Interaction between exopolysaccharide and

Journal of Fish Biology (2008) 73, 186–195 doi:10.1111/j.1095-8649.2008.01924.x, available online at http://www.blackwell-synergy.com

Interaction between exopolysaccharide and oxygenation levels on habitat selection in the sole Solea solea (L.) C. S. C OUTURIER *†, L. N ONNOTTE ‡, G. N ONNOTTE ‡ AND G. C LAIREAUX ‡ *Centre de Recherche sur les Ecosyste`mes Littoraux Anthropis es, Place du S eminaire, BP 5, L’Houmeau, 17137 France and ‡Unit e de Physiologie Compar ee et Int egrative, Universit e de Bretagne Occidentale, UFR Sciences et Technologies, 6 avenue Le Gorgeu, Brest, 29285 cedex-3 France (Received 15 June 2007, Accepted 7 April 2008) A habitat selection experiment was conducted to examine the behavioural response of sole Solea solea to a combination of sediment quality (exopolysaccharides, EPS-free, i.e. 0 mg l1 and EPSrich, i.e. 4 mg l1) and water oxygenation level (100 and 35% air saturation). The distribution of sole was influenced differently by the type of substratum depending on the water oxygenation level. In normoxia, sole settled preferentially on sand whereas under hypoxic conditions, sole settled preferentially on the muddy substratum. In order to explain these apparently counterintuitive observations, it is proposed that, via cutaneous respiration, young sole are able to take advantage of the large quantities of oxygen produced by microphytobenthic organisms present in # 2008 The Authors the upper few millimetres of muddy substratum. Journal compilation # 2008 The Fisheries Society of the British Isles

Key words: EPS; habitat selection; hypoxia; Solea solea; substratum; water viscosity.

INTRODUCTION The French Atlantic coast comprises a series of large intertidal mudflats which are colonized by juveniles of numerous flatfish species including the common sole Solea solea (L.) (Le Pape, 2003). These ecosystems are heavily exploited by the shellfish farming industry (Goulletquer & Le Moine, 2002) which is responsible for substantial biodeposition. For instance, in the Marennes-Oleron Bay the culture of oysters entails the production of 600 t dry mass of biodeposits per cultivated km2 per day (Sornin et al., 1986). Directly (faeces and pseudofaeces) or indirectly (diatoms biofilm), this biodeposition contributes to increase the amount of exopolymers, mainly exopolysaccharides (EPS), at the interface between the water column and the seabed (Dinet et al., 1990; Barille & Cognie, 2000; Wotton & Gordon, 2005; Couturier et al., 2007). These long-chain molecules are organized in colloids and they are known to influence

†Author to whom correspondence should be addressed. Tel.: þ33 5 46 50 06 48; fax: þ33 5 46 50 06 48; email: [email protected]

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the rheological properties of water and in particular its viscosity (Jenkinson & Biddanda, 1995; Couturier et al., 2007). EPS-induced changes in viscosity have been shown to affect ventilatory water flow through the gill cavity of juvenile fishes (Jenkinson & Arzul, 1998, 2002). As viscosity increases, mechanical constraints are exerted on the ventilatory water flow, entailing supplementary opercular workload, and eventually, resulting in ventilatory distress. Accordingly, it has been shown that increased water EPS load is associated with a sizedependant reduction in juvenile sole ability to face increased metabolic demand or reduced oxygen availability (Couturier et al., 2007). A species-dependant effect was also demonstrated, species with high metabolic demand, and presumably, larger gill surface area being more susceptible to EPS load than less active species (Couturier et al., 2007). Biodeposits degradation gives rise to high rates of oxygen consumption by benthic micro-organisms. For instance, it has been shown that the recycling of organic matter in an oyster-culture zone can result in long-term (in the order of days) hypoxia with, in some instances, oxygenation level 005). Air bubbling ensured water saturation between 90 and 100% during normoxic periods. WATER EPS CONTENT

Water EPS concentrations were significantly higher at the interface with the muddy substratum than at the interface with sand (Fig. 2; ANOVA, d.f. ¼ 3 and 20, P 0001). EPS concentrations in the compartments containing sand were not significantly different from zero. HABITAT SELECTION

On average, 5750% of fish were positioned on sand in normoxia and 4237% in hypoxia (Fig. 3). The Bartlett’s test showed homogeneity of variance between hypoxic and normoxic groups (Bartlett’s test, d.f. ¼ 1, P > 005). The effect of oxygenation level (100 v. 35%) upon fish distribution was significant (bilateral paired t-test, d.f. ¼ 5, P < 005). DISCUSSION The objectives of the current study were to examine the behavioural response of sole in environmental situations combining different sediment qualities (EPS-free sand or EPS-rich mud) and water oxygen levels (100 or 35% air saturation). Sole distribution was influenced differently by the type of substratum

5 4

* *

EPS (mg l–1)

3 2 1 0 Mud A

Mud B

Sand A

Sand B

FIG. 2. Exopolysaccharides concentration (EPS) in water at the interface with sediment for both compartments of tanks A and B (*, P < 005).

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100

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Percentage of occurence ( )

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0 0 100

5

10

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5

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35

20

25

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35

(b)

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Time (h) FIG. 3. Occurrence (mean of tanks A and B) of sole on a sandy substratum as a function of time under (a) normoxia and (b) hypoxia (35% air saturation) (number of fish group ¼ 6).

depending on the water oxygenation level. In normoxia, sole settled preferentially on sand whereas under hypoxic conditions, sole occupied preferentially the muddy substratum. The grain size of the sand used in the experiment was c. 200–300 mm whereas grain size of mud was c. 10 mm (Couturier et al., 2007). Previous studies have shown that flatfishes do select their habitat based on sediment grain size (Gibson & Robb, 1992). Flatfish distribution is believed to reflect size-dependant burial capabilities (Stoner & Abookire, 2002). Field observations, however, have revealed that juvenile sole are equally found on sediment such as fine sand, mud or mixed sand and mud (Rogers, 1992; Gibson, 1997; Amezcua & Nash, 2001; Le Pape, 2003). During the experiment, no observation was made to suggest that burial behaviour might have been different between sediment types. On that basis, sediment grain size was not considered as the main driving force of habitat selection in the present experiment. # 2008 The Authors Journal compilation # 2008 The Fisheries Society of the British Isles, Journal of Fish Biology 2008, 73, 186–195

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As expected, EPS load was significantly higher in compartments containing the muddy substratum. EPS are known to influence water viscosity (Jenkinson & Biddanda, 1995). Moreover, these molecules have been shown to affect juvenile sole ventilatory activity (Couturier et al., 2007). This later study

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particularly showed that, EPS concentrations >3 mg l1 resulted in ventilatory distress, presumably caused by the inability of the opercular and buccal muscles to compensate for the associated increase in water viscosity. In the present experiment, EPS concentrations in compartments that contained mud were >3 mg l1, potentially explaining why sole tended to avoid the muddy substratum under normoxic conditions. Contrary to what was initially hypothesized, however, avoidance of the EPS-rich sediment was not aggravated by hypoxia. Conversely, under such conditions sole preferentially settled on mud. In order to elucidate this rather counter-intuitive result, two complementary points may be raised. First, through photosynthesis microphytobenthic communities are able to generate sufficient oxygen to generate hyperoxic conditions within the surface layer of the sediment. For example, Brotas et al. (2003) report field observations of hyperoxic conditions (600 mM, i.e. 300% air saturation) in the first 05 mm of a muddy sediment colonized by benthic microalgae (diatoms). Similarly, under laboratory conditions Revsbech & Jørgensen (1983) measured oxygen concentration up to 1300 mM (475% saturation) in the upper layer of a diatom-rich sediment. These authors also showed that oxygenation level in the first 1 mm layer of the sediment was more than twice that found in water directly above it. Field studies also suggest that oxygen production by microphytobenthos is enhanced in shellfish farming areas. For instance, Barranguet et al. (1996) showed that oxygen production is maximal below mussel tables, where the sediment grain is the finest and the microphytobenthic biomass the highest. The second point that can be raised relates to the fact that in sole, cutaneous oxygen uptake by the blind side (107 nmol cm2 min1) is nearly three times that of the eyed side (38 nmol cm2 min1) and represents up to 30% of fish routine oxygen requirement (Nonnotte & Kirsch, 1978). As shown in Fig. 4; the remarkable efficiency of the skin as a respiratory organ results both from a very thin epidermis (three or five cells layers between sea water and blood) and from a dense subepidermis vascular network, in particular on the blind side. On the basis of the above points, it is tempting to hypothesize that under hypoxic conditions, sole preferentially selected the muddy substratum to take advantage of the oxygen resources made available by the microphytobenthic photosynthetic activity. Although appealing, this hypothesis leads to yet another delicate point. If sole are able to exploit microphytobenthic oxygen production under hypoxic conditions, why do they not do so under normoxic conditions? There is no conclusive explanation for this observation but the

FIG. 4. Structural organization and subepidermis vascular network of the sole skin (eyed side and blind side). Skin patches were fixed in Bouin’s fluid (Gabe, 1968), dehydrated in ethanol and embedded in paraplast for histological investigation. Sections of 5 mm were stained by Gabe’s (1968) modification of Prenant’s stain. The subcutaneous vascular network was analysed after injection of silicone resin (Microfil, MV 117 orange; Canton Biomedical Products Inc., Boulder, CO, U.S.A.) by using the technique of Laurent & Dunel (1980). (a) General organization of skin on the eyed side (100), (b) details of the epidermis on the eyed side (1200), (c) vascular capillary subepidermis network on the eyed side after injection of silicon (40), (d) details of the epidermis on the blind side (1200) and (e) vascular capillary subepidermis network on the blind side after injection of silicon (40). CD, dense conjunctive tissue; CM, mucous cell; CS, spongious conjunctive tissue; E, scale; EP, epidermis; V, blood vessel.

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following situation can be suggested. Under normoxic conditions, fish preferentially selected the sandy substratum in order to avoid EPS-related impairment of their ability to move water across their gills. In hypoxia, on the other hand, as the oxygen diffusion gradient across the gill epithelium was reduced, it may be energetically beneficial to reduce ventilatory activity while moving to the hyperoxic muddy substratum where cutaneous oxygen uptake is possibly more efficient. It is most likely, however, that although sitting on an hyperoxic microlayer, sole are then faced with limitations in their ability to sustain high metabolic demand. This trade-off between short-term survival and scope for activity may be the reason why, under normoxic conditions, sole occupy preferentially the sandy sediment. Clearly, further investigations are required to elucidate all these points. The authors would like to thank R. Batty and P. Richard for discussing some of the issues raised by the current paper and D. Chabot for his valuable comments on statistic analyses. The authors also acknowledge the technical assistance of L. Joassard, Y. Descatoire M. Prineau and N. Lachaussee. C.S.C. was the recipient of a PhD fellowship from the Conseil General de Charente Maritime. The financial support by the European Union, Directorate Fisheries, through contract QLRS-2002-00799, Project ETHOFISH, is also acknowledged.

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