout (Oncorhynchus mykiss). Gen. Comp. Endocrinol. 150: 462-472., Fox et al. 2010Fox B.K., Breves J.P., Davis L.K., et al. 2010. Tissue-specific regulation of the growth hormone/insulin-like growth factor axis during fasting and re-feeding: Importance of muscle expression of IGF-I and IGF-II mRNA in the tilapia. Gen. Comp. Endocrinol. 166: 573-580.), rather than concentrating on tissues that may modulate this status (e.g. intestine). It then appears that contrasting gene expression of growth-related genes in an essential relay to growth such as the liver and an osmoregulatory organ such as the intestine could provide a snapshot of energy investment toward growth and ion homeostasis.

The aim of this study was to investigate whether juveniles of the euryhaline European sea bass may be composed of a mixture of cryptic phenotypes exhibiting different physiological and fitness costs during long-term FW stress. Phenotypes were tested by analysing levels of expression for the GHR and IGF-1 genes in the liver and intestine of juvenile individuals in SW and FW conditions over a long-term experiment (~5 months) allowing stress to accumulate in individuals. In parallel, changes in body mass taken as a proxy of fitness were also investigated.

Materials and MethodsTop

Fish rearing conditions and treatments

In June 2008, young juveniles (n=800; body mass 14.8±2.0 g; age: approx. six months) of D. labrax issued from a mass spawning protocol were obtained from the Écloserie Marine de Gravelines (France). Fish were randomly divided in eight groups of size n=100 and reared in eight 0.5-m3 tanks at the Station Méditerranéenne de l’Environnement Littoral in Sète (France; 43°23’33”N, 3°39’51”E). Fish were randomly assigned to two treatments: (i) fish maintained in SW (37‰, filtered seawater from the neighbouring Thau lagoon; four tanks each seeded with n=100 fish); and (ii) fish acclimated to FW (0.5‰; four tanks seeded with n=100 fish). Following Nebel et al. (2005)Nebel C., Romestand B., Nègre-Sadargues G., et al. 2005. Differential freshwater adaptation in juvenile sea-bass Dicentrarchus labrax: involvement of gills and urinary system. J. Exp. Biol. 208: 3859-3871., fish were progressively acclimated to FW during a two-week period with an increment of 2‰ every day. They were reared at prevailing seasonal photoperiod and temperatures (range: 20 to 27.5°C) over the study period (early June to late October 2008), and were fed with commercial pellets according to Varsamos et al. (2006)Varsamos S., Xuereb B., Commes T., et al. 2006. Pituitary hormone mRNA expression in European sea bass Dicentrarchus labrax in seawater and following acclimation to fresh water. J. Endocrinol. 191: 473-480..

Sampling

Fish that survived in the SW and the FW treatments will be designed as successful seawater (SSW) and successful freshwater (SFW), respectively. At the end of the experiment (October 23, 2008), 20 SSW and 20 SFW were randomly sampled weighed, killed, dissected and organs (liver, and approx. 1.5 cm of intestine after the last pyloric cæcum) were stored at −80°C in 1 ml RNA Later® (Ambion, Austin, TX, USA). Pieces of fin and/or white muscle were collected for each individual and stored in 90% ethanol for DNA analyses of SFW and SSW individuals. As in Nebel et al. (2005)Nebel C., Romestand B., Nègre-Sadargues G., et al. 2005. Differential freshwater adaptation in juvenile sea-bass Dicentrarchus labrax: involvement of gills and urinary system. J. Exp. Biol. 208: 3859-3871., sea bass that demonstrated abnormal behaviour (e.g., swimming out of the shoal, with little or no response to external disturbance) during the freshwater acclimation were categorized as unsuccessfully adapted to freshwater (UFW). They began to appear ~10 days after exposure to full FW (Nebel et al. 2005Nebel C., Romestand B., Nègre-Sadargues G., et al. 2005. Differential freshwater adaptation in juvenile sea-bass Dicentrarchus labrax: involvement of gills and urinary system. J. Exp. Biol. 208: 3859-3871., Boutet et al. 2007Boutet I., Lorin-Nebel C., De Lorgeril J., et al. 2007. Molecular characterisation of prolactin and analysis of extrapituitary expression in the European sea bass Dicentrarchus labrax under various salinity conditions. Comp. Biochem. Physiol. D2: 74-83.). It has been demonstrated that such individuals generally die within 48 h after such behaviours appear (Nebel et al. 2005Nebel C., Romestand B., Nègre-Sadargues G., et al. 2005. Differential freshwater adaptation in juvenile sea-bass Dicentrarchus labrax: involvement of gills and urinary system. J. Exp. Biol. 208: 3859-3871.). This abnormal behaviour was not recorded for sea bass maintained in SW, and a fourth category of fish could not be investigated. We therefore randomly sub-sampled UFW individuals found dying over the course of the experiment (n=30; with ten fish sampled around July 1, August 27 and October 23, 2008, respectively; see below for justification) for the gene expression study. Due to the low sample size considered in each sampling period, testing for a tank effect is not feasible and results in low statistical power. Individuals found as already dead were never sampled for tissues. UFW fish were immediately dissected. The liver and anterior intestine were extracted and stored at −80°C in 1ml of RNA Later® for further molecular analysis. Pieces of fin were also collected for the 30 UFW individuals and stored in 90% ethanol for DNA analyses. A total of 70 fishes were then included in molecular analyses. Because of the bad conservation of tissues for UFW individuals sampled on ca. July 16, none of the individuals sampled at this date was included in the gene expression analysis.

The variation in body mass of SW, FW and UFW fish was monitored at three times during the course of the experiment (July 1, July 16; August 27; note that SW and FW fish cannot be qualified as SSW of SFW on those dates, as those acronyms are valid only for fish that survived at the end of the experiment), and at the end of the experiment (October 23). Thirty SW and FW fish per group were sampled on the first three dates; then 20 SFW and SFW individuals also used for the gene expression analysis were collected at the end of the experiment to investigate changes in body mass. SW and FW fish sampled on intermediate dates were not sacrificed. The first date (July 1) was retained as it matched the estimated date when the first UFW fish may appear (i.e. the two-week acclimation period followed by ~10 days after exposure to full FW; Nebel et al. 2005Nebel C., Romestand B., Nègre-Sadargues G., et al. 2005. Differential freshwater adaptation in juvenile sea-bass Dicentrarchus labrax: involvement of gills and urinary system. J. Exp. Biol. 208: 3859-3871.). Other dates in July and August corresponded to dates on which higher rates of appearance of UFW fish (i.e. mortality peaks) were recorded during the experiment (not shown). For those dates, we grouped UFW individuals that were found dying three days before or after those dates and performed tests to check for significant body mass differences among UFW, SW, and FW fish during the experiment. The duration of three days was retained as it allowed a sufficient number of UFW individuals in each sample for reliable testing. The last UFW fish labelled as ‘October 23’ were fish that were collected over the ten days before this date as no mortality peak was observed over this period. Despite its ad hoc status, this sampling was the only way to obtain an estimate of body mass of UFW fish at the end of the experiment. Mortality peaks decreased sea bass density and, accordingly, food input was proportionally decreased in FW tanks to be kept roughly constant over the course of the experiment. Tanks were regularly cleaned for non-ingested food. However, regular control of feed intake was not feasible in this study. UFW fish randomly retained for body mass monitoring were not all included in the study of gene expression.

Total RNA extraction

Total RNA was extracted from liver and anterior intestine tissues from all samples collected in sea bass using TRIzol (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions; the phase preparation step was done twice in the intestine tissue to remove excess lipids. A treatment with DNAseI (Invitrogen) was applied to all RNA samples to prevent genomic DNA (gDNA) contamination. The DNase was further removed by phenol chloroform extraction. The quantity and quality (A260/230 and A260/280) of total RNA were determined using a Nanodrop spectrophotometer (Nanodrop Technologies, Wilmington, DE, USA). Only RNA samples with A260/280 ratios above 1.6 and A260/230 ratios above 1.8 indicating minimal protein contaminants were used for further analysis.

Reverse transcription and quantitative real-time polymerase chain reaction (qPCR)

In each tissue, we looked at the expression of GHR and IGF-1 by first generating their cDNAs with the Protoscript® II RT-PCR kit (NE BioLabs® Inc, Ipswish, MA, USA) according to the manufacturer’s recommendations. Expression of GH was not investigated as it is only expressed in the pituitary, and variation of GH gene expression was formerly studied in Varsamos et al. (2006)Varsamos S., Xuereb B., Commes T., et al. 2006. Pituitary hormone mRNA expression in European sea bass Dicentrarchus labrax in seawater and following acclimation to fresh water. J. Endocrinol. 191: 473-480.. Primers for qPCR (Bustin et al. 2009Bustin S.A., Benes V., Garson J.A., et al. 2009. The MIQE Guidelines: minimum information for publication of quantitative real-Time PCR experiments. Clin. Chem. 55: 611-622.) are reported in Table 1, including primers for elongation factor-1 (EF1a) retained as reference gene in this study (rationale below). Gene amplifications were carried out with a LightCycler®480 (Roche Applied Science, Mannheim, Germany), using the PCR kit suggested by the manufacturer (LightCycler®480 SYBR Green I Master; Roche Applied Science; Mannheim Germany). In this study, all qPCRs, including reference genes (see below), were performed using three technical triplicates per individual in order to assess the intra-individual variability of gene expression, and then the reproducibility of individual gene expression. EF1a was preliminarily retained as a reference gene as it had been previously used in studies investigating gene expression variation in response to salinity in sea bass (e.g. Varsamos et al. 2006Varsamos S., Xuereb B., Commes T., et al. 2006. Pituitary hormone mRNA expression in European sea bass Dicentrarchus labrax in seawater and following acclimation to fresh water. J. Endocrinol. 191: 473-480., Boutet et al. 2007Boutet I., Lorin-Nebel C., De Lorgeril J., et al. 2007. Molecular characterisation of prolactin and analysis of extrapituitary expression in the European sea bass Dicentrarchus labrax under various salinity conditions. Comp. Biochem. Physiol. D2: 74-83., Giffard-Mena et al. 2008Giffard-Mena I., Lorin-Nebel C., Charmantier G., et al. 2008. Adaptation of the sea-bass (Dicentrarchus labrax) to fresh water: Role of aquaporins and Na+/K+-ATPases. Comp. Biochem. Physiol. A150: 332-338.). Another reference gene traditionally used in salinity-based studies of gene expression in fish was also screened (b-actin; e.g. Tine et al. 2008Tine M., De Lorgeril J., D’Cotta H., et al. 2008. Transcriptional responses of the black-chinned tilapia Sarotherodon melanotheron to salinity extremes. Mar. Genomics 1: 37-46.). In order to retain a reference gene, we adopted a criterion developed in Avarre et al. (2014)Avarre J.C., Dugué R., Alonso P., et al. 2014. Analysis of the black-chinned tilapia Sarotherodon melanotheron heudelotii reproducing under a wide range of salinities: from RNA-seq to candidate genes. Mol. Ecol. Res. 14: 139-142.. Briefly, we randomly sampled a set of 15 fish (five fish per condition: SSW, SFW and UFW) and measured their cycle of quantification value (Cq, i.e. the cycle at which fluorescence from amplification exceeds the background fluorescence) with the EF1a and b-actin genes. We used the NormFinder software (Andersen et al. 2004Andersen C.L., Jensen J.L., Ørntoft T.F. 2004. Normalization of real-time quantitative reverse transcription-PCR data: a model-based variance estimation approach to identify genes suited for normalization, applied to bladder and colon cancer data sets. Cancer Res. 64: 5245–5250.) to investigate the repeatability (i.e. stability of gene expression) of their Cq values by measuring the standard deviations of Cq (SDCq) over the 15 individuals (EF1a: SDCq=0.337; b-actin: SDCq=0.646). Avarre et al. (2014)Avarre J.C., Dugué R., Alonso P., et al. 2014. Analysis of the black-chinned tilapia Sarotherodon melanotheron heudelotii reproducing under a wide range of salinities: from RNA-seq to candidate genes. Mol. Ecol. Res. 14: 139-142. set up a criterion of SDCq<0.5 to qualify genes as ‘good’ reference genes. We did not use other reference genes in this study as there is no rationale to increase the number reference genes when investigating for gene expression variation at only two target genes. Hence, results are hereby presented for EF1a, which recovered good efficiencies (97.5%) and higher repeatability of Cq values.

Table 1. – Specific primers used for real-time PCR analyses (S, sense strand; AS, antisense strand).

Gene

Sequence

IGF-1

S

5’-ACCTAAGGTTAGTACCGCAG-3’

A

5’-CTGATGCACTTCCTTGAAGG-3’

GHR

S

5’-ACAACAGGAAAAGTTGATGG-3’

A

5’-GTTGTTGTACAGCTCTGGC-3’

EF1a

S

5’-AGGTCAATCTGTGGAGATG-3’

A

5’-TTCAGGATGATGACCTGGGC-3’

Primers used for IGF-1 were designed according to the full genomic sequence of IGF-1 provided by Quéré et al. (2010)Quéré N., Guinand B., Kuhl H., et al. 2010. Genomic sequences and genetic differentiation at associated tandem repeat markers in growth hormone, somatolactin and insulin-like growth factor 1 genes of the sea bass, Dicentrarchus labrax. Aquat. Living Resour. 23: 285-296. (Genbank accession number: GQ924783). Note that IGF-1 primers used in this study were preferred to IGF-1 primers used in former sea bass gene expression studies (Genbank accession: AY800248; e.g. Varsamos et al. 2006Varsamos S., Xuereb B., Commes T., et al. 2006. Pituitary hormone mRNA expression in European sea bass Dicentrarchus labrax in seawater and following acclimation to fresh water. J. Endocrinol. 191: 473-480., Terova et al. 2007Terova G., Rimoldi S., Chini V., et al. 2007. Cloning and expression analysis of insulin-like growth factor I and II in liver and muscle of sea bass (Dicentrarchus labrax, L.) during long-term fasting and refeeding. J. Fish Biol. 70: 219-233., Mazurais et al. 2008Mazurais D., Darias M.J., Gouillou-Coustans M.F., et al. 2008. Dietary vitamin mix levels influence the ossification process in European sea bass (Dicentrarchus labrax) larvae. Am. J. Physiol. – Regul. Integr. Comp. Physiol. 294: R520-R527.), as they retrieved, e.g., better repeatability of Cq values and efficiencies (details not reported). GHR expression was not previously investigated in sea bass. Two transcripts (Genbank accessions: AF438177 and AY642116) were available in databases as GHR1 and GHR2, respectively. Fukamachi and Meyer (2007)Fukamachi S., Meyer A. 2007. Evolution of receptors for growth hormone and somatolactin in fish and land vertebrates: lessons from the lungfish and sturgeon orthologues. J. Mol. Evol. 65: 359-372. demonstrated that sequences available in databases and reported as GHR (1 or 2) could be somatolactin receptors (SLR). We performed alignments of sequences, and unambiguously linked the sea bass GHR sequence to the transcript with accession AY642116 (GHR2, the alternate transcript being SLR; details not shown). This transcript was then used to design GHR primers (Table 1). Gene amplifications were also carried out with a LightCycler®480. The amplification of each sample was performed in a total final volume of 5 μl (2 μl cDNA diluted to 1/4, 0.25 μl of each primer [10 μM], and 2.5 μl of buffer 2X SYBR Green I Master). The PCR was done in triplicate for each individual with a denaturing cycle of 95°C/10 min, followed by 45 (50 for GHR) cycles of 95°C/10 s, 63°C (58°C for GHR)/15 s (10 s for GHR), 72°C/10 s (8 s for GHR). For each gene, a number of previous trials were carried out in order to determine their Cq value, while remaining within the linear PCR amplification limits. Agarose gel electrophoresis was performed to verify amplicon size and absence of primer-dimers, and to check the absence of cross-amplification of SLR (GHR1) (results not shown). Amplification products were also checked for the shape of their melting curves. In addition, to determine the qPCR efficiencies (E) of each primer pair used, standard curves were generated using five serial dilutions (1/2, 1/4, 1/8, 1/16, 1/32). We also evaluated the intra-assay variability of triplicate qPCRs at different transcript levels (no significant within-individual differences were detected; not shown). Negative controls (i.e. reverse transcriptase and RNA free samples) were also included to assess the reliability of results.

Statistical analyses of gene expression data

The relative expression of both GHR and IGF-1 normalized to the retained reference gene was assessed following a method presented by Pfaffl (2001)Pfaffl M. 2001. A new mathematical model for relative quantification in real-time RT-PCR. Nuc. Acids Res. 29: e45.. Relative expression (RE) is determined using the equation:

RE = [(Etarget)ΔCqtarget (control-sample)] / [(Eref)ΔCqref (control-sample)]

where Etarget is the amplification efficiency of the target (i.e. gene of interest) and Eref is the amplification efficiency of EF1a. The corresponding qPCR efficiency of one cycle in the exponential phase of amplification was calculated according to the equation E = 10[-1/slope] (Pfaffl 2001Pfaffl M. 2001. A new mathematical model for relative quantification in real-time RT-PCR. Nuc. Acids Res. 29: e45.). The qPCR assays were optimized with linear standard curve with R2≥0.98 (EF1a: R2=0.99; GHR: R2=0.98; IGF-1: R2=0.99). A Kruskal-Wallis test was first used to compare gene expression differences between the three samples of UFW individuals sampled on July 1, August 27 and October 23 in each organ. Then, parametric analysis of variance was used to compare mean levels of gene expression among fish categories (SSW, SFW, UFW) within each organ. Analyses were performed with the R software (v2.8.1; www.r-project.org). Post-hoc corrections for multiple tests were applied when necessary.

Genotype-phenotype relationship

Quéré et al. (2010)Quéré N., Guinand B., Kuhl H., et al. 2010. Genomic sequences and genetic differentiation at associated tandem repeat markers in growth hormone, somatolactin and insulin-like growth factor 1 genes of the sea bass, Dicentrarchus labrax. Aquat. Living Resour. 23: 285-296. developed a microsatellite marker labelled as µIGF-1 and located in the IGF-1 gene in sea bass. Polymorphism of all SSW, SFW and UFW individuals was screened at locus µIGF-1 according to PCR protocol and primer reported by these authors. Amplifications were performed on a PTC-200 (MJ Research). Genotyping of individuals was performed by allele sizing on an ABI PRISM® 3130xl Genetic Analyser (Life Technologies, St Aubin, France), using 5’-labelled reverse primers and the GeneScanTM 600 LIZ® Internal Line Standard (Life Technologies) as internal size standard. Allele scoring was performed using the GeneMapper software v.4.0 (Life Technologies). Genetic differentiation at locus µIGF-1 among SSW, SFW and UFW individuals was estimated using FST (Weir and Cockerham 1984Weir B.S., Cockerham C.C. 1984. Estimating F-statistics for the analysis of population structure. Evolution 38: 1358-1370.), and significance tested by permutation (1000 replicates) in Genetix v4.05 (kimura.univ-montp2.fr). For each organ, we further investigated the genotype-phenotype relationship of IGF-1 gene expression variation using a nested analysis of variance (ANOVA) with mixed effects in which SSW, SFW and UFW fish were used as the main factor (treatment) and genotypic classes as the nested factor. Individuals were pooled in fixed genotypic classes according to size of their alleles to get sufficient number of individuals in each genotypic class. The procedure to pool genotypes is described in the ‘Results’ section. It was based on allele size as, for microsatellite and other types of loci linked to functional candidate genes (e.g. IGF-1), gene expression variation was found to correlate with the respective size of the alleles (Gemayel et al. 2010Gemayel R., Vinces M.D., Legendre M., et al. 2010. Variable tandem repeats accelerate evolution of coding and regulatory sequences. Ann. Rev. Genet. 44: 445-477.; for fish see, e.g. Streelman and Kocher 2002Streelman J., Kocher T. 2002. Microsatellite variation associated with prolactin expression and growth of salt-challenged tilapia. Physiol. Genomics 9: 1-4.). Analyses were performed with the R software v2.8.1.

Results

Survival and body mass differences

The mean mortality rate of fish that stayed in SW was 22.25% [range: 13-32% over the four SW tanks] while that of fish that were submitted to FW was 53.25% [range: 39-76% over the four FW tanks]. Ranges do not overlap, and differences in mean survival rates between the SW and FW experiments was therefore ca. 30%, a gross estimate of UFW fish present in our study. However, variability of survival rates among SW tanks was similar to the difference among treatments, possibly indicating a batch effect due to undetected reasons such as sub-optimal husbandry. After five months of acclimation, the mean (±SD) masses of the two final experimental groups had more than tripled, being 45.15±9.42 g and 53.82±12.60 g in SSW and SFW, respectively (t-test: P=0.41). Hence, no significant cost to FW was demonstrated between groups of surviving sea bass in this study.

Similarly, UFW did not demonstrate significantly lower body mass than fish sampled in FW on the four dates used for this study (Fig. 1). While not significant (lowest observed p-value: P=0.092 on August 27), the mean difference in body mass increased during the experiment, indicating a trend toward impaired growth in UFW compared with SFW fish (Fig. 1). Individuals that stayed in seawater showed an intermediate body mass to SFW and UFW on each date (Fig. 1), suggesting they could be a mixture of other phenotypes.

sm3992fig1.jpg

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Fig. 1. – Evolution of mean body mass (±SE) of UFW sea bass (Dicentrarchus labrax) compared with the body mass of 30 SW and FW fish (July 1, July 16, August 27), or 20 SSW and SFW fish (October 23) (see text for details). Samples of UFW fish represent pools of individuals that demonstrated signs of abnormal behaviour three days before and after those dates, except for the final date October 23, with UFW individuals collected over the 10 days before this date because no mortality peak was observed at that time. Sample sizes of UFW pools are indicated in brackets. No significant differences in body mass were found among groups of fish at each date. Only the lowest observed p-values recorded between UFW fish and fish sampled in FW during the last two surveys are indicated. Note that (S)SW fish always have an intermediate average body mass compared with other fish categories, suggesting that they could be a mixture of cryptic phenotypes.

Gene expression variation

The Kruskal-Wallis test revealed no significant changes for gene expression of GHR and IGF-1 among sub-samples of UFW individuals taken on July 1, August 27 and October 23 (all P’s>0.05; 2 df; details not shown). This suggested that identical changes in gene expression occurred on several occasions during development. To increase statistical power and for comparison with gene expression of GHR and IGF-1 in SFW and SSW fish sampled on October 23, individual gene expression data of the three UFW sub-samples (each n=10) were gathered in a single sample (n=30) for each organ. The Kolmogorov-Smirnov (K-S) tests were previously performed to check the normality of gene expression data in UFW, SSW (n=20), and SFW (n=20) for each gene and each organ. K-S tests were not significant except for the expression of GHR in liver of SSW fish (P<0.01). Despite this observation, gene expression variation was then analysed using a parametric set-up.

Results pertaining to the gene expression of both IGF-1 and GHR in the two tissues after pooling of UFW sub-samples are reported in Figure 2. Use of triplicates reported no significant intra-individual differences in gene expression for each gene, whatever the category of fish (SSW, SFW and UFW) or organ (details not shown). Measurements of gene expression were therefore reproducible for each individual.

IGF-1 - There was expression of IGF-1 in both the liver and the intestine (Fig. 2). Furthermore, fishes from the FW exposure expressed IGF-1 differently in the liver and in the intestine. On the one hand, SFW had significantly greater expression (mean±SD) in the liver than in the intestine (2.04±1.07 and 1.17±0.55, respectively; P<0.05), whereas the inverse relationship was found in UFW, with lower expression levels in liver than in the anterior intestine (0.29±0.25 and 1.12±0.59, respectively; P<0.001). SSW individuals showed no significant difference in IGF-1 expression between liver and intestine (1.60±1.36 and 1.20±0.95, respectively; not significant [NS]). No significant difference was found in intestinal IGF-1 expression among groups of fish.

sm3992fig2.jpg

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Fig. 2. – Real-time PCR analysis of mRNA expression of (A) IGF-1 and (B) GHR in liver and intestine of European sea bass (Dicentrarchus labrax) for SSW (n=20), SFW (n= 0) and UFW (n=30). Each value represents means + SD. Significant differences in gene expression among tissues within each group of fishes are indicated by a horizontal grey line with the significance level of each test. When tests between tissues were not found significant, this line is not reported (SSW). Different letters indicate significant differences in gene expression among the three groups of fishes. For each gene, different boldface characters report significance among SSW, SFW and UFW for liver, whereas standard characters are for the intestine (P<0.001 in each case, except for GHR in liver: P<0.05).

For liver, a significantly lower expression of IGF-1 was detected in UFW individuals than in SSW and SFW individuals (P<0.001 in each case).

GHR - No significant difference in GHR gene expression (mean±SD) was recorded between intestine and liver for the SSW individuals (2.18±0.35 and 1.15±1.69, respectively; NS). There was significant over-expression of GHR in the liver of SFW individuals compared with their intestine (3.66±2.56 and 0.92±0.64, respectively; P<0.001). Conversely, the mean relative expression of GHR was significantly lower in the liver than in the intestine of UFW (1.04±0.86 and 4.16±2.72, respectively; P<0.001).

In the liver, there were significant GHR gene expression differences between SFW and UFW individuals (P<0.001) and between SFW and SSW individuals (P<0.05), but not between SSW and UFW individuals. In the intestine, UFW demonstrated higher relative GHR gene expression than the other two groups (P<0.001 in both cases).

Genotype-phenotype relationship

Six distinct µIGF-1 alleles were found in this study with allele sizes being 232, 240, 246, 248, 254 and 258 base pairs (bp). Allele 240 showed the highest overall allele frequency of the 60 individuals considered in this study (38.25%; details not shown). No significant genetic differentiation was recorded among SSW, SFW, and UFW individuals at locus µIGF-1 (fw=0.007; P=0.617). Individuals were grouped into four distinct genotypic classes with respect to allele size and observed distribution of genotypes: 240/240, 240/240, 240/240+ and 240+/240+ (i.e. heterozygous individuals with one allele with size <240 bp, homozygous individuals for the 240 bp allele, heterozygous individuals with one allele of size >240 bp, homo- or heterozygous individuals with no 240bp allele, respectively). Taking these genotypic classes into account, no significant genotype-phenotype relationship was found in the nested ANOVA, either in liver (among treatments P=0.022, among genotypic classes P=0.181), or in the intestine (among treatments P=0.612, among genotypic classes P=0.255). As a nested factor, genotypes were not significantly involved in observed gene expression variation in IGF-1. The significant p-value for IGF-1 expression in liver among treatments (SSW, SFW, UFW) refers to the previously mentioned under-expression of IGF-1 in UFW individuals (see above, and Fig. 2).

DiscussionTop

Hidden phenotypic diversity is undoubtedly present in sea bass and the results challenged the unconditional euryhalinity recognized in this species. Individuals with the SFW phenotype appear to be euryhaline individuals able to withstand large salinity variation, while UFW sea bass can be seen as representing a cryptic phenotype that can be first perceived when the salinity stress is long enough to cause acclimation failure (i.e. ~3 weeks after initiation of the salinity challenge; Nebel et al. 2005Nebel C., Romestand B., Nègre-Sadargues G., et al. 2005. Differential freshwater adaptation in juvenile sea-bass Dicentrarchus labrax: involvement of gills and urinary system. J. Exp. Biol. 208: 3859-3871., Giffard-Mena et al. 2008Giffard-Mena I., Lorin-Nebel C., Charmantier G., et al. 2008. Adaptation of the sea-bass (Dicentrarchus labrax) to fresh water: Role of aquaporins and Na+/K+-ATPases. Comp. Biochem. Physiol. A150: 332-338.). However, other mortality peaks were observed, and juvenile UFW sea bass were not shown to die during a single mortality episode. These additional mortality peaks were not previously observed in sea bass, because experiments were too short for this observation be reported (~2 months; Nebel et al. 2005Nebel C., Romestand B., Nègre-Sadargues G., et al. 2005. Differential freshwater adaptation in juvenile sea-bass Dicentrarchus labrax: involvement of gills and urinary system. J. Exp. Biol. 208: 3859-3871., Boutet et al. 2007Boutet I., Lorin-Nebel C., De Lorgeril J., et al. 2007. Molecular characterisation of prolactin and analysis of extrapituitary expression in the European sea bass Dicentrarchus labrax under various salinity conditions. Comp. Biochem. Physiol. D2: 74-83., Giffard-Mena et al. 2008Giffard-Mena I., Lorin-Nebel C., Charmantier G., et al. 2008. Adaptation of the sea-bass (Dicentrarchus labrax) to fresh water: Role of aquaporins and Na+/K+-ATPases. Comp. Biochem. Physiol. A150: 332-338.). Multiple mortality peaks hence question the homogeneity of the UFW phenotype itself. Overall, the mortality rate associated with UFW individuals in this study is ~30%, suggesting that UFW individuals may represent a significant proportion of juvenile sea bass.

How phenotypic variation affect fitness may be studied using the differential expression of the genes (Larsen et al. 2011Larsen P.F., Schulte P.M., Nielsen E.E. 2011. Gene expression analysis for the identification of selection and local adaptation in fishes. J. Fish Biol. 78:1-22.). It is, however, impossible to establish causal links between fitness and gene regulation with the study of two genes and two organs. Multiple causalities are likely to be at work. In sea bass, Giffard-Mena et al. (2008)Giffard-Mena I., Lorin-Nebel C., Charmantier G., et al. 2008. Adaptation of the sea-bass (Dicentrarchus labrax) to fresh water: Role of aquaporins and Na+/K+-ATPases. Comp. Biochem. Physiol. A150: 332-338. demonstrated that gene expression difference of two aquaporin genes (AQP1, AQP3) in gut was probably involved in the death of UFW-like individuals. Boutet et al. (2007)Boutet I., Lorin-Nebel C., De Lorgeril J., et al. 2007. Molecular characterisation of prolactin and analysis of extrapituitary expression in the European sea bass Dicentrarchus labrax under various salinity conditions. Comp. Biochem. Physiol. D2: 74-83. reported a similar observation for relative gene expression of prolactin (PRL) in the gills and to a lesser extent in the intestine of SFW- and UFW-like fish. We hence do not speculate that results reporting variability in gene expression of GHR and IGF-1 summarize the complexity of the regulation of the GH–IGF-1 axis in fish, or other molecular, cellular, histological and physiological mechanisms leading UFW individuals to death. Nevertheless, it is clear that changes in gene expression and gene expression dysfunction may reveal differential selection and fitness differences in fish (Larsen et al. 2011Larsen P.F., Schulte P.M., Nielsen E.E. 2011. Gene expression analysis for the identification of selection and local adaptation in fishes. J. Fish Biol. 78:1-22.), and that such results have recently been common in sea bass (Nebel et al. 2005Nebel C., Romestand B., Nègre-Sadargues G., et al. 2005. Differential freshwater adaptation in juvenile sea-bass Dicentrarchus labrax: involvement of gills and urinary system. J. Exp. Biol. 208: 3859-3871., Boutet et al. 2007Boutet I., Lorin-Nebel C., De Lorgeril J., et al. 2007. Molecular characterisation of prolactin and analysis of extrapituitary expression in the European sea bass Dicentrarchus labrax under various salinity conditions. Comp. Biochem. Physiol. D2: 74-83., Giffard-Mena et al. 2008Giffard-Mena I., Lorin-Nebel C., Charmantier G., et al. 2008. Adaptation of the sea-bass (Dicentrarchus labrax) to fresh water: Role of aquaporins and Na+/K+-ATPases. Comp. Biochem. Physiol. A150: 332-338.). As illustrated by Côté et al. (2007)Côté G., Perry G., Blier P., et al. 2007. The influence of gene-environment interactions on GHR and IGF-1 expression and their association with growth in brook charr, Salvelinus fontinalis (Mitchill). BMC Genet. 8: 87. in brook charr (Salvelinus fontinalis), changes in the expression of IGF-1 and GHR may provide a snapshot of this complexity and illustrate how, at a molecular level, SFW and UFW fish could manage the essential trade-off between growth and ion homeostasis during osmotic stress as the liver represents an essential relay for growth while the intestine is involved in osmoregulation. This trade-off was not considered in the aforementioned studies on sea bass, which concentrated only on osmoregulatory organs. In this study, SFW individuals preferentially expressed both IGF-1 and GHR in liver, while UFW individuals showed higher expression in the intestine. IGF-1 and GHR expressions are down-regulated in the liver of UFW compared with SFW fish, while GHR is over-expressed in the intestine. The difference in gene expression patterns between SFW and UFW fish may illustrate increased physiological investment toward osmoregulation in UFW compared with SFW. Nevertheless, patterns of GHR expression observed in the study in the gut of SFW and UFW fish are intriguing. Indeed, increase in GHR abundance in osmoregulatory organs was observed when salmonids colonized SW and not the reverse (Sakamoto and Hirano 1991Sakamoto T., Hirano T. 1991. Growth hormone receptors in the liver and osmoregulatory organs of rainbow trout: characterization and dynamics during adaptation to seawater. J. Endocrinol. 130: 425-433., see also Lerner et al. 2012Lerner D.T., Sheridan M.A., McCormick S.D. 2012. Estrogenic compounds decrease growth hormone receptor abundance and alter osmoregulation in Atlantic salmon. Gen. Comp. Endocrinol. 179: 196-204.). The patterns found in UFW (increased GHR expression in FW compared with SW) and SFW (stable GHR expression in FW compared with SW) contradict what is observed in salmonids. This must be investigated further, but, e.g. Bodinier et al. (2009)Bodinier C., Lorin-Nebel C., Charmantier G., Boulo V. 2009. Influence of salinity on the localization and expression of the CFTR chloride channel in the ionocytes of juvenile Dicentrarchus labrax exposed to seawater and freshwater. Comp. Biochem. Physiol. A153: 345-351. already reported differences or commonalities in gene expression patterns between sea bass and other euryhaline marine fish species, indicating that gene expression variation might be strongly species-dependent. Studies regarding more genes and organs must be performed to investigate the detailed mechanisms of the physiological imbalance observed in UFW.

It is worth noting that the results were made possible because sea bass were submitted to stress over months. Duration of studies regarding metabolic or gene expression variation in response to an osmoregulatory stress often span from a few days to ca. one month (Havird et al. 2013Havird J.C., Henry R.P., Wilson A.E. 2013. Altered expression of Na+/K+–ATPase and other osmoregulatory genes in the gills of euryhaline animals in response to salinity transfer: A meta-analysis of 59 quantitative PCR studies over 10 years. Comp. Biochem. Physiol. D8: 131-140.; for specific studies regarding the genes considered in this study see, e.g. Riley et al. 2003Riley L.G., Hirano T., Gray E.G. 2003. Effects of transfer from seawater to fresh water on the growth hormone/insulin-like growth factor-I axis and prolactin in the tilapia, Oreochromis mossambicus. Comp. Biochem. Physiol. B136: 647-655., Magdeldin et al. 2007Magdeldin S., Uchida K., Hirano T., et al. 2007. Effects of environmental salinity on somatic growth and growth hormone/insulin-like growth factor-I axis in juvenile tilapia (Oreochromis mossambicus). Fish. Sci. 73: 1025-1034., Link et al. 2010Link K., Berishvili G., Shved N., et al. 2010: Seawater and freshwater challenges affect the insulin-like growth factors IGF-I and IGF-II in liver and osmoregulatory organs of the tilapia. Mol. Cell. Endocrinol. 327: 40-46., but see Côté et al. 2007Côté G., Perry G., Blier P., et al. 2007. The influence of gene-environment interactions on GHR and IGF-1 expression and their association with growth in brook charr, Salvelinus fontinalis (Mitchill). BMC Genet. 8: 87.). This observation also holds true for studies searching for a better understanding of the mechanisms promoting euryhalinity (e.g. Scott et al. 2008Scott G.R., Baker D.W., Schulte P.M., et al. 2008. Physiological and molecular mechanisms of osmoregulatory plasticity in killifish after seawater transfer. J. Exp. Biol. 211: 2450-2459.). Such studies are undoubtedly relevant for deciphering the mechanistic basis of osmoregulation and for studying short-term acclimation performance (Havird et al. 2013Havird J.C., Henry R.P., Wilson A.E. 2013. Altered expression of Na+/K+–ATPase and other osmoregulatory genes in the gills of euryhaline animals in response to salinity transfer: A meta-analysis of 59 quantitative PCR studies over 10 years. Comp. Biochem. Physiol. D8: 131-140.). However, these studies poorly illustrate how the long-term fitness of individuals might be affected by a long periods of salinity stress and how gene expression differences translate in adaptive differences. In a rare study monitoring response of individuals to salinity up to five months as performed herein for sea bass, Côté et al. (2007)Côté G., Perry G., Blier P., et al. 2007. The influence of gene-environment interactions on GHR and IGF-1 expression and their association with growth in brook charr, Salvelinus fontinalis (Mitchill). BMC Genet. 8: 87. demonstrated heritable variation of liver gene expression both for IGF-1 and GHR in brook charr. This takes gene expression from an acclimation (short-term) to an adaptive (long-term) context, explaining the success of distinct phenotypes in distinct habitats. Such long-term studies should be promoted.

Concurrently, if differential gene expression was observed between UFW and SFW individuals, no significant osmoregulatory cost between fish was observed in this study, when body mass was used as a proxy. Rubio et al. (2005)Rubio V.C., Sánchez-Vázquez F.J., Madrid J.A. 2005. Effects of salinity on food intake and macronutrient selection in European sea bass. Physiol. Behav. 85: 333-339. and Giffard-Mena et al. (2008)Giffard-Mena I., Lorin-Nebel C., Charmantier G., et al. 2008. Adaptation of the sea-bass (Dicentrarchus labrax) to fresh water: Role of aquaporins and Na+/K+-ATPases. Comp. Biochem. Physiol. A150: 332-338. already reported no significant difference in specific growth rate for juvenile sea bass that experienced FW or SW in ~4 and ~1.5 month study, respectively. While non-significant, a trend toward increased growth rate or body mass in freshwater or brackish water similar to the one observed in this study has been demonstrated in several studies in sea bass (e.g. Chervinski 1975Chervinski J. 1975. Sea basses Dicentrarchus labrax (Linné) and D. punctatus (Boch) (Pisces: Serranidae), a control fish in fresh-water. Aquaculture 6: 249-256, Alliot et al. 1983Alliot E., Pastoureaud A., Thébault H. 1983. Influence de la température et de la salinité sur la croissance et la composition corporelle d’alevins de Dicentrarchus labrax. Aquaculture 31: 181-194., Corti et al. 1996Corti M., Loy A., Cataudella S. 1996. Form changes in the sea bass, Dicentrarchus labrax (Moronidae: Teleostei), after acclimation to freshwater: an analysis using shape coordinates. Env. Biol. Fishes 47: 165-175., Saillant et al. 2003Saillant A., Fostier E., Haffray P., et al. 2003. Saline preferendum for the European sea bass, Dicentrarchus labrax, larvae and juveniles: effect of salinity on early development and sex determination. J. Exp. Mar. Biol. Ecol. 287: 103-117., Rubio et al. 2005Rubio V.C., Sánchez-Vázquez F.J., Madrid J.A. 2005. Effects of salinity on food intake and macronutrient selection in European sea bass. Physiol. Behav. 85: 333-339., Giffard-Mena et al. 2008Giffard-Mena I., Lorin-Nebel C., Charmantier G., et al. 2008. Adaptation of the sea-bass (Dicentrarchus labrax) to fresh water: Role of aquaporins and Na+/K+-ATPases. Comp. Biochem. Physiol. A150: 332-338.; but see Dendrinos and Thorpe 1985Dendrinos P., Thorpe J.P. 1985. Effects of reduced salinity on growth and body composition in the European bass Dicentrarchus labrax (L.). Aquaculture 49: 333-358., Conides and Glamuzina 2006Conides A.J., Glamuzina B. 2006. Laboratory simulation of the effects of environmental salinity on acclimation, feeding and growth of wild-caught juveniles of European sea bass Dicentrarchus labrax and gilthead sea bream, Sparus aurata. Aquaculture 256: 235-245.). The observation of mortality induced by gene expression dysfunction in the absence of significant body mass response to osmoregulation illustrates the absence of a straightforward correlation between a phenotype monitored at the molecular level (gene expression, which reveals a costly situation and specialist phenotypes regarding response to a salinity challenge), and one monitored at the organismic level (body mass, which instead illustrates a no-cost, generalist phenotype). More generally, assessing the physiological costs of salinity at the organismic level seems difficult in sea bass (Claireaux and Lagardère 1999Claireaux G., Lagardère J.P. 1999. Influence of temperature, oxygen and salinity on the metabolism of the European sea bass. J. Sea Res. 42: 157-168.). It may be suggested that this dichotomy between inferred costs at the molecular and organismic levels is only nascent, and has not yet reached a new physiological equilibrium because of recent divergence between UFW and SFW sea bass phenotypes. This has been suggested for stickleback (Gasterosteus aculeatus) colonizing the freshwater and the estuarine environments from the ancestral sea environment (McCairns and Bernatchez 2010McCairns R.J.S., Bernatchez L. 2010. Adaptive divergence between freshwater and marine sticklebacks: insights into the role of phenotypic plasticity from an integrated analysis of candidate gene expression. Evolution 64: 1029-1047.).

The existence of intraspecific variation in FW tolerance through two distinct cryptic phenotypes may be linked to alternative life strategies or opportunities to face different environmental challenges in sea bass. The role of variation in osmoregulatory performances in shaping fish distributions is documented at the interspecific level (e.g. Lasserre and Gallis 1975Lasserre P, Gallis JL. 1975. Osmoregulation and differential penetration of two grey mullets, Chelon labrosus (Risso) and Liza ramada (Risso) in estuarine fish ponds. Aquaculture 5: 323-344., Plaut 1998Plaut I. 1998. Comparison of salinity tolerance and osmoregulation in two closely related species of blennies from different habitats. Fish Physiol. Biochem. 19: 181-188., Rigal et al. 2008Rigal F., Chevalier T., Lorin-Nebel C., et al. 2008. Osmoregulation as a potential factor for the differential distribution of two cryptic gobiid species, Pomatoschistus microps and P. marmoratus in French Mediterranean lagoons. Sci. Mar. 72: 469-476.). Lagoons, estuaries and lower parts of rivers act as nurseries withstanding large salinity variation (Vasconcelos et al. 2010Vasconcelos R.P., Reis-Santos P., Maia A., et al. 2010. Nursery use patterns of commercially important marine fish species in estuarine systems along the Portuguese coast. Estuar. Coast. Shelf Sci. 86: 613-624.), possibly promoting intraspecific variation in sea bass. Inter-individual variation in habitat use has been shown to favour the emergence of alternative phenotypes in order to increase niche width and/or resilience of populations to environmental impact (Räsänen and Hendry 2008Räsänen K, Hendry A.P. 2008. Disentangling interactions between adaptive divergence and gene flow when ecology drives diversification. Ecol. Lett. 11: 624-636.). Purely environmental, epigenetic and genetic mechanisms may be responsible for alternative phenotypes (Gienapp et al. 2008Gienapp P., Teplitsky C., Alho J.S., et al. 2008. Climate change and evolution: disentangling environmental and genetic responses. Mol. Ecol. 17: 167-178., Angers et al. 2010Angers B., Castonguay E., Massicotte R. 2010. Environmentally induced phenotypes and DNA methylation: how to deal with unpredictable conditions until the next generation and after. Mol. Ecol. 19: 1283-1295.). In sea bass, several studies have demonstrated a possible genetic basis related to habitat use. Indeed, lagoon individuals were found to be genetically differentiated from individuals inhabiting the buffered open sea environment (Allegrucci et al. 1997Allegrucci G., Fortunato C., Sbordoni V. 1997. Genetic structure and allozyme variation of sea bass (Dicentrarchus labrax and D. punctatus) in the Mediterranean Sea. Mar. Biol. 128: 347-358., Lemaire et al. 2000Lemaire C., Allegrucci G., Naciri M., et al. 2000. Do discrepancies between microsatellite and allozyme variation reveal differential selection between sea and lagoon in the sea bass (Dicentrarchus labrax)? Mol. Ecol. 9: 457-467.). Further studies of other marine, coastal, euryhaline fish species by, e.g., Blel et al. (2010Blel H., Panfili J., Guinand B., et al. 2010. Selection footprint at the first intron of the Prl gene in natural populations of the flathead mullet (Mugil cephalus, L. 1758). J. Exp. Mar. Biol. Ecol. 387: 60-67.; Mugil cephalus), Chaoui et al. (2012Chaoui L., Gagnaire P.A., Guinand B., et al. 2012. Microsatellite length variation in candidate genes correlates with habitat in the gilthead sea bream Sparus aurata. Mol. Ecol. 21: 5497-5515.; Sparus aurata), González-Wangüemert and Pérez-Ruzafa (2012González-Wangüemert G, Pérez-Ruzafa Á. 2012. In two waters: contemporary evolution of lagoonal and marine white seabream (Diplodus sargus) populations. Mar. Ecol. 33: 337-349.; Diplodus sargus) and González-Wangüemert and Vergara-Chen (2014González-Wangüemert M, Vergara-Chen C. 2014. Environmental variables, habitat discontinuity and life history shaping the genetic structure of Pomatoschistus minutus. Helgol. Mar. Res. 68: 357-371.; Pomatoschistus minutus) reported similar (but still not fully understood) observations. However, the absence of recognized polymorphic markers in GHR, and the non-significant relationship between gene expression level of IGF-1 and genotypes at the µIGF-1 locus in UFW, SSW and SFW individuals does not allow us to go any further with regard to a possible genotype-phenotype relationship in sea bass. However, targeted genome scans must be performed in sea bass, following, e.g. the study by Shikano et al. (2010)Shikano T., Ramadevi J., Merilä J. 2010. Identification of local- and habitat-dependent selection: scanning functionally important genes in nine-spined sticklebacks (Pungitius pungitius). Mol. Biol. Evol. 27: 2775-2789. on nine-spined stickleback (Pungitius pungitius) inhabiting contrasted freshwater and marine habitats.

The molecular basis of freshwater tolerance/adaptation must be investigated further in sea bass. Indeed, many gene networks may have shifted their expression profiles. The observed changes in IGF-1 and GHR may be part of such failures, downstream effects or parallel effects and not necessarily a direct response to low salinity or the cause of the observed deaths. The genomic resources now available for sea bass (Kuhl et al. 2010Kuhl H., Beck A., Wozniak G., et al. 2010. The European sea bass Dicentrarchus labrax genome puzzle: comparative BAC-mapping and low coverage shotgun sequencing. BMC Genomics 11: 68.; Magnanou et al. 2014Magnanou E., Klopp C., Noirot C., et al. 2014. Generation and characterization of the sea bass Dicentrarchus labrax brain and liver transcriptomes. Gene 544:56-66.) and dedicated molecular techniques such as microarrays (Ferraresso et al. 2010Ferraresso S., Milan M., Pellizzari C., et al. 2010. Development of an oligo DNA microarray for the European sea bass and its application to expression profiling of jaw deformity. BMC Genomics 11 : 354.) and RNA sequencing (Wang et al. 2009Wang Z., Gerstein M, Snyder M. 2009. RNA-Seq: a revolutionary tool for transcriptomics. Nat. Rev. Genet. 10: 57-63) may help to achieve this goal at scales far above the levels of expression of a few genes (e.g. Norman et al. 2011Norman J.D., Danzmann R.G., Glede B., et al. 2011. The genetic basis of salinity tolerance traits in Arctic charr (Salvelinus alpinus). BMC Genet. 12: 81., Avarre et al. 2014Avarre J.C., Dugué R., Alonso P., et al. 2014. Analysis of the black-chinned tilapia Sarotherodon melanotheron heudelotii reproducing under a wide range of salinities: from RNA-seq to candidate genes. Mol. Ecol. Res. 14: 139-142.). This should especially motivate future studies (i) in cultured fish for which selective breeding programmes have been initiated (Chatain and Chavanne 2009Chatain B., Chavanne H. 2009. La génétique du bar (Dicentrarchus labrax, L.). Cah. Agric. 18: 249-255.) and which may need to control for cryptic phenotypes, and (ii) in wild juvenile sea bass distributed naturally over a large range of salinity conditions, but also trophic or temperature conditions that also greatly vary between habitats, to investigate molecular mechanisms involved in the lagoon/estuarine life compared with the coastal/pelagic life in this species.

AcknowledgementsTop

The authors thank G. Sposito for kindly making available the fish used in this study and M. Cantou and L. Libicz for their help in fish husbandry at the aquaculture facilities of the Station Méditerranéenne de l’Environnement Littoral. Comments by two anonymous reviewers improved the manuscript. Molecular data were produced with the technical facilities of the qPHD and SeqGen platforms (LabEx CeMEB, Montpellier).

ReferencesTop

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