Josipa Ferri
email: josipa.ferri@unist.hr

Natalija Topić Popović

Rozelinda Čož-Rakovac

Blanka Beer-Ljubić

Ivančica Strunjak-Perović

Frane Škeljo

Margita Jadan

Mirela Petrić

Josip Barišić

Miljenko Šimpraga

Blanka Beer- Ljubić

Rino Stanić

The

Keywords: Oblada melanura, Aquaculture, Food source, Blood chemistry, Liver histology, Effects-physiology, Adriatic

est destinée au dépôt

Introduction

Many fish species, from larvae to adults, are attracted to a high variety of natural and artificial objects [START_REF] A N U S C R I P T A C C E P T E D Accepted Manuscript | Fish aggregation device (FAD) research: gaps in current knowledge and future directions for ecological studies[END_REF]. One of the most important artificial structures in Mediterranean pelagic systems are floating fish farms, whose strong aggregative effect on wild fish may be explained by increased food availability [START_REF] Castro | A general theory on fish aggregation to floating objects: An alternative to the meeting point hypothesis[END_REF]Tuya et al., 2006). The phenomenon is widespread and dense aggregations around fish farms have been described across the Mediterranean Sea [START_REF] Sanchez-Jerez | Ecological relationship between wild fish populations and Mediterranean aquaculture in floating fish cages. Impact of mariculture on coastal ecosystems[END_REF][START_REF] Fernandez-Jover | Seasonal patterns and diets of wild fish assemblages associated to Mediterranean coastal fish farms[END_REF]. Although ecological impacts of fish farming on marine ecosystems have been widely documented [START_REF] A N U S C R I P T A C C E P T E D Accepted Manuscript | Effect of aquaculture on world fish supplies[END_REF], impacts on behaviour and physiology of attracted wild fish species are scarcely reported. Attracted fish change diet, using uneaten food pellets as the main resource, which increases their biological condition but also affects the fatty acid composition [START_REF] Sanchez-Jerez | Ecological relationship between wild fish populations and Mediterranean aquaculture in floating fish cages. Impact of mariculture on coastal ecosystems[END_REF] and blood chemistry profile [START_REF] Coz-Rakovac | Cage culture effects on mullets (Mugilidae) liver histology and blood biochemistry profile[END_REF]. Better understanding of the ecological effects of farming on wild fish populations is necessary, as the production from sea-cages continues to expand (FAO, 2008).

The saddled bream, Oblada melanura (Linnaeus, 1758), is common throughout the Mediterranean (including the Adriatic) and the eastern Atlantic, inhabiting littoral waters above rocky bottoms and Posidonia beds, down to 30 m deep [START_REF] Bauchot | Sparidae, in: Fishes of the North-eastern Atlantic and the Mediterranean[END_REF]. [START_REF] Sanchez-Jerez | Ecological relationship between wild fish populations and Mediterranean aquaculture in floating fish cages. Impact of mariculture on coastal ecosystems[END_REF] and [START_REF] Fernandez-Jover | Coastal fish farms are settlement sites for juvenile fish[END_REF] reported that O. melanura is one of the most abundant species found closely associated with the cages (south-eastern Spain), which are the settlement sites for juvenile saddled bream individuals. In the eastern Adriatic, aggregations of this opportunistic predator around cage installations are recently reported by fish farmers (personal observation), while wild populations of mullets, sea bass and sea bream are already documented as regular feeders in such areas [START_REF] Coz-Rakovac | Cage culture effects on mullets (Mugilidae) liver histology and blood biochemistry profile[END_REF]2009).

Measurement of blood biochemistry parameters is commonly used diagnostic tool in biomonitoring and according to [START_REF] Coz-Rakovac | Cage culture effects on mullets (Mugilidae) liver histology and blood biochemistry profile[END_REF] it presents good indicator of the artificial feed effects. In order to document and better understand variability and dynamics of

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blood chemistry parameters in general, few studies were conducted on the sea bass, Dicentrarchus labrax [START_REF] Coz-Rakovac | Blood chemistry and histological properties of wild and cultured sea bass (Dicentrarchus labrax) in the north Adriatic Sea[END_REF], sea bream, Sparus aurata [START_REF] Coz-Rakovac | Classification accuracy of algorithms for blood chemistry data for three aquacultureaffected marine fish species[END_REF], bluefin tuna, Thunnus thynnus [START_REF] Topic Popovic | Plasma metabolites and enzymes of bluefin tuna, Thunnus thynnus and liver histology[END_REF]2008), mullets [START_REF] Coz-Rakovac | Cage culture effects on mullets (Mugilidae) liver histology and blood biochemistry profile[END_REF] and salmonids [START_REF] Coz-Rakovac | Aminotransferase responses of coho salmon (Oncorhynchuss kisutch, Walbaum) to sea water challenge test in relation to temperature and salinity[END_REF] in the eastern Adriatic.

Although there is a lack of the basic data for the saddled bream, some baseline values of haematological parameters are available for other sparid species [START_REF] Bollard | Effects of artificially elevated plasma cortisol levels on blood parameters in the teleost fish Pagrus auratus (Sparidae)[END_REF][START_REF] Carragher | Plasma levels of sex steroids during sexual maturation of snapper, Pagrus auratus (Sparidae), caught from the wild[END_REF][START_REF] Coz-Rakovac | Blood chemistry and histological properties of wild and cultured sea bass (Dicentrarchus labrax) in the north Adriatic Sea[END_REF]2009). Therefore, there is a need for the basic blood chemistry values of O. melanura, as well as for the evaluation of the impact of floating fish farms on this species population.

The aim of this study was to investigate eleven blood biochemistry parameters (enzymes and metabolites) of the saddled bream captured in remote waters and nearby the fish farm in the central-eastern Adriatic. Moreover, measured blood parameters were correlated to different feeding patterns of the investigated species sampled from two localities, and could serve as biomarkers to infer the influence of floating fish farms to the local fish community. In order to assess the impact of different feeding regimes, the liver histomorphology of cage-associated and natural/control specimens was evaluated in detail.

This paper presents the first data on the baseline blood chemistry values of O. melanura, species of a commercial interest in the Mediterranean and the Adriatic.

Materials and methods

Fish sampling

The saddled bream (O. melanura) specimens were collected during daylight hours using handline and purse seine. Fish samples were obtained from two localities in the centraleastern Adriatic located about 30 km apart, from the remote waters (between islands Orud and Stipanska) and nearby the sea bass (D. labrax) and sea bream (S. aurata) rearing cages (Maslinova Bay). The farm at Maslinova Bay was at a depth of 30 to 40 m, with 22 cages of 12.5 m diameter and 8 m depth, 12 cages of 20 m diameter and 10 m depth and 5 cages of 30 m diameter and 10 m depth, producing almost 300 t yr -1 . A total of 71 O. melanura were collected: 28 specimens caught in the immediate vicinity of the cages (cage-associated) using handline and 43 specimens caught in the remote waters (natural/control) using a purse seine.

Cage-associated and control fishes were caught on two different random days but during the same period (June 2009) to avoid seasonal variation in further analyses. Sampled saddled

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bream did not manifest any external trauma, scars or injuries. Stress was minimized in duration, due to the small groups of fish that were selected for sampling at the time period, and rapid handling.

Blood sampling and biochemical analyses

Blood samples were collected from all sampled fish by caudal vein puncture, immediately after their capture. Samples were collected in tubes coated with anticoagulant lithium heparin, centrifuged at 12 000 g for 90 seconds and the resultant plasma was frozen at -20ºC for storage until the analysis two weeks later. The biochemical analyses of parameters, such as aspartate aminotransferase (AST), gamma glutamyl transferase (GGT), creatine kinase (CK), lactate dehydrogenase (LDH), alkaline phosphatase (ALKP), glucose (GLU), urea (URE), creatinine (CRE), total proteins (TP), triglyceride (TRIG) and plasma cholesterol (CHOL), were carried out using a SABA-18 auto-biochemistry analyzer (AMS Analyzer Medical System, Rome, Italy).

Liver collection and histological analyses

Saddled bream were euthanized by severing the spinal cord, total length (TL, measured to the nearest 0.1 cm) and weight (W, measured to the nearest 0.1 g) were recorded and sex was determined macroscopically by gonad observation. Total length of collected cage-associated and control saddled bream specimens ranged from 18.2 to 28.6 cm and from 24.0 to 30.5 cm, respectively. Cage-associated fish ranged from 142.0 to 381.0 g weight while control fish ranged from 62.8 to 272.8 g weight. During necropsy, pieces of liver tissues were dissected and fixed in 10% buffered formalin for at least two weeks. After completion of fixation, tissue was removed from the fixative, dehydrated in a graded ethanol series (from 70% to 100%), cleared in xylene and embedded in paraffin. Histological sections were cut at 5 µm, stained with haematoxylin-eosin and mounted permanently for microscopic analysis.

All slides were examined 'blind' (to eliminate bias in interpretation) for abnormalities, such as lipid infiltration, degeneration and/or necrosis of hepatocytes. Pictures were loaded in Olympus cell^A Imaging Software to measure area (µm 2 ) and perimeter (µm) (average ± SD) of hepatocytes and nuclei.

Statistical analyses

Blood parameter values were tested for normality (Kolmogorov-Smirnov test of normality) and homogeneity of variance (Levene's test). Because these assumptions were not

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met by most of the plasma biochemical values, all data were log(x+1) transformed before performing any statistical analysis. Relationships between fish length, weight and blood parameters of both sampled fish populations were tested using Spearman rank correlation. Two sample t-test was used to evaluate differences in condition index (CI = (100×W)/TL 3 ), blood parameters and area/perimeter of hepatocytes and nuclei between cage-associated and control fish population. A P value < 0.05 was considered to be significant, while a P value < 0.01 was considered to be highly significant.

In order to elucidate which combination of the plasma biochemical parameters best discriminates between two sampled saddled bream populations, eight parameters (meeting the assumptions of discriminant analysis) were subjected to the stepwise discriminant-function analysis (F to enter = 1.0; F to remove = 0.0) (statistical package STATISTICA, version 8.0.).

The basic principle of discriminant analysis is that groups of specimens can be divided according to functions that are linear combinations of the classifying variables (in this case, the biochemical parameters). We employed stepwise discriminant-function analysis approach, where at each step all variables are reviewed and evaluated to determine which one will contribute most to the discrimination between groups. That variable will then be included in the model, and the process starts again.

Plasma biochemical parameters of the saddled bream were log(x+1) transformed and employed to construct a Bray-Curtis similarity matrix using the PRIMER 5 multivariate statistical package (v. 5.2.9; PRIMER-E Ltd). Resulting rank similarity matrix was subjected to non-metric multidimensional scaling (MDS) ordination, and subsequent one-way analysis of similarity (ANOSIM) to test whether the measured biochemical parameters differed significantly between cage-associated and control saddled bream populations.

Results

Necropsy of cage-associated fish revealed large amounts of visceral fat and yellowish liver while no macroscopic changes were found in control fishes. Moreover, during necropsy we observed the presence of commercial artificial feed in the digestive tract of most cageassociated specimens (21 out of 28), while guts of control fishes usually contained only small amounts of natural food. Significant differences existed in the condition index between studied fish groups. Cage-associated saddled bream had a higher CI (average 1.23 ± SD 0.13) than the control fish (average 1.14 ± SD 0.06; P = 0.001).

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Blood chemistry

Because no significant differences were found in blood parameter values between males and females of cage-associated and normal/control saddled bream, sexes were pooled within each population. Values of GLU and CHOL showed strong departure from normality even when log(x+1) transformed so they were excluded from all statistical tests assuming normal data distribution. Moreover, GGT values were 0.00 in all but one case, so they were also excluded from further analyses.

As shown in Table 1, four plasma biochemical parameters varied significantly, with the exception of CK, CRE, TP and TRIG concentrations which displayed insignificant variation between cage-associated and control fishes. The levels of LDH, ALKP and URE in cage-associated saddled bream were significantly lower than those in control individuals. In contrast to that, the concentration of AST in cage-associated fish was higher than in the control population.

Spearman rank correlation of fish length, weight and plasma chemistry was conducted separately for control (Table 2) and cage-associated (Table 3) saddled bream. For control population, ALKP values significantly correlated (P < 0.05) with fish length, weight, CK, TP, TRIG and CHOL. AST increased with GLU, TP and CHOL, while CK increased with ALKP, CRE and TRIG (P < 0.05 in all cases) (Table 2). For the cage-associated population, different pattern in correlation between parameters was observed (Table 3). The levels of ALKP increased with TP, TRIG and GLU while CK increased together with AST and GLU (P < 0.05 in all cases). Within the cage-associated population, significant negative correlation was estimated between GLU and LDH, CHOL and fish length, and URE and fish weight (Table 3).

Eight parameters were subjected to the stepwise discriminant function analysis, which included URE, AST, LDH and ALKP in the model, whereas CK, CRE, TP and TRIG were excluded (F to enter = 1.0). The main variables contributing to the discrimination between cage-associated and control group were URE and AST (Table 4). Four included variables produced clear distinction between analysed groups, as indicated by a final Wilks' Lambda of 0.23. The overall classification success of the discriminant analysis was 96%, with correct affiliation of 93.33% in the cage-associated and 97.14% in the control group (Table 5).

Although these values are very high, they were obtained by classifying cases from which the discriminant functions were computed, thus capitalizing on chance. A lesser accuracy can be expected when classifying new specimens based just on these four parameters.

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One-way ANOSIM (analysis of similarity) based on blood parameters showed high significant difference between cage-associated and natural/control population (R = 0.697; P < 0.01) (Fig. 1).

Liver histomorphology

Microscopic analysis of the liver sections demonstrated that control saddled bream specimens had no lipid accumulations, and hepatocytes were characterized with granular cytoplasm and central and round nucleus (Fig. 2A). The average area (µm 2 ) of hepatocytes and nuclei were 67.63 (± SD 10.15) and 14.26 (± SD 2.09), respectively (Table 6). The main alterations found in liver sections of cage-associated saddled bream were: irregular shaped nuclei, cytoplasmatic and nuclear degeneration, and melanomacrophages (Fig. 2B and Fig. 2C) which were identified as aggregates of cells containing dark-yellowish granules, normally close to the vessels. In general, hepatocytes of cage-associated fish contained large cytoplasmatic clear spaces which present excessive accumulation of fat in the hepatocyte cytoplasm (Fig. 2C). When comparing sizes of hepatocytes and nuclei between cageassociated and control saddled bream, all sizes were significantly larger in cage-associated saddled bream (P < 0.01 in all cases) (Table 6).

Discussion

The saddled bream, O. melanura is a common member of the aggregated assemblage at the studied Adriatic fish farm (personal observation). However, small migrations of this species influence their abundances around cages throughout a year. Fish farmers observed the highest abundance of the saddled bream during summer and the lowest during winter months, when they are almost completely absent, and this corresponds to the data for this species around the fish farms in the western Mediterranean [START_REF] Sanchez-Jerez | Ecological relationship between wild fish populations and Mediterranean aquaculture in floating fish cages. Impact of mariculture on coastal ecosystems[END_REF]. The wild saddled bream feeds on a wide range of prey and could be considered as an opportunistic predator [START_REF] Pallaoro | Feeding habits of the saddled bream, Oblada melanura (Sparidae), in the Adriatic Sea[END_REF]. The results of the present observational study showed a change in the feeding behaviour of O. melanura around the cages because the majority of fish had pellets in their guts. Beside this qualitative observation of fish digestive tracts, future studies should quantify actual amounts of commercial feed in cage-associated fish. The changes in diet can in turn affect the condition index [START_REF] Sanchez-Jerez | Ecological relationship between wild fish populations and Mediterranean aquaculture in floating fish cages. Impact of mariculture on coastal ecosystems[END_REF], what is confirmed in this research. Cage-associated saddled bream had significantly higher condition index than control

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fish, most probably due to a diet based on the lost food pellets and a relative lack of exercise because of abundant, low-metabolic cost feeding opportunity. The same results were reported for the Mediterranean horse mackerel, Trachurus mediterraneus [START_REF] Fernandez-Jover | Changes in body condition and fatty acid composition of wild Mediterranean horse mackerel (Trachurus mediterraneus, Steindachner, 1868) associated to sea cage fish farms[END_REF] and saithe, Pollachius virens [START_REF] Skog | Salmon farming affects the fatty acid composition and taste of wild saithe Pollachius virens L[END_REF]. Furthermore, observed change of the feeding pattern of the saddled bream around cages influenced its blood indices when compared to active feeders of the natural population. A wide range of most blood chemistry values in both studied populations indicated high intraspecies variability. This characteristic feature of fish has been attributed to factors such as genetic variation, nutritional status, sex, age, and stress due to capture, handling and sampling procedures [START_REF] Tavares-Dias | Comparative study on hematological parameters of farmed matrinxã, Brycon amazonicus Spix and Agassiz, 1829 (Characidae: Bryconinae) with others Bryconinae species[END_REF].

Moreover, because some blood chemistry variables are responsive to multiple endogenous and exogenous influences, the physiological significance of changes in blood chemistry can be uncertain (Wagner and Congleton, 2004) and caution must be taken when interpreting the results.

The lack of data of the aquaculture's impact on fish blood chemistry makes it difficult to evaluate our results. Some similarities can be noted with the work of Coz-Rakovac et al.

(2008) who studied cage culture effect on blood chemistry profile of mullets. Results of both studies indicated that cage-associated fish exhibited a distinctly different response from the wild fish of the control population. In our study, URE and AST contributed the most to variations between two studied populations. According to Walsh et al. (2003), URE is present in all fish, and while the liver is the primary organ of its production, gills are the main organ of its excretion. The relatively low urea levels, as observed in cage-associated specimens, are usually the consequence of low protein diet [START_REF] Topic Popovic | Plasma metabolites and enzymes of bluefin tuna, Thunnus thynnus and liver histology[END_REF], which we find unlikely according to the protein-rich diet of cage-associated fish. The enzyme AST is found in many fish tissues and therefore presents non-specific indicator of damage (Wagner and Congleton, 2004). According to [START_REF] Coz-Rakovac | Cage culture effects on mullets (Mugilidae) liver histology and blood biochemistry profile[END_REF], the activity of this enzyme rises with fish starvation in natural/control population of mullets. However, this was not the case in our study because higher AST levels were observed in well fed specimens around cages. In addition, the fact that non-aggregated individuals are not eating a high-energy diet it does not mean that they starve. The increased AST activity in fish may reveal the possible leakage of this enzyme across damaged plasma membranes and/or the increased synthesis of the AST by the liver [START_REF] Coz-Rakovac | Blood chemistry and histological properties of wild and cultured sea bass (Dicentrarchus labrax) in the north Adriatic Sea[END_REF]. The levels of AST showed positive correlation with the weight of specimens around cages indicating that an increase of transamination processes occurred with the increase of their respective body mass. The impact of the different living

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conditions and feeding regimes is evident, if we consider the significant differences of both parameters (URE and AST) between two studied populations.

Previous studies have shown that TP, CHOL and ALKP are responsive to changes in fish nutritional status. [START_REF] Storebakken | Nutrient partitioning in rainbow trout at different feeding rates[END_REF] found that plasma concentrations of TP were positively correlated with feeding level in rainbow trout (Oncorhynchus mykiss), while other studies have shown declines in fish plasma TP during fasting [START_REF] Navarro | Fasting and starvation[END_REF]Wagner and Congleton, 2004). On the contrary, [START_REF] Coz-Rakovac | Cage culture effects on mullets (Mugilidae) liver histology and blood biochemistry profile[END_REF] found no correlation between TP values and the fish feeding regime. The observed lower TP values in well fed O. melanura around cages are similar to those estimated for the aquaculture-affected sea bream [START_REF] Coz-Rakovac | Classification accuracy of algorithms for blood chemistry data for three aquacultureaffected marine fish species[END_REF], but any conclusions that are not supported by significant results would be very speculative. Moreover, although a difference in plasma CHOL between sampled populations was observed, its significance could not be determined due to the strong departure from normality of even log-transformed values. According to [START_REF] Mensinger | Blood Biochemistry of the Oyster Toadfish[END_REF], CHOL levels can indicate disorders of lipid and lipoprotein metabolism and especially liver dysfunction. The polyfunctional enzyme ALKP is located primarily in the cell membrane, and has no known function in the blood, while plasma activities depend on the rate of leakage from cells and the rate of removal from the plasma (Wagner and Congleton, 2004). As well as TP plasma values, higher activity of ALKP was observed in specimens of control population, for which the metabolic activity of this enzyme increases as the fish grows (in both length and weight). Contrary to our results, high activity of ALKP is directly related to the increased feeding activity of fish, as suggested by Wagner and Congleton (2004). Positive correlation between these two parameters (ALKP and TP) was observed in both sampled populations and therefore pointed on their metabolic linkages.

Concentrations of GLU are also accepted as indicators of fish nutritional status.

However, plasma GLU is highly responsive to stressors, so its variation in blood is likely to be more closely correlated with other stress indices than with nutritional status (Wagner and Congleton, 2004). A noticeable difference in plasma GLU levels between sampled fish populations was observed, but its significance could not be determined due to the strong departure from normality and equality of variance of even log-transformed GLU values.

However, we can just point out that observed GLU values of both saddled bream populations were lower than those estimated for the aquaculture-affected sea bream [START_REF] Coz-Rakovac | Classification accuracy of algorithms for blood chemistry data for three aquacultureaffected marine fish species[END_REF]. The levels of TRIG are considered to be major indices of the health status of teleost, but they are not a reliable indicator of fish nutritional status (Wagner and Congleton, 2004).

On the contrary, [START_REF] Kavadias | Seasonal variation in steroid hormones and blood parameters in cage-farmed European sea bass (Dicentrarchus labrax L.)[END_REF] demonstrated that high concentrations of plasma

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TRIG depend on the feeding rate while low levels can be explained by fish low-energy diet.

However, no significant differences in the TRIG values between cage-associated and control saddled bream were observed in this study so conclusions can not be drawn.

Beside AST, CK and LDH are also useful indices of tissue damage because they may be released quite readily when only minor changes occur in the morphology and function of cells (Wagner and Congleton, 2004). The enzyme CK, which is concentrated in muscle and heart tissue, is an indicator of damage to one or both of these tissues and plays a key role in the energy homeostasis of cells. Elevated CK levels can be caused by many stressful events [START_REF] Shahsavani | Determination of normal values of some blood serum enzymes in Acipenser stellatus Pallas[END_REF] but no significant differences in this enzyme values between the two sampled populations were observed. According to [START_REF] Shahsavani | Determination of normal values of some blood serum enzymes in Acipenser stellatus Pallas[END_REF], LDH catalyzes the conversion of lactate to pyruvate which is an important step in energy production of cells.

High levels of LDH could be connected with hepatic necrosis, hepatic parenchyma lesions, fatty liver, skeletal muscle lesions, myocardial infraction, tumours [START_REF] Topic Popovic | Plasma metabolites and enzymes of bluefin tuna, Thunnus thynnus and liver histology[END_REF] and many other possibilities. Interestingly, in our study, statistically higher LDH values were noted for control specimens, and not for cage-associated, which showed a fatty degeneration of liver hepatocytes. In general, the mean values of LDH of both populations were much higher than those reported for freshwater tilapia (Oreochromis niloticus) or juvenile red pacu (Piaractus brachypomus), which did not exceed 240 U/L [START_REF] Leamaster | Haematologic and blood chemistry values for Sarotherodon melanotheron and a red hybrid tilapia in freshwater and seawater[END_REF][START_REF] Sakamoto | Blood chemistry values of juvenile red pacu (Piaractus brachypomus)[END_REF]. [START_REF] Luskova | Yearly dynamics of enzyme activities and metabolite concentrations in blood plasma of Chondrostoma nasus[END_REF] observed significant correlation between LDH and CK levels in the nase, Chondrostoma nasus, but we have not found metabolic linkage of these parameters for the saddled bream.

Almost all CRE is excreted by the kidney so increased concentrations may reflect kidney dysfunction due to the structural damage [START_REF] Burtis | Tietz fundamentals of clinical chemistry[END_REF]. However, we evaluated our results only according to different feeding behaviour, and the results showed positive correlation between CRE concentrations and the feeding level of the studied species.

Detectable levels of GGT in plasma could not be established in previous studies [START_REF] Manera | Assessment of blood chemistry normal ranges in rainbow trout[END_REF][START_REF] Topic Popovic | Plasma metabolites and enzymes of bluefin tuna, Thunnus thynnus and liver histology[END_REF], what is in agreement with our data if we exclude only one individual with detected value (maybe due to the sample artefact).

The MDS analysis showed that blood chemistry profile of O. melanura aggregated around fish farm was to some degree variable than that of the specimens caught in the wild.

This could be the indicator of the recent arrival of new individuals at the farm. Moreover, different residence time at farms or short migrations of individual fish may explain differences in some parameters among the cage-associated fish. Unfortunately, we do not know the exact residence time of the sampled fish which could be helpful in results

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evaluation, but we can assume that O. melanura could have been resident for a maximum four months prior to our sampling in June (personal observation).

Comparative histological studies of liver of the saddled bream from two sampled populations provided an index to their general condition. Observed liver modifications in cage-associated specimens confirmed an evident effect of artificial feed and are reaction to imbalanced feeding or a nutritional pathological process [START_REF] Tacon | Lipid nutritional pathology in farmed fish[END_REF][START_REF] Caballero | Histological alterations in the liver of sea bream, Sparus aurata L., caused by short-or long-term feeding with vegetable oils. Recovery of normal morphology after feeding fish oil as the sole lipid source[END_REF].

Anomalies such as irregular shaped hepatocytes and cytoplasmatic vacuolation were also described in populations of mullets [START_REF] Coz-Rakovac | Cage culture effects on mullets (Mugilidae) liver histology and blood biochemistry profile[END_REF] and sea bass [START_REF] Coz-Rakovac | Blood chemistry and histological properties of wild and cultured sea bass (Dicentrarchus labrax) in the north Adriatic Sea[END_REF] aggregated around fish farms. In general, this observed morphological pattern found in liver of cage-associated O. melanura is known as steatosis and was documented as a significant problem in cultured fish, specifically in sea bream [START_REF] A N U S C R I P T A C C E P T E D Accepted Manuscript Benedito-Palos | High levels of vegetable oils in plant protein-rich diets fed to gilthead sea bream (Sparus aurata L.): growth performance, muscle fatty acid profiles and histological alterations of target tissues[END_REF].

The fatty degeneration of hepatocytes resulted in their significantly larger sizes when compared to hepatocytes of control specimens. Moreover, an increase in the density of the melanomacrophages, as observed for cage-associated fish in our study, is usually related to important hepatic lesions, such as degenerative and necrotic processes [START_REF] Pacheco | Biotransformation, genotoxic and histopathological effects of environmental contaminants in European eel (Anguilla anguilla L.)[END_REF][START_REF] Agius | Melano-macrophage centres and their role in fish pathology[END_REF].

In conclusion, O. melanura aggregated around fish farm in the eastern Adriatic underwent physiological changes. Qualitative observations during necropsy confirmed that their diet differed from control fish, and this affected their condition index, blood chemistry values and liver histomorphology. Reported data from this observational study are useful for evaluation of the health status of the wild saddled bream in both control and cage-associated conditions. However, further studies should include more fish farms and control locations, provide exact information of the residence time of the aggregated saddled bream around cages and the magnitude of their migrations in order to determine the scale of the influence of seacage fish farms.

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Tuya, F., Sanchez-Jerez, P., Dempster, T., Boyra, A., and Haroun, R., 2006. Changes in demersal wild fish aggregations beneath a sea-cage fish farm after the cessation of farming. Journal of Fish Biology 69, 682-697.

Valle, C., Bayle-Sempere, J.T., Dempster, T., [START_REF] Fernandez-Jover | Changes in body condition and fatty acid composition of wild Mediterranean horse mackerel (Trachurus mediterraneus, Steindachner, 1868) associated to sea cage fish farms[END_REF] Temporal variability of wild fish assemblages associated with a sea-cage fish farm in

Figure 1 .

 1 FIGURE LEGENDS

Table 1 .

 1 Comparative biochemical analysis of plasma parameters (AST -aspartate aminotransferase, GGT -gamma glutamyl transferase, CK -creatine kinase, LDH -lactate dehydrogenase, ALKP -alkaline phosphatase, GLU -glucose, URE -urea, CRE -creatinine,

	TABLES
	TP -total proteins, TRIG -triglyceride, CHOL -cholesterol) of natural/control and cage-
	associated O. melanura
	Parameters	natural/control population Average ± SD Range	cage-associated population Average ± SD Range	P
	AST (U/L)	6.55 ± 5.90	0.17-21.30	87.63 ± 132.34	1.21-443.40	P < 0.01
	GGT (U/L)	-	0.00-0.00	3.21 ± 17.01	0.00-90.00	-
	CK (U/L)	62.17 ± 86.72	4.00-400.00	451.14 ± 1367.51 6.00-5186.00 P > 0.05
	LDH (U/L)	1047.06 ± 505.56 128.00-1762.00 428.00 ± 321.56 96.00-1134.00 P < 0.01
	ALKP (U/L)	125.75 ± 34.70	86.00-170.00	86.13 ± 39.87	40.00-176.00	P < 0.05
	GLU (mmol/L)	5.75 ± 4.82	0.02-15.48	2.03 ± 2.47	0.11-6.71	-
	URE (mmol/L)	1.99 ± 0.73	0.10-3.20	0.05 ± 0.16	0.00-0.60	P < 0.01
	CRE (µmol/L)	41.08 ± 8.57	7.92-54.46	39.22 ± 13.86	22.56-84.44	P > 0.05
	TP (g/L)	45.78 ± 28.73	26.00-118.00	35.80 ± 15.59	16.00-56.00	P > 0.05
	TRIG (mmol/L)	2.74 ± 2.17	0.09-8.72	4.70 ± 6.67	0.04-19.59	P > 0.05
	CHOL (mmol/L)	4.31 ± 3.74	0.00-10.16	7.62 ± 3.82	3.23-15.43	-

Table 2 .

 2 Spearman rank correlation of fish length (TL), weight (W) and plasma chemistry variables (AST -aspartate aminotransferase, CK -creatine kinase, LDH -lactate dehydrogenase, ALKP -alkaline phosphatase, GLU -glucose, URE -urea, CRE -creatinine, TP -total proteins, TRIG -triglyceride) of natural/control O. melanura (*P < 0.05)

		TL	W	AST	CK	LDH ALKP GLU	URE CRE	TP	TRIG
	W	0.99*
	AST	0.28	0.33
	CK	-0.05 -0.02 -0.06
	LDH	-0.27 -0.29	0.15	0.13
	ALKP 0.76* 0.74*	0.45	0.97* 0.29
	GLU	0.04	0.06	0.61* -0.25 -0.10 -0.38
	URE	-0.06 -0.00 -0.09	0.29	-0.33	0.14	0.21
	CRE	0.00	0.04	0.09	0.35* 0.03	0.25	0.22	0.17
	TP	0.27	0.35	0.78*	0.65	0.43 0.81* -0.34	0.00	0.15
	TRIG	0.03	0.13	0.35	0.56* 0.26 0.85*	0.01	0.36	-0.07 0.47
	CHOL 0.08	0.12	0.64*	0.06	0.06 0.87* 0.70* 0.60* 0.21	0.50 0.78*

Table 3 .

 3 Spearman rank correlation of fish length (TL), weight (W) and plasma chemistry variables (AST -aspartate aminotransferase, CK -creatine kinase, LDH -lactate dehydrogenase, ALKP -alkaline phosphatase, GLU -glucose, URE -urea, CRE -creatinine, TP -total proteins, TRIG -triglyceride) of cage-associated O. melanura (*P < 0.05)

		TL	W	AST	CK	LDH ALKP GLU URE CRE	TP TRIG
	W	0.87*
	AST	0.51	0.71*
	CK	0.15	0.18	0.85*
	LDH	-0.38	-0.25	-0.32 -0.28
	ALKP	-0.02	0.18	0.16 0.47	-0.28
	GLU	0.08	0.15	0.10 0.55* -0.61* 0.65*
	URE	-0.42	-0.59* -0.06 0.17	0.04	-0.41 -0.28
	CRE	0.04	0.16	0.12 -0.02	0.06	0.10 -0.05 0.10
	TP	-0.17	0.04	-0.25 -0.03	-0.14	0.68* 0.30 -0.45 -0.20
	TRIG	-0.09	0.16	0.02 0.41	-0.25	0.53* 0.60* -0.35 -0.33 0.75*
	CHOL -0.69*	-0.63	-0.07 0.37	-0.21	0.48	0.10	-	-0.35 0.45 0.72*

Table 4 .

 4 Discriminant function analysis summary for the included plasma parameters (UREurea, AST -aspartate aminotransferase, LDH -lactate dehydrogenase, ALKP -alkaline phosphatase) of natural/control and cage-associated O. melanura

	Parameters	Wilks' Lambda	Partial Lambda	F-remove (1.45)	P-level	Tolerance	R 2
	URE	0.502005	0.444692	56.19358	0.000000	0.958767	0.041233
	AST	0.294230	0.758720	14.31042	0.000455	0.877331	0.122669
	LDH	0.242332	0.921207	3.84898	0.055979	0.992306	0.007694
	ALKP	0.236512	0.943877	2.67571	0.108867	0.913318	0.086682

Table 5 .

 5 Classification matrix of the discriminant analysis between natural/control and cageassociated O. melanura, based on the blood parameters (URE -urea, AST -aspartate aminotransferase, LDH -lactate dehydrogenase and ALKP -alkaline phosphatase)

	Predicted group

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Research highlights -Oblada melanura around the cages used food pellets as the food resource -change of the feeding pattern affected its blood indices (some values differed significantly between two studied populations) and liver histomorphology -morphological pattern in liver, known as steatosis, occurred in cage-associated fish


Suzanne Faxneld
email: suzanne@ecology.su.se

Tove Lund Jörgensen

Ngai D Nguyen

Magnus Nyström

Michael Tedengren

Differences in physiological response to increased seawater temperature in nearshore and offshore corals in northern Vietnam

Keywords: Increased water temperature, Galaxea fascicularis, Porites lutea, Disturbance, Physiological responses, High latitude reefs, Vietnam

Effects of elevated seawater temperature show high spatial heterogeneity and variation within and among coral species. The objective of this study was to investigate how two coral species, Porites lutea and Galaxea fascicularis, from two high latitude reefs differently exposed to chronic disturbance, respond to elevated seawater temperatures.

Corals were collected from reefs nearshore, (i.e. subjected to high sediment load, higher chlorophyll  concentrations, turbidity etc.) and offshore, (i.e. less exposed). The corals were exposed in the lab to gradually increasing temperatures (25.5 -33.5 °C) for 72 h after which they were allowed to recover to ambient temperature (25.5 °C) for 24 h. Production and respiration were measured after 24, 48, 72 and 96 hours. The results show that P. lutea from nearshore reefs suffered an initial decrease in gross primary production/respiration (GP/R) ratio after 24 h, after only a moderate temperature increase (+ 2 °C, from 25.5 to 27.5 °C), while there was no difference in GP/R ratio between heatexposed and controls the other days, indicating that the chronic disturbance in the nearshore reef had no effect on their thermotolerance. Furthermore, P. lutea from the offshore reef showed a decrease in GP/R ratio both after 24 h and 72 h (33.5 °C) of exposure.

In comparison, G. fascicularis showed a decrease in GP/R ratio after 48 h, 72 h and 96 h of exposure for the nearshore corals. Also, after 72 h these corals had withdrawn their polyps. There were no differences between heat-treated and controls for the offshore G.

fascicularis. This implies that the chronically disturbed G. fascicularis had lower thermotolerance when exposed to a temperature increase.

Introduction

Coral reefs have shown a remarkable persistence, despite reoccurring natural disturbance events (Wood, 1995). However, reef-building corals have a very restricted thermal tolerance [START_REF] Hoegh-Guldberg | Climate change, coral bleaching and the future of the world's coral reefs[END_REF]. If temperature exceeds a given thermal limit corals may lose their colour, i.e. they "bleach". This decolouration is due to the expulsion of the endosymbiotic microalgae (zooxanthellae) found in the polyp tissue, making the white calcium carbonate skeleton underneath visible (Douglas, 2003). Since corals receive up to 95% of their energy from their zooxanthellae via photosynthesis [START_REF] Muscatine | The role of symbiotic algae in carbon and energy flux in reef corals[END_REF] bleaching may cause significant mortality, unless the coral host is repopulated with symbionts, in due time (species specific) to allow recovery [START_REF] Glynn | Coral reef bleaching: facts, hypotheses and implications[END_REF]Diaz-Pulido and McCook, 2002). Hence, in the face of climatic changes and increased sea surface temperatures [START_REF] Ipcc | Climate Change 2007: The physical science basis. Contribution of working group 1 to the fourth assessment report of the intergovernmental panel on climate change[END_REF] there is great concern for the future health of coral reefs [START_REF] Hughes | Climate change, human impacts, and the resilience of coral reefs[END_REF][START_REF] Sheppard | Sea surface temperature 1871-2099 in 38 cells in the Caribbean region[END_REF][START_REF] Hoegh-Guldberg | Coral reefs under rapid climate change and ocean acidification[END_REF]. In tandem coral reefs are exposed to direct human impacts, such as increased sedimentation, increased nutrient loading, pollution etc., which may reduce their capacity to respond to environmental changes caused by changes in climate.

Extensive coral mortality due to climate-induced bleaching events (i.e. mass coral bleaching) has occurred repeatedly over the past decades across all oceans (Wilkinson, 2006). These events are predicted to increase in frequency and magnitude in the future [START_REF] Hoegh-Guldberg | Climate change, coral bleaching and the future of the world's coral reefs[END_REF]Donner et al., 2005). The observed patterns of bleaching, however, show great spatial variability (e.g. [START_REF] Mcclanahan | Western Indian Ocean coral communities: bleaching responses and susceptibility to extinction[END_REF]. A multitude of variables may explain different thermal tolerances, including coral morphology M A N U S C R I P T A C C E P T E D ACCEPTED MANUSCRIPT 5 [START_REF] Mcclanahan | Bleaching damage and recovery potential of Maldivian coral reefs[END_REF][START_REF] Spencer | Coral bleaching in the Southern Seychelles during the 1997-1998 Indian Ocean warm event[END_REF]Edwards et al., 2001), taxa [START_REF] Mcclanahan | The relationship between bleaching and mortality of common corals[END_REF][START_REF] Mcclanahan | Comparing bleaching and mortality responses of hard corals between southern Kenya and the Great Barrier Reef, Australia[END_REF]Ulstrup et al., 2006), depth [START_REF] Brown | Adaptations of reef corals to physical environmental stress[END_REF]Warner et al., 1999;[START_REF] Spencer | Coral bleaching in the Southern Seychelles during the 1997-1998 Indian Ocean warm event[END_REF]Craig et al., 2001), distribution of zooxanthellae clades (i.e. genotypes) [START_REF] Glynn | Coral bleaching and mortality in Panama and Ecuador during the 1997-1998 El Nino-Southern Oscillation event: spatial/temporal patterns and comparisons with the 1992-1993 event[END_REF][START_REF] Baker | Coral's adaptive response to climate change[END_REF][START_REF] Rowan | Thermal adaptation in reef coral symbionts[END_REF], and local temperature variations due to local current regimes [START_REF] Mcclanahan | Effects of geography, taxa, water flow, and temperature variation on coral bleaching intensity in Mauritius[END_REF]. Furthermore, it has also been suggested that reduced water quality can amplify the impacts of elevated seawater temperature [START_REF] Hughes | Climate change, human impacts, and the resilience of coral reefs[END_REF][START_REF] Pandolfi | Are U.S. coral reefs on the slippery slope to slime?[END_REF][START_REF] Mora | A clear human footprint in the coral reefs of the Caribbean[END_REF]. For example, experimental studies suggest that thermal tolerance in corals can be reduced by exposure to heavy metals [START_REF] Nyström | Simultaneous and sequential stress from increased temperature and copper on the metabolism of the hermatypic coral Porites cylindrica[END_REF], increased nutrients [START_REF] Nordemar | Effects of elevated seawater temperature and nitrate enrichment on the branching coral Porites cylindrica in the absence of particulate food[END_REF] sedimentation [START_REF] Lambo | Continued post-bleaching decline and changed benthic community of a Kenyan coral reef[END_REF] and decreased salinity (Faxneld et al., 2010).

Besides climate change, increase in sediment load due to human activities, is considered a major threat to coral reefs [START_REF] Bryant | Reefs at risk: a map-based indicator of threats to the world's coral reefs[END_REF][START_REF] Hughes | Climate change, human impacts, and the resilience of coral reefs[END_REF]Wilkinson, 2004). Sedimentation can lead to reduced coral species diversity and increased mortality, reduced calcification-and growth rates, decreased net production and increased respiration [START_REF] Rogers | Responses of coral reefs and reef organisms to sedimentation[END_REF]Crabbe, 2005;Fabricius, 2005;[START_REF] Jupiter | Linkages between coral assemblages and coral proxies of terrestrial exposure along a cross-shelf gradient on the southern Great Barrier Reef[END_REF].

Approximately 50% of the reefs in Vietnam are threatened by sedimentation [START_REF] Burke | Reefs at risk in Southeast Asia[END_REF]. Tuan (2000) reported that the proportion of reefs with less than 25% live coral cover increased from 25% before 1998 to 50% of the reefs by 1999 in the Halong Bay and Cat Ba Island (the study area). This has mainly been attributed to sedimentation [START_REF] Chou | Status of Southeast Asian reefs[END_REF]. During that time, Acropora spp. suffered significant mortality on many reefs while Galaxea fascicularis was basically unaffected. In 2002 G. fascicularis was still the dominating species on many reefs in Halong Bay with up to 40% The aim of this study was to investigate potential differences in physiological responses to elevated temperature depending on exposure to earlier disturbance regimes.

For this purpose two common coral species, Porites lutea and Galaxea fascicularis were used. Corals were collected from two nearshore reefs, more exposed to sedimentation, high chlorophyll  concentrations and nutrients etc. (Fig. 2a and Table 1) and two offshore (less disturbed) reefs (Fig. 1). Two opposite outcomes were predicted beforehand: (1) corals exposed to chronic disturbance will have reduced capacity to withstand further thermal stress, since these corals may already be close to their physiological tolerance limit or (2) corals exposed to chronic disturbance will have enhanced capacity to withstand further stress, due to a more developed acclimatory ability.

Differences in physiological responses within and between species from different disturbance regimes can be relevant for identifying sensitive and tolerant coral species when exposed to additional stress and may have implications when deciding future establishment of marine protected areas.

Material and methods

Study area

Halong Bay M A N U S C R I P T A C C E P T E D ACCEPTED MANUSCRIPT 7
Halong Bay is a relatively shallow turbid bay exposed to high nutrient loading [START_REF] Latypov | Reef-building corals of Vietnam as a part of the Indo-Pacific reef ecosystem[END_REF]. Most coral reefs in Halong Bay are also affected by sedimentation and coastal pollution, especially nearshore reefs [START_REF] Lang | Sedimentation and water exchange on coral reefs in the Ha Long -Cat Ba area, Quang Ninh Province, North Viet Nam[END_REF]. Furthermore, shipping in and out of the bay stirs up the sediments from the bottom thus affecting the reefs, more so in the nearshore sites [START_REF] Tran | An overview of the geological values and sedimentary environment of Ha Long Bay[END_REF]. Due to the high turbidity in the bay corals are restricted to shallow waters and their maximum depth distribution is limited to 3-4 m on nearshore-and to 5-7 m on offshore reefs [START_REF] Lang | Sedimentation and water exchange on coral reefs in the Ha Long -Cat Ba area, Quang Ninh Province, North Viet Nam[END_REF]. Water temperature fluctuates dramatically over the year with a winter (December-February) minimum of 16 °C and normal summer (June-August) maximum around 31.5 °C. Halong Bay is a relatively species-poor coral community, and it is dominated by massive coral species, such as Galaxea fascicularis and Porites lutea. Branching genera, such as Acropora and Pocillopora are relatively scarce [START_REF] Latypov | Reef-building corals of Vietnam as a part of the Indo-Pacific reef ecosystem[END_REF]; Nguyen D Ngai unpubl.).

Collection of corals

Pieces of corals were collected at 2-3 m depth from two reef areas in May 2007 (Fig. 1).

The disturbed (i.e. nearshore) reef area (Cat Ba Island National Park) (including site A and B) is situated close to the coast, in a semi-enclosed area with limited water exchange and receives run-off water from several rivers, whereas the offshore area (Long Chau Islands) (including site C and D) is located approximately 20 km south of the nearshore reef area. The latter location is an open area with good water exchange and it is less affected by land run-off water (e.g. less sediments, nutrients and lower chlorophyll  concentrations (a proxy for water clarity, see LaJeunesse et al., 2010a and2010b).

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Although several environmental parameters differ between the near-and offshore area, temperature and salinity regimes were almost identical between the areas throughout the year (see Table 1 and Fig. 2a and2b); and the ambient temperature and salinity during collection was 25.5 °C and 32 psu at all sites. Corals from the two reef areas thus have very similar thermal histories. Within each area, two reef sites were sampled. In the nearshore area, Ba Trai Dao Island (site A, N 20˚47' 20'', E 107˚ 06' 13'') and Van Boi Island (site B,N 20˚ 45' 35'',E 107˚ 04' 24'') were selected and in the offshore area, coral specimens were collected from Vung Cay Bang Island (site C, N 20˚ 37' 27'', E 107˚ 09' 41'') and Vung Tau Island (site D, N 20˚ 37' 21'', E 107˚ 08' 08'') (Fig 1). The corals were collected using a hammer and chisel. Two massive species, Porites lutea and Galaxea fascicularis, were selected and at each of the four sites 10 coral pieces (approximately 85 cm 2 and 120 cm 2 for P. lutea and G. fascicularis respectively) of each species were collected, except for one nearshore site (Van Boi) where no G. fascicularis and only 6 pieces of P. lutea could be found. Hence, 36 pieces of P. lutea and 30 pieces of G. fascicularis were collected in total. Immediately after collection, corals were transported to the outdoor laboratory at the Tram Bien Research Station in Do Son under low light condition and continuous aeration. After epiphytes and boring organisms had been carefully removed, the corals were put in aerated aquaria (20 litre each) with filtered and UV-treated seawater. The aquaria were placed in larger water-filled tanks (1 m 3 ) in order to minimise temperature fluctuations [START_REF] Nyström | Simultaneous and sequential stress from increased temperature and copper on the metabolism of the hermatypic coral Porites cylindrica[END_REF][START_REF] Nordemar | Effects of elevated seawater temperature and nitrate enrichment on the branching coral Porites cylindrica in the absence of particulate food[END_REF]. The corals were allowed to acclimate to the set control temperature of 25.5 °C and 32 psu (ambient at site of collection) and thereafter kept at that temperature for 48 h before the experiment was started.

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The corals were evenly distributed in eight 20-litre aquaria using a randomized block design. Four of the aquaria were then placed in a heating tank (1 m 3 ) representing temperature "exposure" and the other four were placed in a control tank (1 m 3 ) with ambient water temperature. Aeration pumps mixed the seawater in both the aquaria and the tanks, thus maintaining high oxygen concentrations and distributing water temperature evenly. The water temperature in the exposure tank was increased using heating regulators (ZEBO 300W or FLUVAL Tronic 200W). The temperature was recorded every hour during daytime (between 10 am to 6 pm) and once in the evening as well as once in the early morning (at 9 pm and 6 am respectively) to verify constant temperature throughout a 24 h period before the experiment was started. Throughout the experiment, temperature was measured continuously during the physiological measurements. Between measurements, temperatures were recorded every second hour during daytime and once in the evening as well as once in the early morning (at 9 pm and 6 am respectively). The experiment lasted for 96 h (4 days) during which the temperature was gradually increased for three days, to mimic a natural gradual temperature increase, and then reduced to ambient for a 24-hour recovery period. It is well known that daily temperatures can undergo drastic changes on nearshore reef flats and in coastal lagoons, e.g. 25-33 °C within hours [START_REF] Semesi | Alterations in seawater pH and CO 2 affect calcification and photosynthesis in tropical coralline alga, Hydrolithon sp. (Rhodophyta)[END_REF]. Normal summer temperature in Halong Bay is 30-31.5 °C [START_REF] Tran | An overview of the geological values and sedimentary environment of Ha Long Bay[END_REF][START_REF] Latypov | Reef-building corals of Vietnam as a part of the Indo-Pacific reef ecosystem[END_REF] (Glynn and D'Croz 1990;Jokiel and Coles 1990). The temperature treatment in the exposure tank was set to: day 1: (27.5 °C), day 2: (30.5 °C), day 3: (33.5 °C) and day 4: (25.5 °C). The maximum exposure temperature of 33.5 °C in the present study was chosen since 2 °C above expected summer maximum is known to induce physiological stress in most coral species. It took approximately four hours before the new temperature was reached.

Hence, the corals were exposed in each temperature for 20 hours before the production and respiration were measured. Temperature in the control tank was maintained at 25.5 °C throughout the study. Due to effective heaters and stirring we were able to keep the temperatures very constant (deviation < 0.2 °C from set temperature) throughout the experiment. The experiment was conducted outdoors in natural sunlight, under low light conditions (414-529 µE m -2 s -1 ), corresponding to recorded light levels on shallow reefs (e.g. [START_REF] Semesi | Alterations in seawater pH and CO 2 affect calcification and photosynthesis in tropical coralline alga, Hydrolithon sp. (Rhodophyta)[END_REF]. Production and respiration measurements were conducted each day for both exposed and control corals.

Physiological measurements

Before net primary production and respiration were measured, each coral was put in a 1.5 litre transparent plastic jar with a tight-fitting lid, without extra supplies of oxygen. Each jar contained water with a temperature identical to the treatment temperature. During the measurements, the temperatures also in the measurement jars were constant (set temp.  0.2 °C). Changes in dissolved oxygen concentration in light (net primary production) and darkness (respiration) were measured according to [START_REF] Moberg | Effects of reduced salinity on the rates of photosynthesis and respiration in the hermatypic corals Porites lutea and Pocillopora damicornis[END_REF] ). An earlier pilot test showed that the most stable and reliable values were obtained after a 30 minutes and 2.5 hours acclimation period in light and darkness for production and respiration, respectively. Dissolved oxygen concentration was initially measured in light and then again after 30 minutes. Corals were then placed in darkness and after 2.5 h the oxygen concentration was measured once more and respiration rates calculated. Before the statistical analyses, production and respiration rates were converted to 1-hour values. Four water controls (jars containing only water) were used for each measurement, two with control water temperature and two with a temperature that corresponded to that of the exposed corals. Net primary production was always measured during mid-day, at 1 pm. During the measurements, the oxygen saturation levels never dropped below 75%. To compensate for volume dependent differences in dissolved oxygen concentration, the volumes of the corals were measured by submerging the coral into a graded beaker filled with a known volume of water. Live coral surface area was determined using the aluminium foil method [START_REF] March | Primary productivity of reef-building calcareous red algae[END_REF]. Net production-and respiration rates were expressed per hour and coral surface area (cm 2 ).

Respiration rate (R) was added to the net production value in order to achieve gross primary production rate (GP). Gross primary production rates were then corrected for temporal differences in light intensities between measurements. This was done to compensate for light dependent differences between measurements, since measurements were performed for four consecutive days. In order to do this, the production values were divided by the light intensity during measurement, and then multiplied with the average light intensity during all measurements, assuming a linear relationship between light intensity and primary production in the narrow light interval in this study. Due to lack of

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12 laboratory facilities, data on coral biomass, zooxanthellae density and chlorophyll concentration could not be obtained. Consequently gross primary production and respiration rates are expressed as per cm 2 coral surface area according to [START_REF] Moberg | Effects of reduced salinity on the rates of photosynthesis and respiration in the hermatypic corals Porites lutea and Pocillopora damicornis[END_REF]. Gross primary production/respiration (GP/R) ratios were also calculated to get a dimensionless estimate of the physiological status of the corals.

Statistical analysis

A two-way ANOVA was used to analyse the effect of site (i.e. nearshore site A vs. B and offshore site C vs. D) and treatment (i.e. heat vs. control) on the gross primary production rate, respiration rate and GP/R ratio of both species. Since there were no differences between the two offshore or nearshore sites (the data from site A and B, and the data from site C and D) the data were pooled into one offshore and one nearshore group for each species. A General Linear Model (GLM) univariate one-way ANOVA was used to analyse the effects of the four different treatments (i.e. offshore / heat, nearshore / heat, offshore / control and nearshore / control) on gross primary production rate, respiration rate and GP/R ratio for G. fascicularis and P. lutea respectively. The analyses were carried out separately for each day and species. Where the one-way ANOVA showed differences, Tukey HSD was used as a post hoc test (p < 0.05). All data were tested for homogeneity of variances using Levene's test (p < 0.05) before the ANOVA was carried out. When necessary, the data were log transformed. When Levene's test showed differences in variance and no transformations proved efficient, the non-parametric

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13 Kruskal-Wallis and Mann-Whitney U test was used. For the statistical analyses SPSS 17.0 for Mac OS X was used.

Results

Galaxea fascicularis

There were significant differences when comparing heat-treated corals and controls in nearshore and offshore areas (Table 2). Corals from the nearshore area showed a significant decrease in GP/R ratio on day 2, 3 and 4 when exposed to elevated temperature compared to the controls (p < 0.05 Tukey HSD, Fig. 3a), whereas no significant differences were observed for heat-treated and controls for the offshore corals (Fig. 3b). The differences in GP/R ratio for the nearshore corals were mainly caused by an increase in respiration rates compared to the control (Table 3a). However, on day 3 the difference was also explained by a decrease in gross primary production rates compared to the control (Table 3b).

Sixty percent of the heat-treated Galaxea fascicularis from the nearshore area had withdrawn their polyps (Fig. 4a) after exposure to 33.5 °C, i.e. on the third day of exposure. Heat-treated offshore-and control corals showed no such response (Fig. 4b).

Porites lutea

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ACCEPTED MANUSCRIPT 14 For P. lutea there were significant differences between heat-treated and control corals from nearshore and offshore areas (Table 4). The GP/R ratio was significantly lower on day 1 for nearshore heat-treated corals compared to the controls (p < 0.05 Tukey HSD, Fig. 5a). This was explained by a significant increase in respiration rates that day (p < 0.01 Tukey HSD, Table 5a). Furthermore, the offshore heat-treated corals displayed a significant decrease in GP/R ratio both on day 1 and day 3 (p < 0.05 Tukey HSD, Fig. 5b). This decrease in GP/R ratio was mainly explained by an increase in respiration rates (Table 5a). There were no differences in gross primary production rates between treatments (Table 5b).

Discussion

For Galaxea fascicularis the GP/R ratio differed between the offshore and nearshore area, except for day 1. Heat-treated corals from nearshore reefs showed a decrease in GP/R ratio throughout the study. On day 3 the corals had also withdrawn their polyps, which might explain the large decrease in gross primary production rate observed that day. A direct effect on zooxanthellae cannot be excluded although no counting of zooxanthellae could be done. Other studies have shown that withdrawal of polyps is a stress response in corals (Coyer et al., 1993;[START_REF] Brown | Tissue retraction in the scleractinian coral Coeloseris mayeri, its effect upon coral pigmentation, and preliminary implications for heat balance[END_REF][START_REF] Jones | Changes in quantum efficiency of Photosystem II of symbiotic dinoflagellates of corals after heat stress, and of bleached corals sampled after the 1998 Great Barrier Reef mass bleaching event[END_REF].

The difference in response between nearshore and offshore G. fascicularis suggests that corals pre-exposed to other stressors (i.e. nearshore corals) are more sensitive to heat stress compared to the offshore (less disturbed) reefs. Similar findings have been reported by [START_REF] Carilli | Local stressors reduce coral resilience to bleaching[END_REF], who investigated skeletal growth rates in Montastrea faveolata before and after the 1998 bleaching event in reefs exposed to

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15 different levels of local stress (e.g. sedimentation and nutrients). The authors found that the growth rate was suppressed for longer time after the bleaching event at the reefs exposed to higher levels of stress, thus they were less resilient to coral bleaching. There were no significant differences in physiological response between Porites lutea from offshore and nearshore reefs on any day of exposure (Fig. 5a and5b). There were only significant differences between heat-treated and control corals for the nearshore P. lutea at day 1 of exposure (Fig. 5a) even though the temperature increase was higher, with a maximum of 8 ºC above ambient temperature on day 3. This indicates that even a small increase in temperature can affect P. lutea initially, but that they are able to "maintain" their physiological status (GP/R ratios indicating maintained autotrophy) over the temperature range tested in the present study. Although all heattreated Porites showed a relative decrease in GP/R ratio compared to the corresponding controls, anthropogenic disturbances (e.g. land run-off, sedimentation) seem not to affect their thermotolerance during heat stress. One possible explanation of this could be a more rapid induction of cellular defence mechanisms (e.g. HSP 70see [START_REF] Brown | Adaptations of reef corals to physical environmental stress[END_REF]) in these chronically exposed specimens, something that has been demonstrated in marine mussels exposed to heavy metal pollution followed by thermal stress (Veldhuizen-Tsoerkan et al., 1990).

Another explanation could be that the coral clades differ between P. lutea and G. fascicularis and/or differ between the two areas, since it has been shown that different corals may host different clades of Symbiodinium [START_REF] Lajeunesse | Diversity and community structure of symbiotic dinoflagellates from Caribbean coral reefs[END_REF] and clades can also differ between regions [START_REF] Baker | Diversity of symbiotic dinoflagellates (zooxanthellae) in scleractinian corals of the Caribbean and Eastern Pacific[END_REF][START_REF] Garren | Patterns of association between Symbiodinium and members of the Montastrea annularis species complex on spatial scales ranging from within colonies to between geographic regions[END_REF]. [START_REF] Chen | Symbiont diversity in scleractinian corals from tropical and subtropical non-reef communities in Taiwan[END_REF][START_REF] Apprill | Visibly healthy corals exhibit variable pigment concentrations and symbiont phenotypes[END_REF][START_REF] Hennige | Acclimation and adaptation of scleractinian coral communities along environmental gradients within an Indonesian reef system[END_REF], which generally is not associated with corals located in areas exposed to high temperatures (e.g. [START_REF] Baker | Coral's adaptive response to climate change[END_REF]. Since no analyses of the clades were conducted in this study, further research comparing clades between nearshore and offshore reefs in the area would be of scientific interest. A further explanation to the different responses between the two species and areas could be that the coral hosts are genetically adapted to the local environmental conditions on these reefs, since the environmental quality do differ between nearshore-and offshore reefs, although the thermal history are identical between the areas in the study presented here.

Reduction of GP/R ratio has been recognized as a common stress response in corals when exposed to elevated seawater temperature [START_REF] Coles | Effects of temperature on photosynthesis and respiration in hermatypic corals[END_REF][START_REF] Hoegh-Guldberg | The effect of sudden changes in temperature, light and salinity on the population density and export of zooxanthellae from the reef corals Stylophora pistillata Esper and Seriatopora hystrix Dana[END_REF]Fitt and Warner, 1995;[START_REF] Porter | The effects of multiple stressors on the Florida Keys coral reef ecosystem: a landscape hypothesis and a physiological test[END_REF]. The observed decrease in GP/R ratio for both P. lutea and G. fascicularis was primarily a result of an increase in respiration rate, which has been demonstrated by other authors [START_REF] Hoegh-Guldberg | The effect of sudden changes in temperature, light and salinity on the population density and export of zooxanthellae from the reef corals Stylophora pistillata Esper and Seriatopora hystrix Dana[END_REF]Faxneld et al., 2010). However, for nearshore G. fascicularis on day 3 the decrease in GP/R ratio was also explained by a decrease in primary production rates, which also has been shown by other authors (Fitt and Warner, 1995;[START_REF] Porter | The effects of multiple stressors on the Florida Keys coral reef ecosystem: a landscape hypothesis and a physiological test[END_REF][START_REF] Nyström | Simultaneous and sequential stress from increased temperature and copper on the metabolism of the hermatypic coral Porites cylindrica[END_REF]. The higher respiration-and production rates, observed for offshore G. fascicularis, controls and heat-treated, is likely to be attributed to the fact that the offshore colonies were generally smallerpresumably younger coloniesthat do have higher metabolic activity.

None of the coral species showed signs of bleaching in our study, which supports others studies showing that both G. fascicularis [START_REF] Marshall | Bleaching of corals on the Great Barrier Reef: differential susceptibilities among taxa[END_REF] 17 et al., 2004) andP. lutea (e.g. Riegl, 1999;[START_REF] A N U S C R I P T A C C E P T E D Accepted Manuscript 25 Loya | Coral bleaching: the winners and the losers[END_REF][START_REF] Obura | Can differential bleaching and mortality among coral species offer useful indicators for assessment and management of reefs under stress[END_REF] are relatively tolerant to elevated seawater temperatures, and suggests that the corals in the present study were not exposed to lethal temperatures, although an increase by 2-3 °C in 4 hours is in the upper range of what can be expected on shallow reefs in the study area.

; McClanahan M A N U S C R I P T A C C E P T E D ACCEPTED MANUSCRIPT
It should be emphasized that of the temperatures tested here, the 27.5 and 30.5 °C treatments are well below the thermotolerance threshold for most coral species and only the 33.5 °C treatment is above the normal summer maximum.

Conclusion

This study shows that the response of corals to elevated seawater temperature varies with species and environmental background history. The study suggests that the physiological response in G. fascicularis was more pronounced in more disturbed nearshore areas compared to their conspecifics from less disturbed environments, thus supporting the hypothesis that corals in stressful environments (e.g. subjected to land-based pollution and sedimentation) are less tolerant to elevated water temperature. An explanation for this could be that nearshore corals are already closer to their physiological tolerance limit and therefore more sensitive to additional disturbances [START_REF] Paine | Compounded perturbations yield ecological surprises[END_REF]. On the other hand, the acclimatory ability may very well differ between coral species. For example, P. lutea from nearshore reefs were not more physiologically affected by the temperature increase than corals from offshore reefs. Consequently, it is difficult to predict general effects on coral reefs if episodes of elevated seawater temperatures become more frequent in the future, since these effects may be area and species specific. One likely consequence, M A N U S C R I P T A C C E P T E D ACCEPTED MANUSCRIPT 18 however, is that due to differences in acclimatory capability among coral species, reefs may suffer losses in biodiversity [START_REF] A N U S C R I P T A C C E P T E D Accepted Manuscript 25 Loya | Coral bleaching: the winners and the losers[END_REF], which in turn may impact the resilience of coral reefs [START_REF] A N U S C R I P T A C C E P T E D Accepted Manuscript 26 Nyström | Coral reef disturbances and resilience in a human-dominated environment[END_REF][START_REF] Bellwood | Confronting the coral reef crisis[END_REF]. The data were acquired from the Giovanni online data system (http://gdata1.sci.gsfc.nasa.gov/daac-bin/G3/gui.cgi?instance_id=ocean_month; downloaded 1 November 2010), which was developed and is maintained by the NASA Goddard Earth Sciences (GES) Data and Information Services Center (DISC). 2. Galaxea fascicularis. Results from the ANOVA where all nearshore and offshore corals have been compared to the controls. P-values < 0.05 demonstrate significant differences between heat-treated and controls. + depicts cases when Kruskal-Wallis was used. Table 3. Galaxea fascicularis. Mean ± SE for heat-treated and controls from nearshore and offshore reefs. a) Respiration rates b) Gross primary production rates. Table 4. Porites lutea. Results from the ANOVA where all nearshore and offshore corals have been compared to the controls. P-values < 0.05 demonstrate significant differences between heat-treated and controls.

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  cover (Nguyen D Ngai unpubl. data), thus indicating that tolerance to sedimentation varies among coral species.

  , and studies have shown that large increases (3-4 M A N U S C R I P T A C C E P T E D ACCEPTED MANUSCRIPT 10 °C above normal summer maximum) over a few days can bleach corals

  G. fascicularis have been found to harbour both clade C and D (LaJeunesse et al., 2004; Done et al., while P. lutea is only hosted by clade C

Fig 1 .

 1 Fig 1. Map showing the collection sites. The nearshore, more disturbed reefs (Ba Trai

Fig 2

 2 Fig 2. a) Monthly averages of chlorophyll  concentrations (a proxy for water clarity)

Fig 3 .

 3 Fig 3. Galaxea fascicularis. GP/R ratio ± SE. a) Nearshore corals a) Offshore corals. The

Fig 4 .

 4 Fig 4. Galaxea fascicularis. Polyp extension. a) Nearshore corals b) Offshore corals when

Table 5 .

 5 Porites lutea. Mean ± SE for heat-treated and controls from nearshore and offshore reefs. a) Respiration rates b) Gross primary production rates. ** p < 0.01. .A., Ambrose, R.F., Engle, J.M., Carroll, J.C., 1993. Interactions between corals and algae on a temperate zone rocky reef: mediation by sea urchins. Journal of Experimental Marine Biology and Ecology 167, 21-37 Crabbe, M.J.C., Smith, D.J., 2005. Sediment impacts on growth rates of Acropora and Porites from fringing reefs of Sulawesi, Indonesia. Coral reefs 24, 437-441 Craig, P., Birkeland, C., Belliveau, S., 2001. High temperatures tolerated by a diverse assemblage of shallow-water corals in American Samoa. Coral reefs 20, 185-189 Diaz-Pulido, G., McCook, L.J., 2002. The fate of bleached corals: patterns and dynamics of algal recruitment. Marine Ecology Progress Series 232, 115-128 Dong, Z-J., Huang, H., Huang, L-M., Li, Y-C., 2009. Diversity of symbiotic algae of the genus Symbiodinium in scleractinian corals of the Xisha Islands in the South China Sea. Journal of Systematics and Evolution 47, 321-326 Donner, S.D., Skirving, W.J., Little, C.M., Oppenheimer, M., Hoegh-Guldberg, O., 2005. Global assessment of coral bleaching and required rates of adaptation under climate change. Global Change Biology 11, 2251-2265 Douglas, A.E., 2003. Coral bleaching-how and why? Marine Pollution Bulletin 46, 385-392 Edwards, A.J., Clark, S., Zahir, H., Rajasuriya, A., Naseer, A., Rubens, J., 2001. Coral bleaching and mortality on artificial and natural reefs in Maldives in 1998, sea surface temperature anomalies and initial recovery. Marine Pollution Bulletin 42, 7-15 Fabricius, K.E., 2005. Effects of terrestrial runoff on the ecology of corals and coral reefs: review and synthesis. Marine Pollution Bulletin 50, 125-146 Faxneld, S., Jörgensen, T.L., Tedengren, M., 2010. Effects of elevated water temperature, reduced salinity and nutrient enrichment on the metabolism of the coral Turbinaria mesenterina. Estuarine, coastal and shelf science 88, 482-487 Fitt, W.K., Warner, M.E., 1995. Bleaching patterns in four species of Caribbean reef corals.

Table 1 .

 1 Environmental data from the offshore (Long Chau Islands) and the nearshore (Cat Ba) sites used in this study. Data from biannual surveys provided by The Institute of Marine Environment and Resources, Hai Phong, Vietnam.

Acknowledgements

This work is supported by Sida/SAREC, and we also thank Stockholm Marine Research Center (SMF) for providing financial support. We are grateful to Dr. Cu for allowing us to use his facilities for conducting this experiment and all the helpful staff at Tram Bien Research Station. We also thank the anonymous reviewers for their pertinent and helpful comments.

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