Changements Morphologiques Et Physiologiques En Lien Avec La Capacité De Nage Chez Les Pétoncles

Changements morphologiques et physiologiques en lien avec la capacité de nage chez les pétoncles

Thèse

Isabelle Tremblay

Doctorat en Biologie Philosophae Doctor (Ph.D.)

Québec, Canada

Résumé

Le système locomoteur relativement simple du pétoncle en fait un modèle animal idéal pour étudier les liens entre la performance locomotrice et les différentes composantes du système locomoteur. Cinq espèces de pétoncles (Amusuim balloti, Placopecten magellanicus, Pecten fumatus, Mimachalmys asperrima, Crassadoma gigantea), présentant une morphologie de la coquille et un comportement de nage variés, ont été comparées au niveau du comportement de nage, des capacités métaboliques du muscle adducteur, des propriétés mécaniques du ligament et de la morphologie de la coquille et du muscle adducteur. Les mesures de force lors d’une réponse de fuite simulée ont révélé que l’utilisation des deux parties du muscle adducteur varie grandement entre les espèces et varie aussi avec la morphologie de la coquille et le mode de vie. Ainsi, les pétoncles avec une coquille hydrodynamique utilisent principalement les contractions phasiques alors que les pétoncles avec une coquille de forme plutôt désavantageuse pour la nage utilisent majoritairement les contractions toniques. Aussi, le patron d’utilisation des deux parties du muscle peut être modifié afin de compenser pour une coquille de forme désavantageuse pour la nage. Les capacités métaboliques du muscle adducteur phasique reflètent le patron d’utilisation du muscle des différentes espèces. La résilience du ligament des pétoncles varie entre les espèces avec P. fumatus ayant la résilience la plus élevée. Les caractéristiques morphologiques de la coquille et du muscle adducteur diffèrent entre les espèces étudiées, mais ne reflètent pas toujours la stratégie de nage. Les analyses en composantes principales ont révélé que l’épaisseur et la masse de la coquille, la masse du muscle adducteur et les attributs morphologiques apparentés, sont étroitement liés à l’endurance de la réponse de fuite. L’intensité de cette réponse est, quant à elle, principalement prédite par l’allongement de la coquille et l’oblicité du muscle adducteur. Les liens fonctionnels et évolutifs entre la performance locomotrice et les différentes composantes du système locomoteur sont le résultat de compromis imposés par le style de vie, le type d’environnement et de prédateurs où évolue le pétoncle. Chez les pétoncles, il est important d’intégrer les différents niveaux d’organisation de l’animal car bien souvent la forme ne révèle pas tout.

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Abstract

Due to the relative simplicity of its locomotor system, scallops are ideal for studying the links between the locomotory performance and the various components of its system. The swimming behaviour, adductor muscle metabolic capacities, and ligament properties as well as the morphology of the shell and the adductor muscle of five scallop species (Amusuim balloti, Placopecten magellanicus, Pecten fumatus, Mimachalmys asperrima, Crassadoma gigantea), each with different shell morphology and swimming behaviour, were compared. Force recording measurements during a simulated escape response revealed that the utilisation of the two parts of the adductor muscle varies markedly between the species and also varies with the shell morphology and the lifestyle of the scallop. Thus, scallop species with hydrodynamic shell shape tend to use mainly phasic contractions, while species with shell shape disadvantageous for swimming rely mostly upon tonic contractions. Also, the use of phasic and tonic muscle can be a way for scallops to compensate for a disadvantageous shell shape. The metabolic capacities of the phasic adductor muscle reflect the muscle use in the different species. The ligament resilience varied between the species, with Pecten fumatus having the highest resilience. Morphological characteristics of the shell and the adductor muscle vary between the scallop species, but do not always reflect the swimming strategy. Principal component analysis revealed that the width and mass of the shell, muscle mass and related morphological attributes, were closely linked with swimming endurance. Swimming intensity was best predicted by the aspect ratio and the obliqueness of the adductor muscle. Functional and evolutive links between locomotor performance and the various components of the locomotor system are the result of compromises imposed by the scallop life style, the type of habitat and predators present where the scallop evolves. It is important to consider and integrate the various level of organisation, as often the form does not reveal everything in scallops.

Table des matières Résumé ...... iii Abstract ...... v Table des matières ...... vii Liste des figures ...... xv Remerciements ...... xxiii Avant-propos ...... xxvii CHAPITRE 1 ...... 1 Introduction générale ...... 1 1. Déterminants de la performance locomotrice ...... 3 1.1 Le muscle ...... 3 1.2 Métabolisme musculaire ...... 6 1.3 Facteurs affectant la performance locomotrice ...... 9 2. Forme et fonction au niveau de la locomotion ...... 10 3. Les pétoncles ...... 12 3.1. Origine de la nage chez les pétoncles...... 13 3.2 Évolution des modes de vie des pétoncles ...... 14 3.3 Locomotion chez les pétoncles ...... 14 4. Les composantes du système locomoteur ...... 19 4.1 Le muscle adducteur ...... 19 4.2. Le ligament ...... 21 4.3. La coquille ...... 24 5. Support métabolique de la nage ...... 25 6. Facteurs influençant la nage des pétoncles ...... 28 6.1 Forces agissant sur le pétoncle ...... 28 6.2 Composantes morphologiques affectant la nage ...... 30 6.3 Composantes physiologiques affectant la nage ...... 32 6.4 Facteurs environnementaux affectant la nage ...... 33 7. Biomécanique de la nage ...... 34 8. Caractérisation de la réponse de fuite des pétoncles ...... 35 9. Objectifs de la thèse ...... 39 CHAPITRE 2 ...... 41

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Swimming away or clamming up: the use of phasic and tonic adductor muscles during escape responses varies with shell morphology in scallops ...... 41 Résumé ...... 42 Abstract ...... 43 Introduction ...... 44 Material and methods ...... 49 Scallop sampling and experimental conditions ...... 49 Experimental design and force recordings ...... 50 Data recordings and analysis ...... 52 Anatomical and morphological measurements ...... 54 Statistical analysis ...... 56 Results ...... 56 Shell and anatomical characteristics ...... 56 Escape response recordings ...... 58 Interspecific comparisons: phasic contractions ...... 58 Interspecific comparisons: tonic contractions ...... 61 Discussion ...... 63 Eco-morphological classification versus phasic and tonic contractions...... 64 Initiation of prolonged tonic contractions: metabolic fatigue or energy saving? ...... 64 Habitat temperature and escape response patterns ...... 67 Potential influence of ontogenetic changes on escape responses ...... 67 Links between shell morphology, lifestyle and escape response behaviour ...... 69 Acknowledgements ...... 72 Funding...... 72 CHAPITRE 3 ...... 75 Scallops show that muscle metabolic capacities reflect locomotor style and morphology ...... 75 Résumé ...... 76 Abstract ...... 77 Introduction ...... 78 Materials and methods...... 82 Sampling and maintenance of the scallops ...... 82 Muscle sampling...... 84 viii

Anatomic and morphological measurements ...... 84 Muscle protein fractions ...... 84 Phosphoarginine concentrations ...... 85 Enzyme assays ...... 85 Myosin ATPase ...... 87 Chemicals ...... 88 Statistical analysis ...... 88 Results ...... 88 Anatomical characteristics of the experimental scallops ...... 88 Phasic muscle protein concentrations ...... 90 Phasic muscle phosphoarginine content ...... 91 Phasic muscle enzyme activities ...... 91 Myosin ATPase ...... 94 Discussion ...... 96 Interspecific differences in phosphoarginine levels and patterns of muscle use ...... 97 Interspecific differences in enzyme activities and patterns of muscle use ...... 98 Interspecific difference of myosin ATPase activity and patterns of muscle use ...... 101 Potential effects of ontogenetic change on metabolic capacities ...... 102 Biochemical attributes, behaviour and morphology ...... 103 Acknowledgements ...... 105 CHAPITRE 4 ...... 107 When behaviour and mechanics meet: Scallop swimming capacities and their hinge ligament .... 107 Résumé ...... 108 Abstract ...... 109 Introduction ...... 110 Material and methods ...... 112 Experimental scallops: shell and behavioural characteristics ...... 112 Muscle force measurements ...... 114 Experimental setup and measurement of ligament resilience ...... 116 Statistical analysis ...... 116 Results ...... 119 Shell characteristics ...... 119

Hysteresis loops and muscle force production ...... 119 Discussion ...... 120 Acknowledgements ...... 127 CHAPITRE 5 ...... 129 Can scallop swimming styles be predicted from shell and adductor muscle morphology? ...... 129 Résumé ...... 130 Abstract ...... 131 Introduction ...... 132 Material and methods ...... 135 Experimental scallops ...... 135 Sinking test ...... 135 Morphological measurements ...... 136 Adductor muscle measurements ...... 139 Statistical analysis ...... 140 Results ...... 141 Shell characteristics ...... 142 Sinking test ...... 142 Muscle morphology ...... 142 Proportions of tonic and phasic adductor muscle ...... 146 Muscle obliqueness: adductor muscle impression on left versus right valves ...... 146 Muscle obliqueness: muscle position on right and left valves ...... 150 Principal component analysis (PCA) of scallop morphology and behaviour ...... 150 Discussion ...... 157 Links between morphology and behaviour ...... 157 Shell characteristics ...... 159 Muscle and body morphology ...... 160 Acknowledgements ...... 163 CHAPITRE 6 ...... 165 Discussion générale ...... 165 Rappel des grandes lignes de l’étude...... 166 À chacun sa combinaison… ...... 168 Considérations ontogéniques ...... 172 x

Même morphologie de la coquille, mêmes adaptations? ...... 173 Bibliographie ...... 175

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Liste des tableaux

Tableau 1.1. Propriétés des fibres musculaires squelettiques phasiques chez les mammifères (Modifié à partir de Hill et al. 2008) ...... 7

Tableau 1.2. Principaux modes de vie chez les pétoncles (Modifié à partir d’Alejandrino et al. 2011) ...... 15

Table 2.1. Shell and anatomical characteristics of experimental scallops...... 57

Table 2.2. Behavioural parameters related to phasic contractions during escape responses by the different scallop species...... 60

Table 3.1. Morphological characteristics, water content and condition index of experimental scallops...... 89

Table 3.2. Phasic adductor muscle enzyme activities in different scallop and oyster species...... 100

Appendix 3.1 Regressions between shell height and phasic adductor mass of the scallops in the behavioural studies used to estimate the phasic adductor muscle mass of the scallops of the biochemical assays...... 106

Table 4.1. Shell and adductor muscle characteristics of the experimental scallops and parameters related to muscle contractile activity during escape responses...... 113

Table 4.2. Forces measured in experimental scallops...... 115

Table 4.3. Correlations between ligament resilience and phasic contractile activity during escape responses by the experimental scallop species ...... 122

Table 5.1. Shell and morphological characteristics of experimental scallops...... 143

Table 5.2. Sinking time adjusted for 90 mm shell height ...... 144

Table 5.3. Links between the morphological and behavioural principal components ...... 157

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Liste des figures

Figure 1.1. Molécule de myosine (Modifié à partir de Geeves 1991)...... 4

Figure 1.2. Évolution dans le temps de la contribution des différentes sources d’énergie pendant l’activité musculaire (Modifié à partir de Berg et al. 2002) ...... 9

Figure 1.3. Schémas représentant le mécanisme de la nage chez les pétoncles. A) Vue du dessus. B) Vue latérale ...... 17

Figure 1.4. Exemple de données recueillies lors des deux premiers claps chez Chlamys hastata. A) Pression dans la cavité du manteau et distance entre les valves (distance mesurée près de la bordure des valves au niveau du muscle adducteur). B) Taux d’écoulement de l’eau vers l’extrérieur de la cavité du manteau. C) Puissance mécanique calculée en prenant le produit de la pression (Pa) et du taux d’écoulement (m3 s-1) divisé par la masse (kg) du muscle adducteur phasique. (Fig. 1 de Marsh et al. 1992)...... 18

Figure 1.5. Partie phasique et tonique du muscle adducteur chez Pecten fumatus (Photo : Isabelle Tremblay) ...... 19

Figure 1.6. Intérieur de la région dorsale de la valve gauche, chez Amusium balloti, montrant le ligament en coupe longitudinale. Échelle en centimètres (Photo : Isabelle Tremblay). B) Diagramme de la section transversale du ligament de Chlamys opercularis montrant le ligament externe ainsi que la région centrale et les régions latérales du ligament interne (Modifié à partir de Trueman 1953a)...... 22

Figure 1.7. Valve supérieure d’un pétoncle (Placopecten magellanicus). La ligne pleine correspond à la hauteur tandis que la ligne pointillée correspond à la longueur. L’allongement de la coquille correspond à la longueur de la valve au carré/l’aire de la valve (Dadswell and Weihs 1990); (Photo : Isabelle Tremblay)...... 25

Figure 1.8. Principales voies de production d’énergie chez le pétoncle ...... 26

Figure 1.9. Forces agissant sur le pétoncle dans la colonne d’eau (Modifié à partir de Gould 1971)...... 29

Figure 1.10. Haut: Dynamomètre utilisé pour l’enregistrement des mesures de force (Pecten fumatus). Bas: Pétoncle (Pecten fumatus) avec la valve inférieure attachée au fond du basin, rempli d’eau salée, et avec un levier, attaché au dynamomètre, inséré sous la valve supérieure (Photos : Isabelle Tremblay)...... 37

Figure 1.11. Production de force du muscle phasique et tonique lors d’une réponse de fuite chez Amusium balloti...... 38

Figure 2.1. Upper valve and side view of experimental scallops. (A) Amusium balloti, (B) Placopecten magellanicus, (C) Pecten fumatus, (D) Mimachlamys asperrima and (E) Crassadoma gigantea. Scale bar is 1cm...... 48

Figure 2.2. Typical force recording during an escape response for each experimental scallop species: (A) A. balloti, (B) P. magellanicus, (C) P. fumatus, (D) M. asperrima and (E) C. gigantea. Sharp peaks correspond to phasic contractions whereas sustained force production indicates tonic contractions...... 53

Figure 2.3. Shell dimensions. A) Solid line corresponds to shell height and dashed line is shell length. B) Solid line corresponds to shell width...... 55

Figure 2.4. Escape response parameters related to phasic contractions. (A) Total number of phasic contractions. (B) Total number of phasic contractions during the first series relative to the total number of phasic contractions. (C) Contraction rate during the first 30 s. (D) Minimum interval between two phasic contractions. (E) Number of phasic contractions before the first tonic contraction. (F) Time to fatigue. Data are means ± S.E. Different letters indicate significant differences (Kruskall-Wallis and multiple comparisons, P<0.05). Sample size: A. balloti N=30, P. magellanicus N=15, P. fumatus N=15, M. asperrima N=16, C. gigantea N=19 ...... 59

Figure 2.5. Escape response parameters related to tonic contractions. (A) Total number of tonic contractions. (B) Mean duration of tonic contractions. (C) Total number of phasic contractions relative to the total number of tonic contractions. (D) Time at the first tonic contraction. (E) Percentage of time spent in tonic contractions. (F) Number of tonic contractions of a duration of 5 s or more. Data are means ± S.E. Different letters indicate significant differences (Kruskall-Wallis and multiple comparisons, P<0.05). Sample size: A. balloti N=30, P. magellanicus N=15, P. fumatus N=15, M. asperrima N=16, C. gigantea N=19...... 62

Appendix 2.1. Top: Force gauge used for monitoring the muscle force in intact scallops (commercial scallop, Pecten fumatus, shown below). Bottom: Scallop (Pecten fumatus) with its lower valve attached to the bottom of the tank and the lever, attached to the force gauge, placed under the upper valve ...... 73

Figure 3.1. Behavioural parameters and side view of experimental scallops. Data are means ± S.E. Sample size: A. balloti N=30, P. magellanicus N=15, P. fumatus N=15, M. asperrima N=16, C. gigantea N=19. Scale bar is 1 cm...... 81

Figure 3.2. Concentration of phosphoarginine (µmol P-Arg ∙ g-1 muscle wet mass) in the phasic adductor muscle. Means ± S.E. (N=10-23). Means with different letters are significantly different (P<0.05), as indicated by Kruskall-Wallis and a posteriori multiple comparison tests...... 92

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Figure 3.3. Enzymatic activities (U ∙ g-1 phasic muscle wet mass) in the phasic adductor muscle measured at 18°C. Values represent means ± S.E. (N=7-10 for AK and N=14- 22 for others). Means with different letters are significantly different (P<0.05), as indicated by Kruskall-Wallis and multiple comparison tests...... 93

Figure 3.4. Activity of the myosin ATPase (U ∙ mg-1 protein) in the phasic adductor muscle measured at 18°C. Values represent means ± S.E. (N=9-20). Means with different letters are significantly different (P<0.05), as indicated by Kruskall-Wallis and multiple comparison tests...... 95

Figure 4.1. Interior of the dorsal region of the left valve of Amusium balloti showing the ligament cut in longitudinal section. Scale is in cm...... 110

Figure 4.2. Experimental setup for the ligament resilience. Emptied shell placed on a fixed base, with an adjacent ruler, under the force gauge used to apply the force on the upper shell of the scallop ...... 117

Figure 4.3. Typical hysteresis loops, for each experimental species, obtained by plotting the force applied on the shell (the proxy for the ligament opening force) on the distance between the two valves. Black circles correspond to the loading curve and empty circles to the unloading curve ...... 118

Figure 4.4. Ligament resilience in experimental scallop species. Data are means ± S.E. Bars with different letters are statistically different as indicated by Kruskall-Wallis and multiple comparisons test (P<0.05). Sample size is A. balloti N=30, P. magellanicus N=15, E. bifrons N=20, P. fumatus N=15, M. asperrima N=15, C. gigantea N=16 ...... 121

Figure 4.5. The ligament resilience plotted against A) the number of phasic contractions during the first series (y=0.76+0.011x-0.0002x2, R2=0.27) and B) the phasic contraction rate during the first 30 s of the escape response for each species (y=0.75+0.23x-0.06x2, R2=0.25). Sample size is A. balloti N=30, P. magellanicus N=15, P. fumatus N=15, M. asperrima N=16...... 123

Figure 5.1. A) Shell dimensions. Full line corresponds to shell height and dash line is shell length on the left image and width on the right image. B) Side view of experimental scallops. Scale is 1cm ...... 137

Figure 5.2. A) Phasic and tonic muscle impressions retraced on the inside of the scallop valves. B) Diagram representing the various measurements taken to determine the position of the phasic and tonic adductor muscle. Valve on the left represents the left or upper valve, whilst the one on the right represents the right or lower valve. (B: From Soemodihardjo 1974) ...... 138

Figure 5.3. Typical phasic (gray) and tonic (black) adductor muscle impressions on left (L) and right (R) valves in experimental scallops. Arrowed line corresponds to scale: 20 mm for all species...... 147

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Figure 5.4. Percentage of tonic adductor muscle calculated from the muscle impression on the right valve. Mean±S.E. Kruskall-Wallis and multiple comparisons (P<0.05). Sample size A. balloti N=30, P. magellanicus N=15, E. bifrons N=20, P. fumatus N=15, M. asperrima N=14, C. gigantea N=15, C. gigas N=13...... 148

Figure 5.5. Ratio of the area of the muscle impression on left valve relative to area of the muscle impression on right valve. Mean±S.E. Different letters indicate significant differences (Kruskall-Wallis and multiple comparisons, P<0.05). Lower case letters refers to phasic muscle while capital letters refer to tonic muscle. The dashed line indicates when the areas on the left and right valves are the same size. Ratios significantly different than unity are identified by a star (Wilcoxon signed-rank test, P<0.05). Sample size A. balloti N=30, P. magellanicus N=15, E. bifrons N=19, P. fumatus N=15, M. asperrima N=14, C. gigantea N=16, C. gigas N=12 ...... 149

Figure 5.6. Scallop adductor muscle obliqueness. Obliqueness is the ratio of the dorso- ventral position of the adductor muscle on the left valve to that on the right valve. Mean±S.E. Different letters indicate significant differences (Kruskall-Wallis and multiple comparisons, P<0.05). Lower case letters refers to phasic muscle while capital letters refer to tonic muscle. The dashed line indicates when the ratio is equal to unity, that is when the insertion of the muscle on the right valve is closer to the hinge. Ratios significantly different than unity are identified by a star (Wilcoxon signed-rank test, P<0.05). Sample size A. balloti N=30, P. magellanicus N=15, E. bifrons N=9, P. fumatus N=15, M. asperrima N=14, C. gigantea N=14, C. gigas N=11 ...... 151

Figure 5.7. The normalised scores for the morphological variables associated with the first 3 principal components. Black bars identify variables assigned to each component. The percentages indicate the proportion of variance explained by the principal component...... 152

Figure 5.8. Scores for each scallop species for each principal component from the PCA. The boundary of the box closest to zero indicates the 25th percentile and the boundary of the box farthest from zero indicates the 75th percentile. The full line within the box marks the median, while the dashed line marks the mean. Whiskers (error bars) above and below the box indicate the 90th and 10th percentiles. Open circles are outlying data. ANOVA and multiple comparisons, P<0.05. Sample size: A. balloti N=30, P. magellanicus N=15, P. fumatus N=15, M. asperrima N=16, C. gigantea N=19 ...... 154

Figure 5.9. Normalised scores of the behavioural variables associated with the first 2 principal components. Black bars identify variables assigned to each component. The percentages indicate the proportion of variance explained by the principal component ...... 155

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Figure 5.10. Scores for the two behavioural principal components from the PCA of our behavioural data (Tremblay et al. 2012). The boundary of the box closest to zero indicates the 25th percentile and the boundary of the box farthest from zero indicates the 75th percentile. The full line within the box marks the median, while the dashed line marks the mean. Whiskers (error bars) above and below the box indicate the 90th and 10th percentiles. Open circles are outlying data. ANOVA and multiple comparisons, P<0.05. Sample size: A. balloti N=30, P. magellanicus N=15, P. fumatus N=15, M. asperrima N=16, C. gigantea N=18 ...... 156

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À ma famille qui a toujours été là pour moi

À mes neveux Florian et Christophe, mes petits soleils

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Remerciements

Cette thèse de doctorat a commencé bien avant que le premier mot fut écrit sur ce document. Ainsi, c’est un gros chapitre de ma vie qui arrive maintenant à sa fin. Au cours de toutes ces années pendant lesquelles j’ai mené à terme ce projet, beaucoups de gens ont contribué chacun à leur façon.

Premièrement ma directrice, Dr Helga E. Guderley, qui a cru en moi dès mes tout débuts en recherche (c’était à l’automne 2001). Merci Helga pour la confiance que tu as en moi et en mes capacités. Je suis très reconnaissante envers toi de m’avoir offert ce projet de doctorat et de m’avoir aidée à le réaliser… c’était un projet avec son lot de défis, mais je n’aurais pas pu rêver de meilleur projet. Merci aussi pour m’avoir guidée tout au cours de mon cheminement et de m’avoir aussi appris à trouver un équilibre entre la vie professionnielle et la vie personnelle. Merci à mon co-directeur, Dr John H. Himmelman, dont la rigueur scientifique a permis d’améliorer mes travaux de recherches et manuscrits. Merci aux membres de mon jury de thèse (Dr Jacques Larochelle, Dr Nadia Aubin- Horth et Dr Sandra E. Shumway) d’avoir accepté de lire et d’évaluer ma thèse de doctorat.

Cette thèse de doctorat est le résultat d’une étude comparative qui a nécessité l’échantillonnage de plusieurs espèces dans divers pays à travers le monde. Sans l’aide et la collaboration de plusieurs personnes et centres de recherches, il m’aurait été impossible de réaliser ce projet. Merci à Paul Palmer, Tim Lucas, Satoshi Mikami et Sizhong (Joe) Wang du Bribie Island Aquaculture Research Centre (Bribie Island, Queensland, Australie) pour m’avoir accueillie et permis d’utiliser les installations du centre de recherche pour faire mes expériences. Merci aussi au Dr. Peter F. Duncan, qui était affilié à University of the Sunshine Coast (Mooloolaba, Queensland, Australie) au moment de mon séjour en Australie, de m’avoir aidée à me procurer les pétoncles pour mes expériences en plus d’avoir contribué à l’organisation de mon séjour et de la collaboration avec le BIARC.

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Merci à Julian Harrington, Dr Craig Mundy et à toute l’équipe du Tasmanian Aquaculture and Fisheries Institute (Hobart, Tasmanie, Australie) pour leur précieuse aide lors de la collecte des pétoncles, la réalisation de mes expériences à leur centre de recherche et aussi pour m’avoir acceuillie chaleureusement pendant mon séjour là-bas. Merci à Bruno Myrand, Madeleine Nadeau et toute l’équipe du MAPAQ aux Îles- de-la-Madeleine (Québec, Canada) qui m’ont permis d’utiliser leurs installations pour la réalisation de mes expériences là-bas. Merci à Mélanie Bourgeois, de CultiMer, qui a fourni les pétoncles. Merci à Brian Kingzett et à toute l’équipe du Centre for Shellfish Research (Nanaimo, Vancouver Island, Canada) : sans leur aide et collaboration il m’aurait été impossible de faire le travail avec C. gigantea (ce pétoncle si particulier).

Finalement, un merci particulier à un groupe de personnes bien particulier : The International Pectinid Workshop. C’est grâce aux collaborations et amitiés développées au fil des années avec les gens participants à ce Workshop que j’ai pu réaliser toutes les étapes de mon projet de doctorat. Que ce soit via des contacts, conseils, informations ou encouragements, vous avez tous contribué à la réalisation de ce projet.

Quand j’ai commencé mon doctorat, il y avait un labo avec des gens : Orlane Rossignol, Hernan Mauricio Cortes-Pérez et Nicolas Martin. On était un petit groupe, mais on était bien ensemble. Puis, Helga et John ont pris leur retraite et sont déménagés à Boutillier Point. Ensuite, chacun est parti à tour de rôle jusqu’au moment où j’étais la seule qui restait au labo. C’est à ce moment que j’ai demandé à Helga de me trouver un coin à Dalhousie University où je pourrais terminer d’écrire ma thèse. Je suis très reconnaissante envers le Dr Brian Hall qui m’a gentiment accueillie dans son labo. Merci au gens du labo : Zabrina, Andrew et Kate (ainsi que leurs enfants Freya et Archie). Graduellement, je me suis fait une vie à Halifax et je me retrouve maintenant enourée de gens que j’apprécie beaucoup à l’Université (Joana, Jantina, Gordon, Mike, Zoé, Lauren) et à l’extérieur de l’Université aussi (Arnold, Emma et Jordon). Enfin, merci à Myriam Samson-Dô qui a fait l’analyse des données du ligament au chapitre 4 dans le cadre de son initiation à la recherche qu’elle a acceptée de faire avec moi. xxiv

Je ne peux pas passer sous silence mes ami(e)s qui font partie de ma vie, même si nous ne sommes pas toujours capables de nous voir sur une base régulière. Chacun à votre façon vous contribuez à enrichir ma vie. Je sais que parfois le temps et la distance nous empêchent d’être ensemble aussi souvent qu’on le voudrait, mais tout cela ne change en rien le lien qui nous unit : Joëlle, Gisèle, Marie-Ève, Nikki, Marianne, Perrine, Gaëlle, Maël et William. Une pensée toute spéciale pour Peter qui est toujours là au fil des années.

Finalement et non le moindre, il y a ma famille. Tout premièrement, il y a mes parents qui m’ont toujours soutenue et encouragée dans tous mes rêves et projets même si cela était parfois difficile pour eux. Je leur dois beaucoup et je les remercie pour tout l’amour qu’ils me donnent. Mon grand frère a toujours été un modèle de détermination et de persévérance pour moi. De nous deux, il est celui qui sait où il va dans la vie et prend le chemin le plus direct pour s’y rendre. De mon côté, je suis celle qui a une idée plutôt large d’où je me dirige dans la vie, et j’ai tendance à prendre quelques détours en chemin. Mon frère et moi sommes différents, mais semblables…on se comprend et se complète. Je pourrais parler de Catherine en tant que belle-sœur, mais cela ne serait pas tout à fait exact. Au fil des années, Catherine est devenue une amie et une confidante pour moi. Martin et Catherine ont eu récemment deux adorables garçons : Florian et Christophe. Ensemble ils forment une merveilleuse famille où je suis toujours la bienvenue et avec qui j’aime aller passer du temps pour me ressourcer. Vous dites souvent que ça ne doit pas être très reposant pour moi quand je suis avec vous et les garçons. Sachez que de partager votre quotidien me permet de me vider l’esprit et de remplir mon cœur de bonheur... Florian et Christophe sont très doués pour cette tâche! Merci pour tout l’amour et le support que vous me donnez! Il y a aussi ma famille élargie : oncles, tantes, cousins, cousines, petits-cousins et petites cousines. Ils sont nombreux, mais tous importants pour moi. J’ai la chance de faire partie d’une grande famille et j’en suis consciente. Comme un phare, ils sont toujours là, inébranlables à travers le temps.

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Avant-propos

Ma thèse comprend 6 chapitres, incluant une introduction générale et une conclusion rédigées en français ainsi que 4 chapitres écrits en anglais sous forme d’articles scientifiques pour publication. Je suis la principale auteure de chacun des articles dont j’ai été responsable de la planification, l’acquisition, l’analyse et l’interprétation des données ainsi que de la rédaction. D’autres auteurs ont également contribué à cette thèse, dont ma directrice Dr Helga E. Guderley, qui est professeure titulaire récemment retraitée au département de biologie de L’Université Laval. Dr Guderley est co-auteure de tous les articles pour son implication au niveau du financement et de la planification du projet en plus de sa supervision lors de la rédaction et la révision des manuscrits.

Le chapitre 2 intitulé « Swimming away or clamming up: the use of phasic and tonic adductor muscles during escape responses varies with shell morphology in scallops » a été publié dans la revue The Journal of Experimental Biology en 2012. Je suis premier auteur de cet article qui a été écrit en collaboration avec Dr Helga E. Guderley et Dr John H. Himmelman. Mon co-directeur de thèse, Dr John H. Himmelman professeure titulaire récemment retraité au département de biologie de L’Université Laval, est co-auteur du chapitre 2 pour son implication au niveau du financement du projet et de la révision du manuscrit.

Le chapitre 3 intitulé « Scallops show that muscle metabolic capacities reflect locomotor style and morphology » a été publié dans la revue Physiological and Biochemical Zoology en 2014. Je suis premier auteur de cet article qui a été écrit en collaboration avec Dr Helga E. Guderley.

Le chapitre 4 intitulé « When behaviour and mechanics meet: Scallop swimming capacities and their hinge ligament » a été soumis à la revue The Canadian Journal of Zoology. Je suis premier auteur de cet article qui a été écrit en collaboration avec Myriam Samson-Dô et Dr Helga E. Guderley. Myriam Samson-Dô est deuxième auteur de cet

xxvii article pour son implication dans l’acquisition d’une partie des données et dans leur analyse, ainsi que pour ses commentaires sur le manuscrit.

Le chapitre 5 intitulé « Can scallop swimming styles be predicted from shell and adductor muscle morphology? » sera prochainement soumis pour publication. Je suis premier auteur de cet article qui a été écrit en collaboration avec Dr Helga E. Guderley.

xxviii

CHAPITRE 1

Introduction générale

Isabelle Tremblay

La mobilité est une caractéristique importante des animaux et la variété des capacités locomotrices existantes est impressionnante. Par exemple, une grenouille peut nager et sauter, un cheval peut marcher, trotter et galoper, les phoques nagent tandis que leur capacité à se déplacer en dehors de l’eau est plutôt limitée. Cette diversité au niveau des capacités locomotrices se retrouve aussi à l’intérieur d’une même classe animale, par exemple les oiseaux. Dans ce groupe, on retrouve des espèces qui volent tandis que d’autres ont perdu, au cours de leur évolution, toute capacité à voler. Parmi les oiseaux qui ne volent pas, certains vont marcher et nager, par exemple les manchots, tandis que d’autres vont seulement marcher comme les kiwis endémiques de la Nouvelle-Zélande. En ce qui concerne le vol, il peut être passif ou actif. On parle de vol passif lorsque l’oiseau ne bat pas des ailes et qu’il fait du vol plané, par exemple un albatros exploitant les forts courants aériens de l’Antarctique. Quant au vol actif, il se caractérise par le battement des ailes pour combattre la gravité et les forces de traînées comme c’est le cas chez le pigeon. Ces différents modes de locomotion sont le résultat de relations complexes entre le style de vie d’un animal, son environnement, sa morphologie et ses capacités métaboliques. Chez les animaux, les fonctions majeures telles que la locomotion et la reproduction peuvent servir de force sélective favorisant certaines morphologies et les mécanismes physiologiques sous-jacents. Par exemple, la performance locomotrice est importante pour les activités telles que la recherche de nourriture, la capture de proies, la fuite face aux prédateurs et la migration. Ces activités sont déterminantes pour la survie, la croissance et le succès reproducteur d’un animal. Ainsi, les variations au niveau des mécanismes physiologiques déterminant la performance locomotrice sont êtres sujettes à la sélection. De plus, chaque organisme présente une combinaison d’attributs morphologiques, physiologiques et biochimiques travaillant de concert afin de permettre à l’organisme de se mouvoir dans son environnement. L’étude de la locomotion représente donc un défi majeur lorsque l’on veut comprendre comment chaque composante du système fonctionne séparément ainsi que dans un ensemble comme un tout intégré.

1. Déterminants de la performance locomotrice

1.1 Le muscle Les mouvements nécessaires à la locomotion chez les animaux sont générés principalement par les contractions musculaires. En effet, les cellules musculaires sont spécialisées dans la contraction. Elles sont regroupées selon leur morphologie et leur fonction en deux grands types cellules: les fibres musculaire striées et lisses. On retrouve les fibres musculaires striées au niveau du muscle squelettique et cardiaque. Le muscle squelettique est responsable des mouvements volontaires alors que le muscle cardiaque se retrouve seulement au niveau du cœur et a pour fonction de pomper le sang et l’hémolymphe dans l’organisme. Pour sa part, le muscle lisse est responsable, du moins chez les vertébrés, des mouvements involontaires comme par exemple au niveau du tube digestif et des parois vasculaires. Le muscle est composé de plusieurs faisceaux de fibres musculaires arrangés de façon parallèle les uns aux autres. Les fibres musculaires se forment lors du développement embryonnaire lorsque plusieurs cellules, appelées myoblastes ou myocytes, se fusionnent pour donner des cellules longues, cylindriques et multinucléées. Chaque fibre musculaire est composée de plusieurs sous-unités contractiles, appelées myofibrilles, arrangées de façon parallèle. Les myofibrilles consistent en une répétition longitudinale d’unités fonctionnelles appelées sarcomères, dont le patron spatial est responsable de l’apparence striée des muscles. Les sarcomères sont composés de filaments épais, de filaments minces et de titine. Les filaments épais sont formés principalement d’une protéine appelée myosine. Chaque molécule de myosine comprend deux chaînes lourdes identiques et plusieurs chaînes légères (Rayment et al. 1993 pour une revue complète). Une région de la molécule de myosine a la forme d’une double tête globulaire, tandis que le reste de la molécule est plutôt de forme longiligne (Fig. 1.1). C’est dans la région globuleuse de la molécule de myosine, appelée la tête, que se produit l’activité ATPasique et que se trouve le site de fixation avec l’actine. La région de la tête est formée de la partie globulaire des deux chaînes lourdes ainsi que deux ou trois (tout dépendant de l’espèce) chaînes légères de myosine (Fig. 1.1). Ces chaînes légères de la tête de la myosine diffèrent selon les types de muscles et influencent la vitesse maximale de contraction du muscle. La partie longiligne

3 de la molécule de myosine se divise en deux parties, appelées le cou et la queue, qui sont formées des parties en hélice α des chaînes lourdes de myosine entortillées les unes autour des autres (Fig. 1.1). Il existe différentes isoformes de chaînes lourdes dans le muscle strié squelettique dont certaines sont associées à une vitesse de contraction rapide (MHC-2a, MHC-2b, MHC-2x) et d’autres à une vitesse de contraction lente (MHC-1). L’expression des différentes isoformes des chaînes lourdes de myosine dépend en partie du type de fibre (rapide versus lente), mais aussi de l’espèce.

chaîne légère S1

Cou et Queue Tête (chaînes légères (chaîne lourdes de myosine) de myosine)

Figure 1.1. Molécule de myosine (Modifié à partir de Geeves 1991).

La titine est une protéine élastique géante qui joue un rôle structural majeur au niveau du sarcomère. En effet, la titine est importante pour l’assemblage et le maintient de la structure du sarcomère en plus de réguler l’élasticité de ce dernier. Dans le muscle, la titine est liées aux filaments de myosine et s’étend de la ligne Z à la ligne M. Les filaments minces sont formés principalement d’une protéine appelée actine et ressemblent à deux colliers de perles enroulés l’un autour de l’autre. Chaque monomère d’actine est une protéine globulaire appelée actine-G. Plusieurs monomères d’actine-G ensembles forment une chaîne d’actine-F (filamenteuse). Ainsi, deux chaînes d’actine-F s’enroulent l’une autour de l’autre pour former la matrice du filament mince. Au niveau

des sillons des chaînes d’actine, on retrouve une molécule filamenteuse appelée tropomyosine. Attaché à intervalles réguliers sur le filament de tropomyosine, on retrouve un complexe globulaire de protéines appelé troponine. La troponine, chez les vertébrés, contient trois sous-unités (TnT, TnI et TnC) qui ensemble établissent la position de la tropomyosine et régulent l’accès aux sites de fixation de la myosine sur l’actine.

1.1.1 La contraction musculaire La production de force mécanique par le muscle est le résultat de l’interaction entre les filaments épais de myosine et les filaments minces d’actine qui glissent les uns sur les autres lors de l’hydrolyse de l’ATP (Huxley 1985). Le cycle de la contraction musculaire requiert la libération de Ca2+ et la présence d’ATP. La contraction du muscle squelettique est déclenchée par l’influx nerveux qui stimule la libération du calcium (Ca2+). Ainsi, le Ca2+ contenu dans le réticulum sarcoplasmique (réseau spécialisé de citernes internes) est libéré dans le cytosol, ce qui résulte en une augmentation locale de la concentration du Ca2+. Le Ca2+ ainsi libéré va se lier à la troponine C et modifier la position du complexe troponine-tropomyosine, exposant les sites de fixation de la tête de myosine. La tête de myosine, portant un ADP et un ion phosphate (Pi), va se fixer à la molécule d’actine-G et changer l’angle de la tête. La fixation de la myosine à l’actine-G va déclencher la libération rapide du Pi et provoquer le mouvement de la tête de myosine, résultant en un déplacement du filament d’actine et un changement de la longueur du sarcomère. Ensuite, la tête de la myosine va relâcher l’ADP et rester attachée fermement à l’actine. C’est la fixation de l’ATP sur cette tête de myosine qui va permettre la séparation des deux filaments. Une fois détachée de l’actine, la myosine va hydrolyser l’ATP (ADP + Pi) et changer l’angle de la tête de myosine maintenant prête à s’attacher de nouveau à l’actine.

1.1.2 Types de fibres musculaires Le muscle squelettique des vertébrés est composé de plusieurs types de fibres musculaires qui se distinguent selon leurs propriétés biochimiques, métaboliques et histologiques. Au niveau du muscle squelettique des vertébrés, quatre groupes majeurs sont reconnus : les fibres toniques et trois types de fibres phasiques. 5

Les fibres toniques se contractent très lentement et ne produisent pas de secousse. Leur cycle de contraction lent permet des contractions soutenues à faible coût énergétique. Ainsi, on les retrouve principalement au niveau des muscles posturaux. Les fibres phasiques oxydatives lentes de type I (Tableau 1.1) se contractent et se fatiguent lentement. Les fibres phasiques oxydatives rapides de type IIa (Tableau 1.1) ont une vitesse de contraction et un niveau d’activation rapide. Grâce à leur niveau élevé de mitochondries, les fibres de type IIa peuvent produire de l’ATP rapidement par la phosphorylation oxydative et se fatiguent lentement. On retrouve les fibres de type IIa au niveau des muscles spécialisés pour les mouvements rapides et répétitifs d’un mode de locomotion vigoureux et soutenu comme par exemple, les muscles utilisés pour le vol de certains oiseaux. Les fibres phasiques glycolytiques rapides de type IIb (Tableau 1.1) se contractent rapidement et se fatiguent vite. Puisque leur contenu en mitochondries est faible, les fibres de type IIb s’appuient sur le métabolisme glycolytique anaérobie qui produit rapidement de l’ATP, mais elles se fatiguent rapidement. On retrouve, par exemple, des fibres glycolytiques de type IIb au niveau des muscles pectoraux du faisan qui sont utilisés pour l’envolée rapide et de courte durée de l’oiseau en fuite.

1.2 Métabolisme musculaire Lors de la contraction musculaire, il existe deux processus majeurs nécessitant l’utilisation d’énergie sous forme d’ATP. Le premier est l’hydrolyse de l’ATP, par la myosine ATPase, lorsque les têtes de myosine se détachent de l’actine. Le second est la réabsorption du Ca2+ à partir du cytosol par le réticulum sarcoplasmique. L’ATP nécessaire peut être produite via trois mécanismes biochimiques : l’utilisation de phosphagènes, le métabolisme anaérobie (glycolyse anaérobie) et le métabolisme aérobie.

Tableau 1.1. Propriétés des fibres musculaires squelettiques phasiques chez les mammifères (Modifié à partir de Hill et al. 2008)

Oxydative lente Oxydative rapide Glycolytique rapide Propriétés (type I) (type IIa) (type IIb) Taux de contraction Lent Rapide Rapide Activité de la myosine ATPase Faible Élevée Élevée Résistance à la fatigue Élevée Intermédiaire Faible Capacité pour la phosphorylation oxydative Élevée Élevée Faible Activité des enzymes de la glycolyse anaérobie Faible Intermédiaire Élevée Quantité de mitochondries Beaucoup Beaucoup Peu Diamètre des fibres Petit Intermédiaire Large Force produite par aire transversale Faible Intermédiaire Élevée

1.2.1 Utilisation des phosphagènes Les phosphagènes sont la principale source d’énergie lors des activités intenses de courte durée qui ne nécessitent pas d’oxygène (Fig. 1.2). En effet, les phosphagènes constituent une réserve d'énergie qui peut être mise très rapidement à la disposition du système contractile. L’hydrolyse du phosphagène va produire de l’ATP. Chez les vertébrés, la phosphocréatine est le seul phosphagène tandis que les invertébrés utilisent d’autres phosphagènes, comme par exemple la phosphoarginine.

1.2.2 Métabolisme anaérobie Le métabolisme anaérobie intervient lorsque l'activité musculaire se prolonge en absence d’oxygène et que la réserve de phosphagènes est presqu’épuisée (Fig. 1.2). Le glucose ou le glycogène servent alors de source énergétique pour la production d’ATP. Chez les vertébrés, il y a aussi production et accumulation d’acide lactique (lactate) et parfois même d’éthanol dans le muscle comme produit secondaire. En effet le pyruvate provenant de la dégradation du glucose est converti en lactate afin de régénérer le NAD+ requis pour la poursuite de la glycolyse. Au lieu de produire du lactate, les invertébrés vont accumuler d’autres produits secondaires tels que l’octopine, la strombine et l’analopine (Hochachka & Somero 2002).

1.2.3 Métabolisme aérobie Chez les vertébrés, une activité musculaire de longue durée dépendra du métabolisme aérobie (Fig. 1.2). Au cours du métabolisme aérobie, il y a dégradation des glucides, des lipides et ultimement des protéines, les trois agissant comme source énergétique pour la production d’ATP. La phosphorylation oxydative au niveau des mitochondries va produire de l’ATP, CO2 et H2O. L'implication de l'oxygène au niveau de la phosphorylation oxydative requiert donc une perfusion sanguine adéquate des tissus musculaires.

Phosphagènes ATP Métabolisme aérobie

Métabolisme anaérobie

Énergie

Secondes Minutes Heures

Temps

Figure 1.2. Évolution dans le temps de la contribution des différentes sources d’énergie pendant l’activité musculaire (Modifié à partir de Berg et al. 2002).

1.3 Facteurs affectant la performance locomotrice La performance locomotrice chez les animaux est affectée par divers facteurs abiotiques et biotiques. Chaque mode de locomotion est tout d’abord affecté par le milieu dans lequel l’animal évolue (aquatique, aérien et terrestre), ainsi que par les différentes forces physique (gravité, portance, poussée et traînée). La forme et la taille corporelle ainsi que la masse d’un individu vont influencer les différentes forces physiques auxquelles l’animal doit faire face. Les propriétés du milieu, telles la température ambiante et la disponibilité en oxygène, sont aussi des facteurs importants à considérer. En plus du niveau d’oxygénation et d’humidité, on doit aussi considérer la salinité en milieu aquatique. La température environnementale est probablement le facteur abiotique qui affecte le plus la performance locomotrice et ce via son influence sur les taux de fonctionnement des processus physiologiques (Bennett 1990). La performance locomotrice n’est pas nécessairement une fonction linéaire de la température et sa dépendance varie selon les espèces. La température ambiante affecte peu les animaux dits endothermes, car ces derniers sont capables de reternir la chaleur et de maintenir une température corporelle

élevée et stable. Pour leur part, les animaux dits ectothermes vont plutôt compter sur leur comportement afin de maintenir leur température corporelle préférée car leur capacité à retenir la chaleur est faible. Le maintien d’une température corporelle optimale va permettre aux ectothermes d'optimiser leurs fonctions physiologiques, ainsi que leur performance locomotrice. Chez plusieurs espèces d’ectothermes, la thermorégulation comportementale requiert souvent un changement important d'habitat. Dans ce cas-là, la capacité de thermorégulation comportementale est plutôt limitée. La capacité d’une espèce à tolérer des changements de température est donc d'une importance centrale pour les ectothermes. Ceci est d'autant plus vrai que la capacité locomotrice d’un animal est très importante pour sa survie et sa reproduction. Enfin, l’état physiologique d’un animal peut aussi influencer la performance locomotrice. Puisque la locomotion est un processus dont le coût énergétique est élevé, l’état reproducteur et la santé générale d’un animal peuvent influencer la performance locomotrice. En effet, la mobilisation des réserves énergétiques au cours de la reproduction d’un animal résulte en une diminution des réserves disponibles pour soutenir la locomotion. Lorsque les réserves énergétiques générales de l’animal sont en mauvaises conditions, tous les processus physiologiques en sont affectés.

2. Forme et fonction au niveau de la locomotion Au niveau des systèmes biologiques, le lien entre la morphologie et la physiologie est fondamental car bien souvent la forme dicte la fonction. Les processus au niveau morphologique, physiologique et biochimique seraient à la base de la locomotion et limiteraient chaque type de locomotion. Chez les vertébrés, la structure osseuse, les capacités cardiovasculaires ainsi que la taille des muscles, le type de fibre musculaire, les réserves énergétiques et les capacités métaboliques reflètent le style de locomotion de chaque espèce. Par exemple, les thons ont un corps de forme hydrodynamique en plus de présenter plusieurs adaptations au niveau physiologique (muscle rouge positionné centralement et endothermie régionale), ce qui leur permet d’augmenter considérablement leur performance de nage (Bernal et al. 2001). Les oiseaux capables de vol soutenu, tels les pigeons, ont des muscles pectoraux dotés d’une grande capacité aérobie (Pennycuick 1968,

Davis & Guderley 1987), tandis que les muscles requis pour l’envolée rapide des faisans ont une grande capacité glycolytique, mais se fatiguent très rapidement (Kiessling 1977, Davis & Guderley 1987, Marden 1994, Askew & Marsh 2002). Les capacités métaboliques du système locomoteur reflètent aussi les capacités locomotrices chez les animaux. En effet, les carburants et l’oxygène doivent être transportés à travers l’organisme (via le système circulatoire et respiratoire) pour se rendre jusqu’aux tissus où ils seront utilisés. Les propriétés intrinsèques des muscles, telles que le taux d’activation et de relaxation, la vitesse maximale de contraction et la production de force, ont aussi une influence sur la performance locomotrice (Else & Bennett 1987, Bennett et al. 1989, Herrel et al. 2007, James et al. 2007, Seebacher & James 2008). Pour leur part, les propriétés contractiles des muscles devraient être liées à l’activité de l’ATPase myofibrillaire ainsi qu’aux capacités des voies métaboliques oxydative et glycolytique à produire l’ATP nécessaire au mouvement (Garland 1984, Geeves 1991, Dickson et al. 1993, Guderley 2004, Alexander 2005). La comparaison d’organismes avec des styles de locomotion très différents montre que les indicateurs de la capacité aérobie sont plus élevés chez les animaux avec une plus grande endurance (Davis & Guderley 1987, Weibel et al. 1996). Les muscles des animaux de capacité aérobie élevée ont une plus grande proportion de fibres de type I et IIa possédant une quantité élevée de mitochondries. D’un autre côté, les animaux spécialisés pour les activités intenses et de courte durée ont des muscles avec une plus grande proportion de fibres de type IIb qui se contractent rapidement et s’appuient sur le métabolisme glycolytique. La série de travaux fait par Weibel et al. (1996) compare la capacité des systèmes de captation, livraison et utilisation de l’oxygène chez le chien et la chèvre, deux mammifères de taille semblable, mais de capacité aérobie bien différente. Ces travaux montrent qu’en général les capacités des systèmes se suivent avec pour seule exception les poumons dont la capacité dépasse celle des autres parties. Quant à l’appui métabolique à l’exercice de ces deux mammifères, l’étude révèle que la capacité d’utilisation des glucides et des lipides augmente parallèlement. Ainsi, plusieurs types d’évidences suggèrent que la co-évolution des éléments du système locomoteur s’étend aussi aux capacités métaboliques musculaires. Les études in vitro chez les vertébrés montrent plusieurs adaptations aux niveaux morphologique, physiologique et métabolique. Les études comparatives ont permis de faire

11 le lien entre l’endurance d’un organisme et sa capacité maximale de consommation d’oxygène, alors que la vitesse et l’intensité de la réponse ont été corrélées aux capacités du métabolisme anaérobie, soit via le catabolisme des phosphagènes ou via la production de lactate (ou d’octopine selon le taxon); (Bennett 1991). Mais qu’est-ce qui se passe in vivo? Qu’est-ce qui se passe au niveau de l’utilisation des voies métaboliques pendant la locomotion? Comment est utilisé le muscle pendant la locomotion?

Quand les différences au niveau de la performance sont grandes, les relations structure-fonction deviennent plus évidentes. Ainsi, nous pouvons comparer différentes espèces présentant des capacités locomotrices très variées ou même, encore mieux, comparer des organismes provenant de la même espèce mais présentant un éventail de capacités locomotrices. Cependant, la biomécanique complexe des vertébrés rend difficile l’intégration de ces connaissances avec les aspects mécaniques de l’appareil locomoteur. Parmi les invertébrés, on retrouve une famille de bivalves avec une grande diversité au niveau de la morphologie de la coquille, accompagnée d’une diversité intéressante au niveau des capacités locomotrices. Cette famille, les pectinidés, a aussi l’avantage d’avoir un système locomoteur relativement simple. Ainsi, les pétoncles sont tout désignés comme modèle animal pour comprendre les liens entre la performance locomotrice et les différentes composantes du système locomoteur.

3. Les pétoncles Les pétoncles font partie de la classe Bivalvia qui comprend aussi l’huître, la moule et la mye. La plupart des bivalves sont sessiles, mais il existe tout de même différents modes de vie. Certains, telle la mye, s’enfouissent dans les sédiments. D’autres, tels l’huître ou la moule, vont se fixer sur des surfaces à l’aide d’un byssus ou directement en secrétant leur coquille. Quelques bivalves peuvent même aller jusqu’à percer des matériaux, tels que le bois ou la pierre, pour s’y réfugier et y vivre au fond d’un trou qu’ils ont creusé. Enfin, les pétoncles sont parmi les seuls bivalves à être capables de nager dans l’eau.

La famille des pectinidés comprend environ 250 espèces (Brand 2006) vivantes occupant une grande variété de niches écologiques et démontrant une grande diversité morphologique et comportementale. De plus, les pétoncles ont l’avantage de présenter un système locomoteur composé d’un seul muscle adducteur, de deux valves et d’un ligament. Cette structure biomécanique relativement simple des pétoncles, combinée à une grande diversité morphologique et comportementale, en font un modèle animal idéal pour comprendre les liens entre la performance locomotrice et les différentes composantes du système locomoteur.

3.1. Origine de la nage chez les pétoncles Un scénario évolutif suggère que l’habilité de nage a été acquise suite à la colonisation des habitats turbides. En effet, les changements morphologiques de la coquille accompagnant la réduction ou la perte du muscle adducteur antérieur, la réduction de l’attachement par un byssus et l’ouverture du manteau (pour augmenter les courants pour la filtration) sont des modifications qui auraient permis aux ancêtres des pétoncles d’avoir accès à des eaux plus profondes et/ou turbides (Yonge 1936). Il y aurait aussi eu perte du siphon rendant ainsi l’évacuation des particules, accumulées dans la cavité du manteau, problématique. Ainsi, on remarque une amélioration du tractus ciliaire facilitant l’évacuation des sédiments accumulés et une augmentation importante du muscle adducteur strié qui permet d’augmenter la vitesse de contraction des valves et des courants transportant les sédiments hors de la cavité du manteau des pétoncles. De plus, le repli interne du manteau s’est élargi dans le but de former une ouverture pour l’entrée et la sortie de l’eau dans la cavité du pétoncle. Ces modifications structurales, qui seraient nécessaires à la nage et aux autres comportements de fuite, se seraient développées chez les ancêtres des pétoncles en réponse à des changements morphologiques accompagnant la colonisation des eaux turbides. Ainsi, les modifications au niveau de la structure de la coquille, du manteau et du muscle adducteur sont considérées comme des adaptations dérivées chez les bivalves monomyaires, soit ceux ayant un seul muscle adducteur (Drew 1906, Yonge 1936). Il y a donc eu une co-évolution de traits complexes permettant une réponse comportementale particulière chez les pétoncles.

3.2 Évolution des modes de vie des pétoncles Au cours de leur développement, les larves véligères de pétoncles nagent librement dans la colonne d’eau pour ensuite se métamorphoser et se fixer au substrat par un byssus et ainsi continuer leur croissance. Éventuellement, le pétoncle va adopter un mode de vie qu’il gardera pour le reste de sa vie adulte. Il y a 6 ou 7 modes de vie, au stade adulte, dans lesquels les différentes espèces de pétoncles sont généralement regroupées. Le tableau 1.2 décrit brièvement ces modes de vie. Les études génétiques ont montré que l’attachement via un byssus est fort probablement le mode de vie ancestral qui aurait donné naissance aux différentes espèces de pétoncles (Alejandrino et al. 2011). Les deux types d’évolution, parallèle et convergente, auraient générées par la suite les différents modes de vie observés chez les pétoncles. Il semble que les différents modes de vie, observés chez les pétoncles au stade adulte, seraient apparus à plusieurs reprises au cours de l’évolution. Les travaux d’Alejandrino ont dénombré 17 transitions entre les différents modes de vie chez les pétoncles (Alejandrino et al. 2011). Alors que la majorité des modes de vie observés chez les pétoncles peuvent être soit un stade ancestral ou transitoire, le mode de vie cimenté apparaît seulement en tant que stade dérivé. Ce biais, au niveau des transitions entre les modes de vie, signale la présence de contraintes dues à la complexité des changements physiologiques et morphologiques nécessaires à la transition d’un mode de vie à un autre. Ainsi il a été suggéré que les pétoncles, avec un mode de vie cimenté, pourraient avoir des traits physiologiques spécifiques qui empêcheraient la transition vers un autre mode de vie.

3.3 Locomotion chez les pétoncles En présence des prédateurs ou lors d’un contact avec ces derniers, le pétoncle peut produire un simple saut, il peut tourner sur lui-même ou il peut se déplacer horizontalement sur une distance plus ou moins longue; c’est ce dernier que l’on appelle la nage (Buddenbrock 1911). La nage chez les pétoncles provient de la propulsion de l’animal grâce à des jets d’eau produits par une succession de contractions où alternent adductions

Tableau 1.2. Principaux modes de vie chez les pétoncles (Modifié à partir d’Alejandrino et al. 2011). Mode de vie Description Exemple d’espèce Références Niché Fixé par un byssus, le pétoncle se retrouve coincé à Yonge 1967 l’intérieur d’un corail vivant qui se développe autour du Pedum spondyloideum pétoncle. Cimenté Attaché de façon permanente à un substrat dur au fur et à Yonge 1951 Crassadoma gigantea mesure que sa coquille se développe. Attaché par un byssus Attaché temporairement au substrat par des filaments de Young & Martin 1989, Mimachlamys asperrima, byssus; il peut toutefois se détacher et nager pour se Gruffydd 1976 Chlamys islandica relocaliser Enfouis Enfouis partiellement ou totalement dans une cavité Pecten maximus, Baird 1958, Vélez et al. 1995 creusée par le pétoncle dans les sédiments mous Euvola ziczac Libre Repose sur les sédiments mous ou sur un substrat dur. Aequipecten opercularis, Belding 1931, Olsen 1955, Equichlamys bifrons, Chapman et al. 1979 Argopecten irradians Planeur Capable de nager sur plus de 5 m lors d’une réponse de Manuel & Dadswell 1993, Amusium balloti, fuite; inclut une phase de nage à l’horizontal avec une Joll 1989 Placopecten magellanicus composante de glisse

(fermetures) et abductions (ouvertures) des deux valves (Drew 1906, Dakin 1909, Buddenbrock 1911). Le pétoncle nage avec l’ouverture des valves à l’avant et la charnière à l’arrière, donnant l’impression qu’il prend des bouchées dans l’eau (Fig. 1.3). Le muscle adducteur est responsable de la fermeture des valves alors qu’un ligament non-calcifié, aux propriétés élastiques, agit comme un ressort pour l’ouverture des valves lorsque le muscle adducteur relaxe (Trueman 1953a, Alexander 1966, Marsh et al. 1976). La taille et l’orientation des jets d’eau sont contrôlées par le manteau musculaire du pétoncle. Ainsi, le pétoncle peut produire un simple jet d’eau dirigé ventralement lorsqu’il veut simplement se détacher d’un prédateur. Lorsque le pétoncle se déplace sur de longues distances, les jets d’eau sont dirigés antérieurement et sortent par les ouvertures latérales de chaque côté de la charnière (Fig. 1.3). La dynamique de fermeture et d’ouverture des valves, ainsi que la production de force, ont été analysées par Marsh et al. (Fig. 1.4) à l’aide d’un transducteur piézo- électrique chez Chlamys hastata et Argopecten irradians (Marsh et al. 1992). Au cours de la phase initiale de fermeture des valves, l’eau est explusée de la cavité du manteau qui n’est pas scellée. La pression à l’intérieur de la cavité du manteau augmente rapidement seulement lorsque la cavité est complètement scellée par le manteau. Durant cette phase du cycle de contraction, le taux d’écoulement de l’eau et la pression à l’intérieur de la cavité du manteau sont corrélés positivement. Lorsque le muscle adducteur relaxe et que les valves s’ouvrent, grâce à la décompression du ligament, la pression dans la cavité du manteau devient négative et l’eau va s’écouler de l’extérieur vers l’intérieur de la cavité. La cavité du manteau ainsi remplie d’eau, le pétoncle est prêt pour la contraction suivante. Marsh et al. a calculé que seulement une faible portion de la force produite par le muscle adducteur est nécessaire pour la compression du ligament et l’accélération des valves et de la masse ajoutée de l’eau.

A) Entrée d’eau Entrée d’eau Ventral

Antérieur Postérieur

Coquille Muscle adducteur

Dorsal Jet d’eau Jet d’eau

B) Entrée d’eau

Direction de la nage

Jet d’eau

Figure 1.3. Schémas représentant le mécanisme de la nage chez les pétoncles. A) Vue du dessus. B) Vue latérale.

Pression (kPa) Pression Distance (mm) Distance

) 1 -

(ml s Écoulement

)

1 -

kg (W Puissance

Temps (ms)

4. Les composantes du système locomoteur

4.1 Le muscle adducteur Les pétoncles sont monomyaires, c’est-à-dire qu’ils n’ont qu’un seul muscle adducteur. Ce muscle est composé de deux parties distinctes, une partie phasique et une partie tonique (Fig. 1.5), séparées par un mince tissu conjonctif (Millman 1967, de Zwaan et al. 1980). Ces deux parties du muscle adducteur diffèrent au niveau de leur structure et de leur fonction (Chantler 2006).

Phasique

Tonique

Figure 1.5. Partie phasique et tonique du muscle adducteur chez Pecten fumatus (Photo : Isabelle Tremblay).

4.1.1 Le muscle phasique Chez la plupart des espèces, le muscle phasique compte pour la plus grande proportion du muscle adducteur. Par exemple, la partie phasique du muscle adducteur correspond à environ 80% du poids total du muscle chez Placopecten magellanicus (de Zwaan et al. 1980). Il existe cependant une certaine variabilité, entre les différentes

19 espèces de pétoncles, au niveau de la proportion totale du muscle adducteur correspondant à la partie phasique et tonique du muscle. Il semble que la partie phasique du muscle adducteur est plus grande chez les espèces de pétoncles qui nagent plus comparativement aux espèces plutôt sédentaires (Yonge 1936, Gould 1971, Soemodihardjo 1974), reflétant ainsi le rôle central du muscle pour les contractions phasiques. Cette partie du muscle adducteur est aussi une importante réserve en glycogène et protéines (Barber & Blake 1991). La partie phasique du muscle est composée de fibres musculaires striées qui se contractent rapidement et c’est cette partie du muscle qui est responsable des fermetures rapides des valves nécessaires à la nage du pétoncle (Lowry 1953, Millman 1967, Millman & Bennett 1976, Nunzi & Franzini-Armstrong 1981, Olson & Marsh 1993). Les pics de tension et de relaxation du muscle phasique des pétoncles sont similaires à ceux observés au niveau des fibres musculaires rapides des vertébrés (Rall 1981, Marsh et al. 1992, Olson & Marsh 1993, Pérez et al. 2009b). Au niveau des filaments épais de myosine du muscle phasique des pétoncles, on retrouve une protéine appelée paramyosine qui forme le cœur central des filaments (Levine et al. 1976). Le contenu en paramyosine du muscle phasique est faible comparativement au muscle tonique (Hardwicke & Hanson 1971, Levine et al. 1976). Chez les pétoncles, les activités motrices et enzymatiques de la myosine sont activées par le Ca2+ qui se fixe directement sur la myosine la rendant ainsi disponible pour interagir avec l’actine (Kendrick-Jones et al. 1970, Kendrick-Jones et al. 1976, Chantler & Szent-Gyorgyi 1980, Chantler et al. 1981). Ce mécanisme régulateur lié à la myosine des pétoncles diffère de celui observé au niveau du muscle squelettique des vertébrés où le mécanisme régulateur est lié à l’actine et qui nécessite la troponine et la tropomyosine (Ebashi et al. 1969, Lehman & Szent-Gyorgyi 1975).

4.1.2 Le muscle tonique Le muscle tonique représente habituellement une faible proportion du muscle adducteur; ce qui correspond à environ 20% du poids total du muscle chez Placopecten magellanicus (de Zwaan et al. 1980). Il est composé de fibres musculaires lisses qui se contractent lentement et qui sont capables de maintenir leur contraction pour une longue durée à un faible coût métabolique (Watabe & Hartshorne 1990). En effet, le muscle

tonique du pétoncle utilise un mécanisme de verouillage (catch) similaire à celui retrouvé au niveau du muscle adducteur antérieur des moules (Nunzi & Franzini-Armstrong 1981, Chantler 2006). Le mécanisme de verouillage (catch) du muscle tonique se caractérise par le maintient de la tension dans le muscle, durant une période de temps prolongée, à la fin de la contraction active (Winton 1937, Twarog 1954, Jewell 1959, Johnson & Twarog 1960, Lowy and Millman 1963). Le muscle tonique est utilisé lors de la fermeture prolongée des valves (Nunzi & Franzini-Armstrong 1981) ou supposément pour le maintien d’une ouverture constante des valves lors de la respiration du pétoncle non perturbé. Le muscle tonique est aussi recruté entre les séries de contractions phasiques et permet ainsi une certaine récupération de la charge énergétique du muscle phasique, ce qui aiderait à maintenir la production de force lors de la poursuite de la réponse de fuite (Pérez et al. 2008b). Les filaments épais du muscle tonique sont riches en paramyosine, comptant jusqu’à 80% de leur masse (Millman 1967, Szent-Gyorgyi et al. 1971, Eliott 1974, Levine et al. 1976). Ces filaments épais sont composés d’un centre dense de paramyosine recouvert d’une fine couche de myosine (Szent-Gyorgyi et al. 1971, Nonomura 1974). On retrouve aussi deux protéines, la « catchine » (Yamada et al. 2000) et la « twitchine » (Siegman et al. 1998), qui seraient toutes deux impliquées dans le mécanisme de verouillage (catch), mais leur rôle n’est pas encore bien compris (Yamada et al. 2000, Tsutsui et al. 2007). En plus de l’activation directe par le Ca2+, il peut y avoir phosphorylation de la myosine afin d’activer cette dernière (Sohma et al. 1985, Castellani & Cohen 1987, Takahashi et al. 1988)

4.2. Le ligament Chez les pétoncles, le ligament est produit par le manteau et est situé dorsalement entre les deux valves de chaque côté du rostre (Fig. 1.6a); (Trueman 1953b). Le ligament est divisé en deux parties nommées respectivement ligament externe et interne. Le ligament externe réunit les deux valves au niveau dorsal le long de la charnière. En plus d’être stratifié en couches quasi parallèles, le ligament externe est flexible et joue le rôle de charnière (Trueman 1953b). Le ligament interne a quant à lui une forme pyramidale et sa

A) Ligament interne

Rostre

Ligament externe

B) Région latérale du ligament interne

Ligament externe

valve

Région centrale du ligament interne

Figure 1.6. A) Intérieur de la région dorsale de la valve gauche, chez Amusium balloti, montrant le ligament en coupe longitudinale. Échelle en centimètres (Photo : Isabelle Tremblay). B) Diagramme de la section transversale du ligament de Chlamys opercularis montrant le ligament externe ainsi que la région centrale et les régions latérales du ligament interne (Modifié à partir de Trueman 1953a).

base ventrale est bombée lorsque les valves sont fermées (Trueman 1953b). Ce ligament est composé d’une large partie centrale non-calcifiée ainsi que deux régions latérales calcifiées qui attachent le ligament à la coquille (Fig. 1.6b); (Trueman 1953b). La partie centrale du ligament interne est composée d’une protéine élastique, l’abductine, qui agit comme un ressort à compression permettant ainsi aux valves de s’ouvrir lorsque le muscle adducteur se relaxe (Alexander 1966, Kelly & Rice, 1967). Au niveau des propriétés mécaniques, le ligament des pétoncles diffère de celui des autres bivalves, laissant supposer une structure efficace pour l’ouverture et la fermeture répétée des valves (Trueman 1953a, Kahler et al. 1976). En effet, le ligament des pétoncles a une résilience plus élevée que celui des bivalves qui ne nagent pas (Kahler et al. 1976). La résilience correspond à la propriété d’un matériel à absorber l’énergie lorsqu’il est déformé de façon élastique et ensuite à libérer cette énergie lors du déchargement. Aussi, le ligament des pétoncles a un moment d’ouverture des valves (valeur du moment appliqué (g mm-1) lorsque les valves commencent juste à s’entrouvrir lors le cycle d’ouverture), par gramme de coquille, plus faible comparativement aux autres bivalves (Kahler et al. 1976). Enfin, lorsqu’un poids est appliqué sur la valve supérieure, le changement d’angle d’ouverture des valves se fait plus rapidement chez le pétoncle Pecten maximus comparativement aux bivalves qui ne nagent pas (Trueman 1953a). Le ligament interne des pétoncles présente aussi des différences au niveau de sa composition biochimique par rapport aux bivalves qui ne nagent pas. En effet, l’abductine du ligament des pétoncles contient plus de glycine et moins de proline et de cystéine que celle qui est habituellement retrouvée chez les bivalves non-nageurs (Kahler et al. 1976). La résilience du ligament des bivalves est corrélée positivement avec le contenu en glycine ce suggérant que plus le ligament s’approche de la polyglycine, plus il fonctionne efficacement (Kahler et al. 1976). Aussi, la résilience du ligament est corrélée négativement à la concentration en cystéine, laissant supposer que la faible quantité de liens entre les molécules résulte en une diminution au niveau des restrictions de mouvement dans le ligament et donc confère une propriété plutôt caoutchouteuse au ligament (Kahler et al. 1976). Enfin, la résilience du ligament est corrélée négativement à la concentration de

CaCO3, ce qui suggère que les cristaux d’aragonite pourraient interfèrer au niveau de la capacité du ligament à récupérer sa forme suite à la compression (Kahler et al. 1976). Ces

23 particularités au niveau de la composition biochimique du ligament chez les pétoncles contribuent fort probablement à rendre ce dernier particulièrement bien adapté pour les mouvements répétés de fermeture et d’ouverture des valves lors de la nage (Kahler et al. 1976). Bien que les différences au niveau de la résilience du ligament soient claires au niveau interspécifique, la valeur adaptative de cette propriété reste plutôt mitigée. Il a été montré que l’abductine du ligament d’Adamussium colbecki a une résilience plus élevée à basse température comparativement aux espèces de pétoncles vivants à des températures tempérées (Denny & Miller 2006). Par contre, les auteurs ont montré que même s’il y avait un changement drastique de la résilience du ligament, l’impact sur la période de résonnance du système coquille-ligament serait faible. De plus, la majeure partie de l’énergie perdue lors de l’oscillation des valves est d’origine hydrodynamique et non due à la résilience du ligament. Ainsi, il est plutôt difficile de juger l’impact qu’aura un changement de la résilience du ligament sur la locomotion du pétoncle.

4.3. La coquille La coquille des pétoncles a deux principaux rôles : la protection contre les prédateurs et la nage. Les pétoncles ayant de bonnes capacités de nage ont tendance à avoir une coquille légère, lisse, avec un allongement élevé et avec la valve supérieure légèrement plus convexe que la valve inférieure (Gould 1971, Soemodihardjo 1974). Pour leur part, les pétoncles qui ne nagent pas ont tendance à avoir une coquille lourde, asymétrique et à couler rapidement dans l’eau (Gutsell 1931, Moore & Marshall 1967, Gould 1971). Les différentes caractéristiques de la coquilles influencent chacune à leur façon la nage chez les pétoncles. Un allongement (longueur de la valve au carré/l’aire de la valve; Fig. 1.7) élevé de la coquille augmente le ratio poussée/traînée, ce qui favoriserait la nage (Gould 1971). Lorsque la valve supérieure est plus convexe que la valve inférieure, il se crée une zone de pression plus faible sur la face supérieure de la coquille ce qui produit une portance additionnelle pour la nage (Stanley 1970). La surface lisse des valves réduit la friction, alors qu’un faible poids de la coquille réduit à la fois la vitesse à laquelle le

hauteur

longueur

Direction de la nage

pétoncle coule et la vélocité nécessaire pour élever le pétoncle dans la colonne d’eau (Gould 1971).

5. Support métabolique de la nage Comme l’hémolymphe des pétoncles ne possède pas de pigment respiratoire et que le muscle adducteur a une perfusion sanguine très limitée, l’activité de nage est appuyée par le métabolisme anaérobie (Grieshaber & Gäde 1977, Thompson et al. 1980, de Zwaan et al. 1980). Le muscle adducteur phasique utilise l’ATP à un rythme élevé afin de réaliser les contractions phasique rapides. La majorité de l’ATP utilisée pour les contractions phasiques (environ 70% chez Placopecten magellanicus et Argopecten irradians concentricus) est générée à partir de la phosphoarginine par l’arginine kinase (de Zwaan et al. 1980, Livingstone et al. 1981, Chih & Ellington 1983, Chih & Ellington 1986); (Fig. 1.8). Ainsi, on observe une relation linéaire négative entre la concentration de 25

Glycogen Pi

Glucose-6-phosphate

Fructose-6-phosphate ATP

ADP

Fructose-1,6-bisphosphate

Glyceraldehyde 3-phosphate + Pi NAD

NADH

Glyceraldehyde 1,3-phosphate ADP ATP

3-phosphoglygerate

Arginine phosphate Phosphoenolpyruvate ADP

ATP ATP Arginine Pyruvate ADP +

NADH

NAD+

Octopine

Figure 1.8. Principales voies de production d’énergie chez le pétoncle.

phosphoarginine dans le muscle phasique et le nombre de contractions phasiques (Livingstone et al. 1981, Bailey et al. 2003). La réponse de fuite des pétoncles par la nage ne peut être maintenue que quelques minutes, soit jusqu’à ce que les stocks de phosphoarginine s’épuisent (Grieshaber 1978). En effet, l’épuisement des stocks de phosphoarginine, ainsi que la diminution de l’énergie libre de l’hydrolyse de l’ATP dans le muscle adducteur, sont probablement à l’origine de l’apparition des contractions toniques prolongées qui sont un signe d’épuisement chez les pétoncles (Bailey et al. 2003, Pérez et al. 2008a). À la fin de la réponse de fuite, la principale source d’ATP pour environ le dernier 30% des contractions phasiques devient la glycolyse anaérobie (Grieshaber & Gäde 1977, Gäde et al. 1978). Au cours de la glycolyse anaérobie, le glycogène est utilisé comme substrat et il y a une formation concomitante d’octopine à partir du pyruvate et de l’arginine (Grieshaber & Gäde 1977, de Zwaan et al. 1980, Livingstone et al. 1981, Chih & Ellington 1983). En parallèle avec la formation du lactate chez les vertébrés, la production d’octopine refait le NAD+ à partir du NADH ce qui permet de maintenir la production d’ATP lorsque les tissus sont peu oxygénés (Grieshaber & Gäde 1977, Livingstone et al. 1981). Éventuellement le pétoncle va s’épuiser, ce qui se manifeste par une absence de réponse même si la stimulation se poursuit (MacDonald et al. 2006). Lorsqu’un pétoncle est épuisé on remarque, chez la majorité des espèces, une fermeture des valves et ce pour une période de durée variable. Durant cette période, l’approvisionnement en oxygène est nul, donc la production d’ATP provient du métabolisme anaérobie. Au cours de cette phase, la formation d’octopine va permettre la récupération partielle de la charge énergétique (Livingstone et al. 1981, Pérez et al. 2008a). La récupération métabolique complète, suite à un épuisement, requiert le métabolisme aérobie (Livingstone et al. 1981). En effet, le pétoncle va finir par ouvrir à nouveau ses valves et commencera une phase de récupération aérobie. Durant cette période, le pétoncle pourra restaurer ses stocks de phosphoarginine et de glycogène du muscle adducteur (Grieshaber 1978, Livingstone et al. 1981, Pérez et al. 2008a). Lors de

27 la récupération aérobie, la consommation d’oxygène du pétoncle dépasse de beaucoup le taux mesuré au repos, soit jusqu’à 12 fois chez Chlamys islandica (Thompson et al. 1980, Tremblay et al. 2006). Il faut cependant compter plusieurs heures avant de retrouver les niveaux initiaux des substrats énergétiques et de performance de fuite : 12-24 h chez P. magellanicus (Livingstone et al. 1981, Pérez et al. 2008a), 2 h chez Aequipecten opercularis (Grieshaber 1978), jusqu’à 18h chez Chlamys islandica matures (Brokordt et al. 2000a), et seulement 35 min chez Euvola ziczac matures (Brokordt et al. 2000b).

6. Facteurs influençant la nage des pétoncles La capacité de nage des pétoncles peut être affectée par plusieurs facteurs abiotiques et biotiques incluant les forces physiques du milieu, les propriétés morphologiques et physiologiques des pétoncles ainsi que les conditions environnementales.

6.1 Forces agissant sur le pétoncle Puisque l’eau et l’air sont des fluides qui partagent des propriétés similaires, les animaux qui nagent ou qui volent doivent faire face à des forces semblables lors de leurs déplacements. Afin de nager, un pétoncle doit produire une poussée suffisante pour vaincre la force de traînée en plus de produire assez de portance pour contrer la gravité (Fig. 1.9). Chez les pétoncles, la poussée est produite par les jets d’eau. Comme pour tous les animaux utilisant la propulsion par jets d’eau, la capacité de nage des pétoncles dépend non seulement de la rapidité de l’animal à expulser l’eau pour produire la poussée, mais aussi sa capacité à remplir d’eau sa cavité. Lorsque la fréquence des cycles de fermeture et d’ouverture des valves est élevée, la poussée produite pour une période de temps donnée et la puissance disponible pour propulser le pétoncle s’accroissent. Une augmentation de la puissance de la poussée peut potentiellement soulever une plus grande masse et permettre au pétoncle de se déplacer plus rapidement.

Portance

Poussé Traînée e

Gravité

Figure 1.9. Forces agissant sur le pétoncle dans la colonne d’eau (Modifié à partir de Gould 1971).

La traînée, qui a pour effet de résister au déplacement horizontal du pétoncle. Chez les pétoncles on observe une traînée de profile ainsi qu’une traînée induite (Gould 1971). Chacune des ces deux traînées peut se décomposer en deux : une traînée de friction (synonyme : traînée de surface) et une traînée de pression (synonyme : traînée de forme). La traînée induite est le résultat de la génération de portance, ainsi elle est influencée principalement par la forme de la coquille, l’angle d’attaque et l’angle de nage du pétoncle dans l’eau ainsi que la vitesse à laquelle le pétoncle nage. La traînée de friction concerne la couche limite, laquelle est soumise à des contraintes de cisaillement suite au glissement forcé des plans moléculaires les uns sur les autres. Ainsi, la traînée de friction est affectée par la surface de la coquille. La traînée de pression est due à la création d’un gradient entre une zone de surpression qui est créée à l’avant de la coquille du pétoncle et une zone de dépression vers l’arrière de la coquille. Donc, la traînée de pression va être influencée principalement par forme de la coquille. La force de gravité est surtout déterminée par la masse de la coquille et la flottabilité du pétoncle. Tandis que les tissus mous du pétoncle ont une densité similaire à l’eau de mer, la densité de la coquille du pétoncle est quant à elle plus élevée.

Une force de portance doit être générée pour contrebalancer la gravité et éviter au pétoncle de couler au fond de l’eau. La portance est influencée par la forme de la coquille, l’angle d’attaque et l’angle de nage du pétoncle dans l’eau ainsi que la vitesse à laquelle le pétoncle nage. Certain pétoncles ont une coquille dont le profil ressemble à une aile d’avion; la valve gauche (supérieure) ayant une convexité plus élevée que la valve droite (inférieure), ce qui serait favorable pour la génération de portance. Par contre, la majorité des pétoncles ont un profil hydrodynamique équiconvexe ou même plano-convexe (valve supérieure plate et valve inférieure convexe).

6.2 Composantes morphologiques affectant la nage Les caractéristiques morphologiques de la coquille affectent les forces agissant sur le pétoncle lorsqu’il nage. Ainsi, la capacité de nage du pétoncle est dépendante de la morphologie. Les principales caractéristiques de la coquille influençant la nage incluent la densité, la taille, la masse, l’épaisseur, la forme, la microstructure, l’allongement ainsi que le bord d’attaque de la coquille (Gould 1971, Morton 1980, Dadswell & Weihs 1990). Les pétoncles, étant reconnus comme étant de bons nageurs, ont habituellement des coquilles légères, lisses, avec un allongement élevé et une valve supérieure plus convexe que la valve inférieure (Gould 1971, Soemodihardjo 1974). D’un autre côté la forme et les caractéristiques de la coquille de certaines espèces semblent désavantageuses pour la nage, comme c’est le cas pour les pétoncles du genre Pecten qui ont une coquille de forme plano- convexe. Il est intéressant de noter que ce type de coquille est capable de produire une portance similaire aux coquilles de forme bi-convexe, mais à un angle d’incidence inférieur (Millward & Whyte 1992). Au niveau de l’anatomie du pétoncle, la proportion et l’arrangement des deux parties du muscle adducteur, ainsi que les propriétés du ligament, pourraient aussi influencer la capacité de nage. En se basant sur des descriptions qualitatives de performance de nage, on suggère que ce comportement de nage varie positivement avec la proportion de la partie phasique du muscle adducteur (Gould 1971, Soemodihardjo 1974) ainsi qu’avec l’oblicité du muscle phasique dans un plan perpendiculaire à la charnière (Thayer 1972). Les propriétés mécaniques du ligament des pétoncles diffèrent des autres

bivalves (Trueman 1953a). En effet, la résilience du ligament est plus élevée chez les pétoncles comparativement aux bivalves qui s’enfouissent ou qui sont sessiles, ce qui rendrait le ligament des pétoncles plus efficace pour les cycles répétées de fermeture et d’ouverture des valves (Kahler et al. 1976). La taille possède une forte influence sur la capacité de nage des pétoncles. Puisque la masse de la coquille augmente de façon exponentielle avec la taille, la puissance requise pour la nage augmente également (Gould 1971, Gruffydd 1976). De plus, une augmentation de taille des pétoncles va entraîner des changements de la forme de la coquille et de la position du muscle adducteur (Merrill 1961, Gould 1971, Dadswell & Weihs 1990). Chez plusieurs espèces, les pétoncles de petite taille sont habituellement plus actifs que ceux de grande taille (Yonge 1936, Olsen 1955, Baird & Gibson 1956, Caddy 1968, Gruffydd 1976, Tremblay et al. 2006, Labrecque & Guderley 2011). Les connaissances concernant la dépendance de la capacité de nage, en fonction de la taille, proviennent principalement des études chez P. magellanicus (Dadswell & Weihs 1990, Manuel & Dadswell 1991, Manuel & Dadswell 1993, Labrecque & Guderley 2011). Ainsi, il a été montré que l’efficacité hydrodynamique et la performance de nage sont maximales chez les pétoncles d’une hauteur de coquille entre 40 et 80 mm (Dadswell & Weihs 1990, Labrecque & Guderley 2011). Les propriétés métaboliques du muscle adducteur changent avec la taille avec l’atteinte de valeurs maximales d’activités enzymatiques et de contenu en phosphoarginine chez les pétoncles d’environ 60 mm (Labrecque & Guderley 2011). Le style de nage, chez P. magellanicus, varie aussi en fonction de la taille. Il semble que les petits pétoncles ont tendance à s’élever dans la colonne d’eau, mais ont peu de succès au niveau du déplacement horizontal. Les pétoncles de taille intermédiaire vont s’élever dans un mouvement rectiligne et peuvent nager sans interruption sur des distances de plusieurs mètres tandis que les pétoncles de grande taille ne se déplacent que sur de courtes distances (Caddy 1968, Dadswell & Weihs 1990, Manuel & Dadswell 1993). Chez A. opercularis, la taille modifie le patron de mouvement (nage versus saut). Ainsi, les A. opercularis de petite taille nagent plus que ceux de grande taille (Schmidt et al. 2008). Enfin, les changements au niveau de la capacité de nage des pétoncles ont été observés au cours de l’ontogénie. Ces changements au niveau du comportement des pétoncles peuvent survenir en réponse aux prédateurs dans l’environnement ou être le reflet

31 de l’investissement reproductif. En effet, les pétoncles semblent atteindre une taille refuge leur donnant une certaine protection face aux prédateurs, ce qui permet aux pétoncles de grande taille de réduire leur réponse de fuite (Barbeau & Scheibling 1994b, Arsenault & Himmelman 1996a, Wong & Barbeau 2003). Enfin, rendus à des tailles adultes, les pétoncles vont investir de plus en plus d’énergie au niveau de la reproduction, au dépend des capacités de la réponse de fuite (Brokordt et al. 2000a, Brokordt et al. 2000b, Kraffe et al. 2008).

6.3 Composantes physiologiques affectant la nage La performance de nage peut être influencée par la condition physiologique et énergétique des pétoncles. En effet, le contenu en glycogène au niveau du muscle adducteur constitue une importante réserve énergétique. Ainsi, des changements au niveau de la disponibilité en nourriture et du statut reproducteur peuvent se refléter au niveau de la performance de nage des pétoncles, plus spécifiquement la récupération et la réponse de fuite suite à un épuisement.

L’investissement reproducteur important des pétoncles peut affecter la réponse de fuite. Lors de la gamétogenèse, les réserves énergétiques au niveau du muscle adducteur et de la glande digestives sont mobilisées et redirigées vers la gonade, réduisant ainsi les niveaux de glycogène et de protéines (Barber & Blake 1991, Brokordt & Guderley 2004a). De même, il y a une diminution de l’activité des enzymes glycolytiques et mitochondriales du muscle adducteur, qui est probablement causée par la diminution de la disponibilité du glycogène qui sert de matrice pour ces enzymes (Brokordt et al. 2000a, Brokordt et al. 2000b, Brokordt & Guderley 2004b). Une augmentation de l’investissement reproducteur va affecter les capacités oxydatives et métaboliques des pétoncles. Chez E. ziczac et C. islandica, les capacités oxydatives des mitochondries du muscle adducteur diminuent lorsque l’investissement reproducteur est élevé (Boadas et al. 1997, Brokordt et al. 2000a, Brokordt et al. 2000b). Des capacités mitochondriales réduites peuvent ralentir la récupération suite à une réponse de fuite. En effet, la récupération se fait plus lentement chez E. ziczac et C. islandica en fin de gamétogenèse ou après la ponte comparativement

aux pétoncles dont les gonades sont immatures (Brokordt et al. 2000a, Brokordt et al. 2000b). Aussi, le taux métabolique standard de P. magellanicus suite à la ponte équivaut à

60% du taux métabolique maximal (VO2 max) alors qu’il est seulement de 30-40% chez les pétoncles dont les gonades sont matures ou sur le point de pondre (Kraffe et al. 2008). L’investissement reproducteur a pour effet d’augmenter le coût de maintien des pétoncles, réduisant le registre aérobie disponible et risquant d’affecter la réponse de fuite suite à un épuisement. En effet, l’investissement reproducteur diminue la réponse de fuite suite à un épuisement chez Argopecten purpuratus et P. magellanicus (Brokordt et al. 2006, Kraffe et al. 2008). Pour sa part, la réponse de fuite initiale est relativement constante au cours de du cycle de reproduction des pétoncles malgré des changements importants au niveau du statut métabolique du muscle adducteur (Brokordt et al. 2000a, Brokordt et al. 2000b, Brokordt et al. 2006).

6.4 Facteurs environnementaux affectant la nage Puisque les pétoncles sont des ectothermes vivants dans l’eau et respirant via des branchies, la température est probablement le facteur environnemental le plus important. En milieu naturel, la température peut changer rapidement avec des modifications du courant ou des remontées des eaux profondes. Les changements de température peuvent aussi se produire graduellement lors des changements saisonniers. Peu importe l’échelle de temps à laquelle la température va varier, ces changements vont influencer la performance de nage des pétoncles directement via un effet sur la cinétique des processus physiologiques ou indirectement via le contenu en oxygène dans l’eau. La sensibilité thermique de la performance de nage des pétoncles varie d’une espèce à l’autre, selon l’habitat et selon l’historique thermique des pétoncles (Guderley et al. 2009). En général, la performance de nage des pétoncles a tendance à augmenter avec la température jusqu’à un optimum pour ensuite diminuer. La température affecte à la fois les propriétés contractiles musculaires et le comportement de nage des pétoncles. Chez Placopecten magellanicus, le taux de fermeture des valves augmente avec la température ambiante (9.5 à 14 ºC); (Manuel & Dadswell 1991). Chez Argopecten irradians et P. magellanicus, les propriétés contractiles reliées au

33 temps (délais de réponse, temps à la tension maximale et temps de relaxation) diminuent avec une augmentation de la température (Olson & Marsh 1993, Pérez et al. 2009b). D’un autre côté, les variables reliées à la production de force sont peu affectées par la température (Olson & Marsh 1993, Pérez et al. 2009a). Un changement brusque de température, de 18 à 8ºC, ralentit le taux des contractions phasiques et la récupération suite à un épuisement chez P. magellanicus (Lafrance et al. 2002). Lors d’une acclimatation thermique, ce sont principalement les propriétés contractiles du muscle adducteur qui sont affectées. Chez Aequipecten opercularis, ces propriétés sont plus sensibles à un changement de température que la durée du cycle de fermeture et d’ouverture des valves (Bailey & Johnson 2005a). La vitesse de nage et la force produite par le muscle adducteur chez A. opercularis augmentent avec la température (Bailey & Johnson 2005b). Il semble que les pétoncles soient capables d’une certaine compensation face aux changements saisonniers de température (Guderley et al. 2009).

7. Biomécanique de la nage Même si le système locomoteur des pétoncles comprend peu de composantes, la mécanique de la nage des pétoncles reste tout de même relativement complexe. Plusieurs études ont examiné la dynamique des valves pendant la nage, la puissance et la production des jets d’eau par le muscle adducteur et les propriétés du ligament. L’analyse la plus détaillée décrivant la biomécanique de la nage du pétoncle a été faite chez le pétoncle géant, Placopecten magellanicus. Cheng et collaborateurs ont développé un modèle dynamique de la nage chez P. magellanicus qui intègre les propriétés du ligament, l’inertie des valves, la réaction de l’écoulement des fluides externes, la pression des fluides dans la cavité du manteau et la contraction musculaire. Lorsqu’un pétoncle nage, la réaction des fluides externes a trois composantes : la masse ajoutée, la pseudo-élasticité due à l’écoulement et la pseudo- viscosité (Cheng & DeMont 1996). Chez un P. magellanicus de 6.5 cm de hauteur, la masse ajoutée est environ 10 fois plus élevée que la masse d’une seule valve tandis que la pseudo-viscosité induite par l’écoulement compense pour environ la moitié de la viscosité du ligament (Cheng et al.1996). Cheng et al. (1996) ont divisé le système locomoteur des

pétoncles en deux parties : une pompe à pression produisant les jets d’eau et un oscillateur impliquant le ligament et les fluides externes. La dynamique de l’oscillateur est principalement déternimée par l’interaction entre les fluides externes et les propriétés du ligament. La fréquence de résonnance de l’oscillateur est près de la fréquence des claps lorsque le pétoncle nage (Cheng et al.1996). Ceci indique que la majorité de l’énergie mécanique produite lors de la contraction du musculaire est utilisée par la pompe à pression pour la production des jets d’eau (Cheng et al.1996) comme cela avait été trouvé dans les analyses in vivo chez Chlamys hastata et Argopecten irradians par Marsh et al. (1992). Enfin, le moment et l’énergie requis par l’oscillateur sont négligeables (environ 1% de la puissance produite chez P. magellanicus de 6.5 cm); (Cheng et al.1996).

8. Caractérisation de la réponse de fuite des pétoncles Le comportement de nage des pétoncles suscite l’intérêt des scientifiques depuis plus d’un siècle et plusieurs chercheurs se sont penchés sur sa caractérisation. Les études les plus simples se sont limitées à la description du comportement des pétoncles dans leur milieu naturel (Drew 1906, Dakin 1909, Buddenbrock 1911, Baird & Gibson 1956) ou en laboratoire (Baird 1958). D’autres études ont quantifié, en milieu naturel, la réponse de fuite en mesurant la distance parcourue, la durée de la réponse ainsi que la direction du mouvement du pétoncle (Caddy 1968, Joll 1989).

Des études plus exhaustives sur la quantification du comportement de nage des pétoncles se sont faites en milieu artificiel, c’est-à-dire en laboratoire. Afin d’induire le comportement de fuite stéréotypé des pétoncles, différentes techniques sont utilisées : petits coups sur la coquille (Joll 1989), vider la coquille de son contenu en eau (Morton 1980), injection d’une solution saline (Bailey et al. 2003) ou d’extrait d’étoile de mer entre les deux valves du pétoncle (Thompson et al. 1980, Bailey & Johnston 2005a) ainsi que le contact physique du prédateur naturel avec le manteau du pétoncle (Thomas & Gruffydd 1971, Manuel & Dadswell 1991). Une fois la réponse de fuite induite, des observations visuelles ou d’enregistrements vidéos sont utilisées afin de quantifier la réponse de fuite en termes de nombre et fréquence des adductions (claps), le nombre d'adductions par série, la durée de la réponse de fuite ainsi que la durée des cycles de fermeture et d’ouverture des

35 valves (Morton 1980, Joll 1989, Manuel & Dadswell 1991, Brokordt et al. 2000a, Brokordt et al. 2000b, Lafrance et al. 2002, Bailey & Johnston 2005a, Tremblay et al. 2006, Kraffe et al. 2008). Des études plus complexes ont utilisé l’imagerie à haute vitesse afin d’étudier le comportement du pétoncle nageant librement (Cheng et al. 1996, Bailey & Johnston 2005a, Bailey & Johnston 2005b). Certaines études ont même été jusqu’à mesurer la production de force durant la nage en utilisant la sonomicrométrie pour évaluer la longueur du muscle adducteur (Marsh et al. 1992). Enfin, Bailey et al. (2003) ont utilisé la spectroscopie par résonnance magnétique afin d’examiner les paramètres physiologiques au cours de la contraction du muscle adducteur. Quoique diverses, ces différentes techniques utilisées pour la caractérisation du comportement de nage des pétoncles nous donne peu d’information quant à l’utilisation des deux parties du muscle adducteur et à la production de force par le muscle. Une technique non invasive, développée il y a déjà quelques années, permet de noter la production de force par le muscle phasique et tonique durant une réponse de fuite (Fleury et al. 2005). Cette technique requiert l’utilisation d’un dynamomètre relié à un ordinateur pour l’enregistrement des données (Fig. 1.10). Une pince à succion est utilisée pour immobiliser la valve inférieure du pétoncle au fond d'un bassin contenant de l'eau de mer. Un levier, attaché au dynamomètre, est placé sous la valve supérieure du pétoncle. Un prédateur, habituellement une étoile de mer, est placé en contact avec le manteau du pétoncle afin de stimuler une réponse de fuite. En réponse au prédateur, le pétoncle va essayer de fermer ses valves et la valve supérieure qui est libre va appliquer une force sur le levier attaché au dynamomètre. Les enregistrements obtenus nous permettent de différencier les contractions du muscle phasique et tonique. Les contractions rapides du muscle phasique correspondent aux pics de force, alors que les contractions lentes et soutenues du muscle tonique correspondent aux plateaux de force (Fig. 1.11). Cette technique permet de faire des études quantitatives et standardisées de la réponse de fuite. De plus, il devient possible de voir et quantifier l’utilisation des deux parties du muscle adducteur lors d’une réponse de fuite. Ainsi, cette méthode est toute désignée pour les études comparatives du comportement de nage des pétoncles.

Figure 1.11. Production de force du muscle phasique et tonique lors d’une réponse de fuite chez Amusium balloti.

9. Objectifs de la thèse Ma thèse a pour but de mieux comprendre les liens entre la performance locomotrice et les différentes composantes du système locomoteur. Pour ce faire, j’ai étudié la réponse de fuite chez les pétoncles. En effet, il existe une grande variabilité tant au niveau morphologique qu’au niveau du mode de vie (Tableau 1.2). J’ai donc choisi pour mon étude 5 espèces de pétoncles (Amusium balloti, Placopecten magellanicus, Pecten fumatus, Mimachlamys asperrima et Crassadoma gigantea) ayant une morphologie coquillaire et des modes de vie différents. Le cœur de ma thèse est divisé en 4 volets dont chacun des aspects suivants est traité: le comportement de nage, les attributs biochimiques du muscle adducteur, la résilience du ligament et les propriétés morphologiques du muscle adducteur et de la coquille du pétoncle. Au niveau des études sur le comportement de fuite des pétoncles, il est souvent question de la capacité de nage des pétoncles. Or, le concept de capacité de nage n’est pas clairement défini et semble référer à des différents paramètres comportementaux selon les travaux dans la littérature. Ainsi, un des objectifs premiers de ma thèse est de caractériser, de façon quantitative et standardisée, le comportement de nage des pétoncles. Pour ce faire, j’ai étudié les patrons d’utilisation du muscle phasique et tonique des pétoncles, au cours d’une réponse de fuite, à l’aide d’enregistrements avec un dynamomètre.

Chapitre 2 Dans ce chapitre, je me suis intéressée au comportement de nage des pétoncles au cours d’une réponse de fuite simulée. L’objectif de cette étude est de quantifier le patron d’utilisation des contractions phasique et tonique des différentes espèces de pétoncles, lors d’une réponse de fuite simulée, afin d’évaluer si ces patrons varient avec la morphologie et le mode de vie des pétoncles. Puisque l’intensité des contractions phasiques influence le développement de la portance et de la poussée, je prédis que le patron d’utilisation du muscle adducteur phasique et tonique varie avec le mode de vie et la morphologie de la coquille chez les pétoncles.

Chapitre 3 Dans ce chapitre je me suis intéressée aux propriétés biochimiques du muscle adducteur phasique des pétoncles. L’objectif est de savoir si le muscle adducteur phasique des pétoncles, ayant une réponse de fuite et une morphologie coquillaire distinctes, diffèrent au niveau de leur capacité métabolique. Je prédis que les attributs biochimiques du muscle adducteur phasique des pétoncles reflètent les patrons d’emploi du muscle lors d’une réponse de fuite.

Chapitre 4 Dans ce chapitre, j’ai étudié le ligament des pétoncles. L’objectif principal est de caractériser les propriétés mécaniques du ligament chez des espèces de pétoncles ayant une réponse de fuite et une morphologie coquillaire distinctes. Puisque le ligament joue un rôle important lors de l’ouverture des valves, je prédis que la résilience du ligament devrait varier avec le comportement de nage des pétoncles.

Chapitre 5 Dans ce chapitre, j’ai examiné les caractéristiques morphologiques de la coquille et du muscle adducteur des pétoncles. L’objectif de ce chapitre est de déterminer si les propriétés morphologiques, du muscle adducteur et de la coquille des pétoncles, reflètent le patron d’utilisation du muscle phasique et tonique chez les différentes espèces de pétoncles. Je prédis que les caractéristiques anatomiques et morphologiques des pétoncles reflètent leur capacité de nage, telle que mesurée par l’emploi du muscle lors d’une réponse de fuite.

CHAPITRE 2

Swimming away or clamming up: the use of phasic and tonic adductor muscles during escape responses varies with shell morphology in scallops

ISABELLE TREMBLAY, HELGA E. GUDERLEY et JOHN H. HIMMELMAN

Publié dans « The Journal of Experimental Biology, 2012, 215:4131-4143»

Résumé Le système locomoteur relativement simple des pétoncles facilite l’étude de l’utilisation du muscle pendant la locomotion. Cinq espèces de pétoncle, présentant différentes morphologies au niveau de la coquille, ont été comparées afin de déyerminer si la forme de la coquille et l’utilisation du muscle changent en parallèle ou si l’utilisation du muscle peut compenser pour les contraintes morphologiques. Les enregistrements de la force déployée pendant une réponse de fuite ont révélé que l’utilisation des contractions phasiques et toniques variait de façon marquée entre les espèces. Les espèces qui sont des nageurs actifs, Amusium balloti, Placopecten magellanicus et Pecten fumatus, font plus de contractions phasiques comparativement aux espèces plutôt sédentaires, Mimachlamys asperrima et Crassadoma gigantea. Les contractions toniques variaient considérablement entre les espèces. Face à un prédateur, les deux espèces plutôt sédentaires ont tendance à débuter leur réponse par une contraction tonique tandis que les espèces actives ont plutôt tendance à faire de courtes contractions toniques entre les séries de contractions phasiques. Le pétoncle Placopecten magellanicus a tendance à faire une utilisation abondante des contractions toniques de courte durée. Le pétoncle Pecten fumatus fait plutôt une intense série de contractions phasiques au tout début de la réponse de fuite, probablement dans le but de compenser pour les contraintes induites par la forme désavantageuse de sa coquille. La fermeture prolongée des valves chez les espèces plutôt sédentaires suggère que la morphologie de leur coquille les protège contre la prédation. La nage chez les espèces actives, quant à elle, s’appuie sur la production de contractions phasiques intenses en plus de caractéristiques favorables au niveau de la coquille.

Abstract The simple locomotor system of scallops facilitates the study of muscle use during locomotion. Five species of scallops, with different shell morphologies were compared to determine whether shell morphology and muscle use change in parallel or whether muscle use can compensate for morphological constraints. Force recordings during escape responses revealed that the use of tonic and phasic contractions varied markedly among species. The species that are active swimmers, Amusium balloti, Placopecten magellanicus and Pecten fumatus, made more phasic contractions than the more sedentary species, Mimachlamys asperrima and Crassadoma gigantea. Tonic contractions varied considerably among these species, with the two more sedentary species often starting their response to the predator with a tonic contraction and the more active species using shorter tonic contractions between series of phasic contractions. The scallop Placopecten magellanicus made extensive use of short tonic contractions. The scallop Pecten fumatus mounted an intense series of phasic contractions at the start of its response, perhaps to overcome the constraints of its unfavourable shell morphology. Valve closure by the more sedentary species suggests that their shell morphology protects them against predation, whereas swimming by the more active species relies upon intense phasic contractions together with favourable shell characteristics.

Introduction Animals use diverse styles of locomotion that rely upon a range of morphological, physiological and biochemical attributes. In vertebrates, bone structure and cardiovascular capacity as well as muscle size, fibre types, energetic reserves, and metabolic capacity reflect the species’ style of locomotion. For example, high-performance swimming fish, such as tuna and lamnid sharks, have a hydrodynamic body shape as well as many physiological characteristics (centrally positioned red muscle, regional endothermy and enhanced tissue oxygen delivery) that increase swimming efficiency (Bernal et al. 2001). The large leg muscles and short wings of deep-diving cormorants compromise flying but favour diving (Watanabe et al. 2011). Sustained flight by pigeons requires highly aerobic flight muscles (Pennycuick 1968, Davis and Guderley 1987), whereas burst flight by pheasants relies upon powerful anaerobic muscles that fatigue rapidly (Kiessling 1977, Davis and Guderley 1987, Marden 1994, Askew and Marsh 2002). Although it is clear that muscle morphology, metabolic capacity and fibre type composition differ with locomotory style, the complex musculoskeletal structures of vertebrates make it difficult to study the use of muscle during movement and to identify how body morphology influences muscle use. The study of simpler locomotor systems could indicate whether body morphology and muscle use change in parallel or whether muscle use can compensate for morphological constraints.

Scallop swimming primarily involves one muscle, one ligament, and the hinged shells. This mode of swimming is shared by scallops exhibiting a wide range of shell morphologies and life styles, ranging from the highly hydrodynamic Amusium balloti to the cemented (non locomotory) rock scallop, Crassadoma gigantea. In response to disturbance, particularly to contact with their predators, scallops swim using water jets produced by successive contractions during which rapid adductions (closure) alternate with abductions (opening) of the valves (Drew 1906, Dakin 1909, Buddenbrock 1911). The phasic adductor muscle is responsible for rapid valve closure (Lowy 1954, Millman 1967), whereas the uncalcified hinge ligament with its rubber-like properties (Alexander 1966, Marsh et al. 1976) acts like a spring to open the valves when the adductor relaxes. The phasic adductor is composed of cross-striated fibres whereas the smaller tonic adductor is

composed of smooth fibres, which contract slowly, allowing prolonged valve closure or low-energy maintenance of a constant valve opening during filter feeding (Lowy 1954, Chantler 2006).

Shell and muscle morphology, as well as the metabolic capacity and use of the adductor muscle, should determine the swimming ability of scallop species. A swimming scallop has to produce thrust to overcome drag, and lift to overcome gravity. Thrust is produced by expulsion of water following contraction of the phasic adductor muscle, drag and lift are influenced by shell shape, swimming angle and swimming speed, whilst gravitational effects are primarily influenced by shell mass and organismal buoyancy. For a given scallop species, size and swimming angle as well as current speed and direction help determine lift production and swimming capacity (Gruffydd 1976, Thorburn and Gruffydd 1979, Millward and White 1992). Scallops with good swimming abilities generally have a high aspect ratio, an upper valve that is slightly more convex than the lower valve, and light valves with smooth surfaces (Gould 1971, Soemodihardjo 1974). Swimming ability varies positively with the ratio of phasic to tonic adductor muscle surface area (Gould 1971, Soemodihardjo 1974) and with the obliqueness of the phasic adductor, in a plane perpendicular to the hinge (Thayer 1972). Shell size and the energetic status of the adductor muscle also influence swimming ability, as the ability of individual scallops to swim and recover from exhausting exercise changes with size, acclimation temperature and reproductive stage (Manuel and Dadswell 1991, Manuel and Dadswell 1993, Brokordt et al. 2000a, Brokordt et al. 2000b, Bailey and Johnston 2005, Guderley et al. 2009, Labrecque and Guderley 2011). Whereas interspecific comparisons of shell morphology and muscle characteristics can predict relative swimming abilities, little is known about how the phasic and tonic adductor muscles are used during activity and of the influence of shell morphology. We reasoned that, as the intensity of phasic contractions should influence the development of thrust and lift, the pattern of utilisation of the phasic and tonic adductor muscles should vary with swimming ability and shell morphology.

Phasic muscle contractions are better understood than tonic contractions, primarily because phasic contractions are visible and more readily quantified. As the phasic adductor relies upon phosphagens for fuel and has limited endurance, it is analogous to fast glycolytic fibres in vertebrates (Grieshaber 1978, de Zwaan et al. 1980, Livingstone et al. 1981). In contrast to the case in vertebrate fast muscle, glycogen breakdown provides limited metabolic support for scallop swimming, but is used during anaerobic and aerobic recovery (Livingstone et al. 1981). Tonic contractions have no direct analogue in vertebrate skeletal muscle. In the context of swimming escape responses, tonic contractions are used in a variety of situations. Prolonged tonic contractions occur once the phasic adductor muscle is fatigued, when most scallop species close their valves. Short tonic contractions separate series of phasic contractions during escape responses in both Placopecten magellanicus (Fleury et al. 2005) and Argopecten purpuratus (Pérez et al. 2009a). Prolonged tonic contractions allow partial metabolic recovery of the phasic adductor (Pérez et al. 2008b), potentially facilitating subsequent phasic contractions.

The objective of this study was to quantify the pattern of utilisation of phasic and tonic contractions by different species of scallops during escape responses to evaluate whether these patterns vary with morphology and life style. Using a force gauge, the phasic and tonic forces generated by different scallop species were recorded during responses stimulated by contact with predators. For each scallop species, the predator that elicited the strongest escape response, as identified from the literature and personal observations, were used. Scallop performance was measured at habitat temperature (12.5 to 18.5°C). The number of phasic and tonic contractions, the duration of tonic contractions and the frequency of phasic contractions were used to characterise the use of the adductor muscle during simulated escape responses. It is predicted that active scallops should make more phasic contractions, have a higher rate of phasic contractions and sustain these phasic contractions for longer periods compared with less active scallops. Less active scallops should rely more on tonic contractions when faced with predators.

The species chosen for this study correspond to the ‘ecomorphs’ established by Minchin (Minchin 2003) according to swimming abilities and lifestyles, ranging from the 46

most to the least active as follows: the saucer scallop Amusium balloti (Bernardi 1861), the sea scallop Placopecten magellanicus Gmelin 1791, the commercial scallop Pecten fumatus Reeve 1852, the doughboy scallop Mimachlamys asperrima (Lamarck 1819) and the purple-hinge rock scallop Crassadoma gigantea (Gray 1825). The morphological characteristics of A. balloti suggest that it should be the most accomplished swimmer (Yonge 1936, Stanley 1970, Gould 1971, Thayer 1972). Its shell is very light, symmetrical and slightly biconvex, with internalised ribs, small auricles and very smooth outer valve surfaces (Fig. 2.1). In its natural habitat, A. balloti is free-living and found exposed or partly recessed on the seabed. As the maximum single-swim distance of A. balloti is greater than that recorded for any other scallop, Joll (1989) suggested that A. balloti is a more active swimmer than P. magellanicus and Amusium pleuronectes. The sea scallop Placopecten magellanicus is an active swimmer (Caddy 1968, Dadswell and Weihs 1990) and lives mainly exposed on the seabed. Its upper valve is more convex than the lower valve, providing a good hydrodynamic shape (Fig. 2.1) (Stanley 1970, Thorburn and Gruffydd 1979). As P. magellanicus grows, its valves become heavier and its swimming performance declines (Manuel and Dadswell 1991). Like other members of the genus Pecten, the upper valve of P. fumatus is flat and the lower one convex (Fig. 2.1), a combination that is disadvantageous for swimming (Stanley 1970, Gruffydd 1976). Usually Pecten spp. lie recessed in the substrate in small depressions with their upper valves covered and roughly level with the seabed. The scallop Pecten fumatus, like other Pecten species, is considered to have a moderate swimming ability, although it is capable of intense swimming when disturbed (Olsen 1955, Baird and Gibson 1956, Thomas and Gruffydd 1971). Like many scallops of the subfamily Chlamydinae, M. asperrima is frequently byssally attached, but is capable of detaching and swimming when touched by its predator (Olsen 1955, Vahl and Clausen 1981, Brand 2006). The valves of M. asperrima are biconvex, covered by external ribs and have asymmetical auricles (Fig. 2.1). The similar shell shape and aspect ratio of Chlamys islandica and M. asperrima suggest that these scallops have a limited swimming ability (Gruffydd 1976). Finally, the purple- hinge rock scallop, C. gigantea, is free-living in early life (<30 mm shell height) and then cements its lower, right valve, to rocky surfaces (Yonge 1951, Lauzier and Bourne 2006). Once cemented, C. gigantea shells become heavily calcified and irregularly shaped, with

Amusium balloti

Placopecten magellanicus

Pecten fumatus

Mimachlamys asperrima

Crassadoma gigantea

Figure 2.1. Upper valve and side view of experimental scallops. Scale bar is 1cm.

shell height generally exceeding length (Fig. 2.1). The scallop Crassadoma gigantea is normally cryptic and its shell is covered by other organisms.

Material and methods

Scallop sampling and experimental conditions Amusium balloti Mature A. balloti (90-113 mm shell height) were collected in August 2007 near Gladstone (Queensland, Australia) and were kept in holding tanks with running seawater (18.5°C, salinity ~35 ppt) at the Bribie Island Aquaculture Centre (Woorim, Bribie Island, Queensland, Australia). Scallops were left undisturbed for 1 week before the escape response experiments.

Placopecten magellanicus Mature P. magellanicus (85-96 mm shell height) were obtained from Cultimer (Cap-Aux-Meules, Îles-de-la-Madeleine, Québec, Canada) in September 2008. Approximately 1 week before the experiments, the scallops were transferred to holding tanks with seawater pumped in from the neighbouring lagoon (salinity ~30 ppt) in the Ministère des Pêcheries et de l’Alimentation du Québec laboratory and were left undisturbed in the tanks. This period allowed them to recover from the transfer and to adjust to the laboratory conditions. Initially, the temperature was 17.5°C, but after a week, it dropped to 14°C and remained constant. Scallops were left another 3 days to habituate to this temperature prior to the escape response experiments.

Pecten fumatus Mature P. fumatus (86-104 mm shell height) were collected by SCUBA near Satellite Island (43º 32’ 491”S and 147º 23’ 297”E, Channel d’Entrecastreux, Tasmania, Australia) in September 2007. Scallops were transferred to the Tasmanian Aquaculture and Fisheries Institute (Taroona, Tasmania, Australia) where they were kept in tanks with

49 running seawater (12.5°C, 34 ppt). Scallops were left undisturbed in the tank for 1 week prior to the escape response tests.

Mimachlamys asperrima Mature M. asperrima (71-95 mm shell height) were collected at the same time and locality as P. fumatus and were transferred to the Tasmanian Aquaculture and Fisheries Institute laboratory where they were kept in tanks with running seawater (12.5°C, 34 ppt). Scallops were left undisturbed for 1 week prior to the escape response experiments.

Crassadoma gigantea Mature C. gigantea were collected in May 2010 from oyster-rearing systems that had been in the water for the past 4-5 years near Espinosa Inlet (Vancouver Island, British Columbia, Canada). After collection, scallops (73-130 mm shell height) were transferred to the Centre for Shellfish Research (Vancouver Island University, Nanaimo, British Columbia, Canada) where they remained in tanks with running seawater (12.5°C, ~28 ppt) for 1 week prior to the escape response experiments.

Experimental design and force recordings Force production of scallops during an escape response was characterized using a modified version (Guderley et al. 2008) of a previously described technique (Fleury et al. 2005). Although referring to the recordings as escape responses, the scallops could not escape as they had their lower valve fixed, while the upper valve was free to move during stimulation by the predator (see appendix 2.1). The lower valve of the scallop was attached to the bottom of a water bath filled with seawater whereas a lever, attached to a force gauge (AFG-50 N, Quantrol Advanced Force Gauge, Dillon, Fairmont, MN, USA), was placed under the ventral edge of the upper valve (appendix 2.1). The force gauge was mounted on a test stand that allowed us to use the lever to separate the valves by a distance corresponding to that observed in scallops at rest and ventilating normally. This distance was previously evaluated for each species. The upper valve was free to move and only downward movements were recorded by the force gauge. In P. magellanicus, this

technique slightly reduces the total number of phasic contractions compared with the number assessed by visual observations of the response of unattached scallops to their predator, but escape performance differences between age groups of P. magellanicus were independent of the measurement technique (H.E.G., unpublished observations). The force gauge method was chosen as it provides considerable information concerning the use of the tonic and phasic adductor muscles. Once attached, the scallop was stimulated to contract by continuously stroking the mantle of the scallop with a predator. Each scallop was stimulated for 355 s, as preliminary tests showed that this was enough to exhaust all scallop species. Water was changed after every second scallop tested to keep the temperature constant. As each species presented specific morphological constraints, certain aspects of the experimental design differed, as detailed below.

Each A. balloti was identified with a permanent marker and the lower valve was attached to the bottom of the waterbath with a suction clamp. Slipper lobsters, Thenus orientalis (~13 cm long) were used to induce escape responses, as this predator induces the strongest escape response in A. balloti (Himmelman et al. 2009).

Sea scallops Placopecten magellanicus were identified individually with a permanent marker. They were attached to the bottom of the waterbath using a suction clamp and the sea star Asterias vulgaris (~15 cm diameter) was used to induce escape responses.

As it was not possible to write on the shell, each P. fumatus had a plastic tag glued on its lower valve for subsequent identification. A nylon screw was fixed on the lower valve using Araldite glue and then the scallops were left at rest for 1 week prior to the experiments. For the escape response experiments, the scallops were attached via a bolt to an aluminum platform attached to lead weights in the waterbath. The escape response was stimulated by the sea star Coscinasterias muricata. As the sea star was too big for the experimental chamber, an arm of the sea star was removed and used to stimulate the scallops.

Each M. asperrima was identified with a numbered plastic tag glued on the lower valve. We attached the scallops to the bottom of the waterbath with a suction clamp and used an arm of the sea star C. muricata to elicit the escape response.

A screw was glued to the lower valve of each C. gigantea using Araldite glue and etched a number in the glue to identify the individuals. For the escape response experiments, we attached the scallops to an aluminum frame that was attached to the bottom of the waterbath. We used the sea star Pycnopodia helianthoides (~20 cm diameter) to elicit the escape response.

Data recordings and analysis Recordings from the force gauge (Fig. 2.2) were stored on a personal computer using Dataplot-X software (Dillon, Fairmont, MN, USA) and were then transferred to an Excel spread sheet for analysis of phasic and tonic muscle performance during the tests. As one person stimulated the scallop with the predator and controlled the computer, manipulations had to be done in a specific sequence. First, the scallop was fixed on the experimental setup and then the computer recording was started. Stimulation of the scallop with the predator started at the fifth second of the recording. Thus, the force recordings from the 5th to the 360th second were analyzed, giving 355 s of force recording for analysis.

Phasic contractions are apparent as sharp peaks in the force recordings whereas sustained force production indicates tonic contractions (Fig. 2.2). The total number of phasic contractions made by the scallop during the test were counted. As the initial intensity of the response is important for survival upon encounters with a predator, the number of phasic contractions during the first series, the minimal interval between two consecutive phasic contractions and the contraction rate during the first 30 s (i.e. 5-35 s) of the test were noted. A series of phasic contractions was defined as consecutive phasic contractions separated by < 3 s. As the capacity to maintain swimming activity may be important when escaping a predator, the number of phasic contractions before fatigue, the number before the first tonic contraction, the percentage time spent in phasic contractions

14 Amusium balloti 12 10 8 6 4 2 0 0 100 200 300

25 Placopecten magellanicus 20 15

10 5 0 0 100 200 300 14 12 10 8 12 Pecten fumatus 6 4 10 2 0 8 20 22 24 26 28 30 6 Force (N) Force 4 2 0 0 100 200 300 Mimachlamys asperrima 8 6

0 0 100 200 300

30 Crassadoma gigantea 25 20 15 10 5 0 0 100 200 300 Time (s)

53 and the time to fatigue were quantified. A scallop was considered as fatigued when it made no phasic contractions during 1 min of stimulation with its predator.

Tonic contractions lead to sustained valve closure. For scallops that cannot close their valves tightly, valve closure does not offer protection against predators. Given the low energetic cost of tonic contractions, valve closure offers metabolic respite to the adductor muscle (Pérez et al. 2008b). On the force recordings, tonic contractions were defined as sustained force production lasting more than 0.5 s (Fig. 2.2). Tonic contractions were characterized by evaluating their total number, the number that lasted ≥ 5 s, the percentage of time spent in tonic contraction, the mean duration of contraction, the time until the initiation of the first tonic contraction and total number of phasic contractions relative to the total number of tonic contractions.

Anatomical and morphological measurements The height, length and width of the shells of each individual were measured using a digital caliper (±0.01 cm). Shell height corresponded to the maximum distance between the dorsal (hinge) and ventral margins, whereas shell length was the maximum distance between the anterior and posterior margins and was perpendicular to shell height (Fig. 2.3). Shell width was measured at the point of maximum convexity with the two valves placed in their natural closed position (Fig. 2.3). Wet tissue mass was obtained after removing all the tissues from the shell and placing them on absorbent paper to remove excess water. The phasic and tonic adductor muscles, gonad and remaining soft tissues separately were weighted using a digital balance (0.01 g). Tissues were subsequently dried for 48 h at 60°C to assess dry mass and percentage water content. The total damp-dried shell mass of each individual was assessed. As the lower shell of P. fumatus and C. gigantea had a screw attached to it with Araldite glue, another sample of shells was used to establish the damp- dried shell mass and the relationship between shell dimensions and damp-dried shell mass.

A) B)

Figure 2.3. Shell dimensions. A) Solid line corresponds to shell height and dashed line is shell length. B) Solid line corresponds to shell width.

The condition index (mg/ml) was expressed as the wet mass of all soft tissues /volume between the two valves (Lafrance et al. 2003). The volume between the two valves was estimated by weighing the upper and lower valves filled with sand (500 µm sifted sand) level with the shell margin. As the mass of a given volume of sand was known, the volume in each valve could be estimated to determine the total volume.

Shell and soft tissue dry masses were adjusted to a standard shell height of 90 mm. The masses were adjusted to a standard scallop measuring 90 mm shell height as this size was present in the samples of all species studied. First, the regressions between the shell height and the mass of the shell and the soft tissues were done. These regressions were used to calculate A1 A2 and A3 whrere A1 was the mass expected for a 90 mm shell height scallop in the allometric series, A2 the mass expected for a scallop with the size of the experimental scallop in the allometric series and A3 the measured mass for the experimental scallop. Finally, the adjusted shell and soft tissue dry masses, at 90 mm shell height, were calculated using the following formula:

Corrected mass = (A1/A2)×A3

Results

Shell and anatomical characteristics The shell height of the different experimental scallop species overlapped, although the M. asperrima studied had a mean shell height of 84.2 mm and were slightly smaller than the other species (Table 2.1). To compare the anatomical characteristics of the species, we adjusted shell and tissue masses to a common shell height of 90 mm. After this adjustment, C. gigantea had the heaviest shell (136.4 g) followed by P. fumatus and P. magellanicus, which had similar shell masses, equivalent to one-third the mass of C. gigantea (Table 2.1). The shells of M. asperrima and A. balloti were the lightest, being respectively 4 and 5 times less heavy than the shells of C. gigantea (Table 2.1). Interspecific differences in the soft tissue dry mass were marked: C. gigantea usually had the largest mass and A. balloti had the lowest (Table 2.1). Tissue water content varied slightly between species with A. balloti having the highest (85.5%) and C. gigantea the lowest (79.2%) values (Table 2.1). Crassadoma gigantea had the highest condition index (0.61 g ml-1) and M. asperrima the lowest (0.41 g ml-1) (Table 2.1). All the species, except A. balloti, had mature gonads and a relatively high gonadosomatic index (Table 2.1).

Table 2.1. Shell and anatomical characteristics of experimental scallops.

Amusium Placopecten Pecten Mimachlamys Crassadoma balloti magellanicus fumatus asperrima gigantea Shell Height, mm 96.3±1.1a 90.4±0.9b,c 94.4±1.7a,c 84.2±2.0b 97.0±4.5a,c Length, mm 95.5±1.4 99.6±1.0 107.0±1.6 82.4±2.1 92.9±3.5 Width, mm 19.8±0.4 27.7±0.4 22.6±0.4 31.3±1.1 34.5±2.0 Aspect ratio 1.01±0.004 a 0.91±0.06 b 0.88±0.0.01b 1.02±0.005 a 1.04±0.03 a Mass at 90 mm height, g 26.5±0.4a 48.8±1.1b 45.9±1.4b 32.7±0.7c 136.4±9.9d N 27 15 15 16 19 Soft tissue dry mass, g Total animal 2.00±0.08a 6.71±0.26b 4.96±0.30c 5.45±0.17c 8.87±0.55d Phasic muscle 0.86±0.05a 3.08±0.13b 2.05±0.16c 1.92±0.09c 3.60±0.24b Tonic muscle 0.05±0.004a 0.25±0.01b 0.23±0.01b 0.17±0.01c 0.29±0.02d N 18 15 15 16 19 Tissue indicators Water content in total animal, % 85.5±0.2a 80.2±0.3b 84.1±0.4c 84.6±0.3c 79.2±0.3d Water content in gonad, % 87.3±0.7 a 86.2±1.0 a 82.8±0.8b 81.9±0.5b 80.3±0.4d Condition index, g ml-1 0.48±0.01a 0.51±0.01b 0.42±0.02c 0.41±0.01c 0.61±0.02d Gonadosomatic index, % 5.4±0.3a 10.5±0.7b,c 11.5±0.7c 17.8±0.9d 9.9±0.6b N 18 15 15 16 20 Data are means ± S.E. Soft tissue dry mass was adjusted for 90 mm shell height. Aspect ratio is the ratio of height/length. Total animal refers to all soft tissues including the adductor muscle. Condition index is the total animal wet mass / shell volume. Gonadosomatic index is calculated as (gonad tissue wet mass / total animal wet mass) ×100. In a given row, different letters indicate significant differences (Kruskall-Wallis and multiple comparisons, P<0.05).

Escape response recordings For A. balloti, phasic contractions were spread throughout the tests, although most were made during the first 120 s (Fig. 2.2A). The first tonic contraction appeared quite late in the test. Tonic contractions were of relatively short duration, becoming longer as the test progressed. Placopecten magellanicus also spread its phasic contractions throughout the test, but alternated them with short tonic contractions (Fig. 2.2B). Placopecten magellanicus made many very short tonic contractions that became longer as the test progressed. Pecten fumatus made an intense burst of phasic contractions at the beginning of the test followed by relatively long tonic contractions (Fig. 2.2C). Mimachlamys asperrima started its response with a short tonic contraction, followed by a few series of phasic contractions interspersed with tonic contractions, ending with a prolonged tonic contraction that lasted until the end of the test (Fig. 2.2D). Mimachlamys asperrima made its phasic contractions at a slower rate than A. balloti and P. fumatus. Crassadoma gigantea usually made a prolonged tonic contraction that lasted throughout the test, but sometimes made a few phasic contractions (Fig. 2.2E).

Interspecific comparisons: phasic contractions The scallop species considered to be active swimmers used their phasic muscle more than the less active scallops. Amusium balloti made the highest number of phasic contractions (41.0, Fig. 2.4A) and spent the greatest proportion of time in phasic contractions (2.8%, Table 2.2). Pecten fumatus and P. magellanicus were next in line, making respectively one-fifth and one-third fewer phasic contractions than A. balloti (Fig. 2.4A) and spending half to a third less time in phasic contractions than A. balloti (Table 2.2). Mimachlamys asperrima only made half the number of phasic contractions compared with A. balloti and spent the same proportion of time in phasic contractions as P. magellanicus (Table 2.2). Finally, C. gigantea primarily kept its valves closed and spent the lowest proportion of time in phasic contraction (Table 2.2). The distribution of phasic contractions during the escape response varied among the scallop species. Amusium balloti and P. magellanicus spread phasic contractions

50 A 1.4 D b a 1.2 40 c 1.0 b 30 0.8 c

d 20 0.6 a,c 0.4 a 10

contractions no. Total phasic 0.2

e (s) Timecontractions between 0 0.0

0.8 B 20 E b c

0.6 15

0.4 a c a a 5 0.2 a,c contractions phasic No. a b b 0.0 0

No. contractions in first contractions in contractions No. series/total

250 F a 0.6 C b a a 0.5 200

)

-1 b 0.4 150 b 0.3 100 b 0.2 c

Time to fatigue (s)

Contraction rate (sContraction 50 0.1 d 0.0 0

A. balloti A. balloti P. fumatus P. fumatus C. gigantea C. gigantea M. asperrima M. asperrima P. magellanicus P. magellanicus

Table 2.2. Behavioural parameters related to phasic contractions during escape responses by the different scallop species.

Amusium Placopecten Pecten Mimachlamys Crassadoma balloti magellanicus fumatus asperrima gigantea Number of phasic contractions before fatigue 39.1±1.5a 26.7±1.7b 32.0±2.8c 13.3±1.4d 1.4±0.7e Percentage of time spent in phasic contractions 2.8±0.1a 1.0±0.1b 1.3±0.1c 0.9±0.1b 0.2±0.1d Phasic parameters data are means ± S.E. Sample size: A. balloti N=30, P. magellanicus N=15, P. fumatus N=15, M. asperrima N=16, C. gigantea N=19. In a given row, different letters indicate significant differences (Kruskall-Wallis and multiple comparisons, P<0.05).

throughout the tests, whereas P. fumatus and M. asperrima tended to concentrate theirs (Fig. 2.2), generally at the beginning of the test. The timing of phasic contractions by C.gigantea was unpredictable (Fig. 2.2E). Most of the scallop species made ~20% of their phasic contractions during the first series (phasic contractions not separated by more than 3 s), but P. fumatus made 66% of its phasic contractions during the first series (Fig. 2.4B). The phasic contraction rate during the first 30 s was highest for A. balloti (0.51 contractions s-1, Fig. 2.4C), slightly less for P. fumatus, and still less for P. magellanicus (Fig. 2.4C). The rate for M. asperrima was approximately one-third that of A. balloti and C. gigantea had the lowest rate (Fig. 2.4C). Amusium balloti and P. fumatus could make phasic contractions in quick succession as shown by the short minimal interval between two successive phasic contractions (0.38 and 0.32 s, respectively, Fig. 2.4D). In contrast, repetition of phasic contractions by P. magellanicus and M. asperrima was much slower and P. magellanicus had the longest gap between phasic contractions of the species studied, except C. gigantea (Fig 2.4D). Given the sporadic nature of phasic contractions by C. gigantea, we did not calculate its minimal interval between contractions.

Interspecific comparisons: tonic contractions The escape response recordings revealed considerable interspecific variation in the use of tonic contractions. Placopecten magellanicus made the highest number of tonic contractions with a mean of 20.9 (Fig. 2.5A). Amusium balloti made approximately half as many and P. fumatus and M. asperrima made about one-third as many (Fig. 2.5A). Placopecten magellanicus and A. balloti made the shortest tonic contractions (mean of 16.2 and 20.9 s, respectively, Fig. 2.5B). The tonic contractions of M. asperrima and P. fumatus were 2-3 times longer than those of P. magellanicus but those of C. gigantea were by far the longest (276.3 s, Fig. 2.5B). For C. gigantea, all tonic contractions lasted longer than 5 s, whereas for the other species half of the tonic contractions lasted 5 s or less (Fig. 5F). The pronounced differences in the number (and duration) of tonic contractions led to considerable variability in the ratio of phasic to tonic contractions; from 5.3 for P. fumatus and 4.0 for A. balloti to 1.6 for M. asperrima and 1.4 for P. magellanicus (Fig. 2.5C).

25 A 40 D d c 20

15 30 a

c a 10 c 10

5 contractions no. tonic Total d

Time at 1st tonic contraction (s) at contraction Time 1st tonic b d d 0 0

B 100 E c c 300 90 b b 80 250 a a 100 70 b (s) duration Contraction 50 b 60 a a

Time spent in tonic contractions (%) contractions tonic spent in Time 0 50

8 C F b 12 a 10 6 a 8 a 4 6 a

b c No. contractions contractions No. 4 d 2 b 2 c

0 0 contractions tonic contractions/no. phasic No. A. balloti A. balloti P. fumatus C. gigantea P. fumatus C. gigantea M. asperrima M. asperrima P. magellanicus P. magellanicus Figure 2.5. Escape response parameters related to tonic contractions. (A) Total number of tonic contractions. (B) Mean duration of tonic contractions. (C) Total number of phasic contractions relative to the total number of tonic contractions. (D) Time at the first tonic contraction. (E) Percentage of time spent in tonic contractions. (F) Number of tonic contractions of a duration of 5 s or more. Data are means ± S.E. Different letters indicate significant differences (Kruskall-Wallis and multiple comparisons, P<0.05). Sample size: A. balloti N=30, P. magellanicus N=15, P. fumatus N=15, M. asperrima N=16, C. gigantea N=19.

Generally, the more active species spent less time in tonic contractions and began them later than the less active species. Pecten fumatus spent the least time in tonic contractions (65%) and A. balloti spent slightly but not significantly longer (Fig. 2.5E). The time spent in tonic contraction was similar for P. magellanicus and M. asperrima (about 82%). This was surprising given that M. asperrima is a less active swimmer (Fig. 2.5E). Crassadoma gigantea spent almost all the test in tonic contractions (93%, Fig. 2.5E). The less active scallops, M. asperrima and C. gigantea, started using tonic contractions when first touched by their predator (Fig. 2.5D). Placopecten magellanicus made their first tonic contraction within seconds of the beginning of the test (Fig. 2.5D). Both A. balloti and P. fumatus made numerous phasic contractions before their first tonic contraction (Fig. 2.2A, C). Amusium balloti took eight times longer than P. magellanicus to make its first tonic contraction and P. fumatus took 30 times longer (Fig. 2.5D).

Discussion Although all scallops use jet propulsion to swim at some point in their lives, the species compared varied considerably in their use of the adductor muscle during escape responses. Both the timing and frequency of phasic contractions varied markedly. The use of tonic contractions varied greatly among the species. Some species made many short tonic contractions throughout the escape response, others only used tonic contractions after a major phasic effort, and still others started the tests with tonic contractions. Effectively, each species adopted a specific combination of phasic and tonic contractions that presumably facilitates escape from predators within the constraints of their morphology, habitat and lifestyle. Our analysis suggests that differential use of the adductor muscle can facilitate swimming despite quite marked differences in shell morphology, including some morphologies that appear hydrodynamically unfavourable. Alternatively, when scallop species can avoid predation by closing their valves, they can use the more energetically efficient tonic contractions, avoiding the costs of phasic contractions.

Eco-morphological classification versus phasic and tonic contractions In general, the eco-morphological classification (Minchin 2003) of our scallop species predicted the patterns of use of phasic contractions during escape response tests. The most hydrodynamic species, A. balloti, relied the most on phasic contractions. The scallop A. balloti was tested in August, during the Australian winter and after spawning. Even during this period of relatively low natural responsiveness (Himmelman et al. 2009), A. balloti showed more phasic activity than the other species. Placopecten magellanicus and P. fumatus were next in sequence for high phasic activity. Presumably because of its hydrodynamically unfavourable shell morphology (Stanley 1970), P. fumatus makes a particularly intense series of phasic contractions at the start of its response to get up off the seabed. By contrast, the timing of the bursts of phasic contractions by M. asperrima was much less predictable than for P. fumatus. Finally, C. gigantea only occasionally made phasic contractions in response to stimulation by their predator, this not being their primary response.

Only some of the interspecific variation in the use of tonic contractions could be predicted from the use of phasic contractions or from the eco-morphological classification. Species making many phasic contractions (A. balloti, P. magellanicus and P. fumatus) made more and shorter tonic contractions than species making few phasic contractions. The more sedentary species, M. asperrima and C. gigantea, often initiated the response to the predator with tonic rather than phasic contractions. The time spent in tonic contraction did not follow any obvious correlation with the eco-morphological classification. For example, P. magellanicus is considered a good swimmer, but its frequent use of short tonic contractions, appearing early in the escape response test, led it to spend a similar proportion of time in tonic contractions to M. asperrima. The numerous brief tonic contractions by P. magellanicus were unexpected and their role remains to be elucidated.

Initiation of prolonged tonic contractions: metabolic fatigue or energy saving? The initiation of prolonged tonic contractions presumably reflects fatigue of the phasic adductor muscle in active scallops, whereas it may be the preferred response to

predators in more sedentary scallops. Fatigue is presumably caused by an inability of the phasic adductor to contract as a result of its metabolic status. Amusium balloti and P. magellanicus made phasic contractions throughout the escape response, and fatigued much later in the tests than the three other species (Fig. 2.4F). By contrast, despite being quite active, P. fumatus fatigued quickly, presumably as a consequence of the intense deployment of phasic contractions in its initial response to the predator. Much like the burst flight of pheasants, the burst of phasic contractions by P. fumatus is needed to propel this awkwardly shaped scallop through the water, but requires an unsustainable power output. Scallops use phosphoarginine as the major source of ATP for phasic contractions (Livingstone et al. 1981, Bailey et al. 2003). Pecten fumatus probably used much of its phosphoarginine in its initial burst of phasic contractions, explaining the rapid fatigue. For the active scallops, A. balloti, P. magellanicus and P. fumatus, it is likely that the initiation of relatively long tonic contractions reflects depletion of phosphoarginine and a decrease in the free energy of ATP hydrolysis in the phasic adductor (Bailey et al. 2003, Pérez et al. 2008b). The less active species, M. asperrima and C. gigantea, made few phasic contractions and often made prolonged tonic contractions during the initial responses to the predator. Such early use of tonic contractions is unlikely to be due to metabolic fatigue of the phasic adductor. This reliance on tonic contractions probably reflects an alternative defence against predation whereby prolonged closure of a robust, tightly closing shell provides adequate protection. The observation of the gaps between the closed valves of the scallops shows differences between experimental species (Fig. 2.1). The species can be ranked from the most to least tightly-closed shells as follows: C. gigantea, M. asperrima, P. fumatus, P. magellanicus and A. balloti. If valve closure is an effective defence against predation, the use of energetically efficient tonic contractions would decrease the need for phosphoarginine-powered phasic contractions. This suggests that interspecific differences in the total number of phasic contractions may be paralleled by differences in the phosphoarginine content of the phasic adductor. In turn, interspecific differences in time to “fatigue” would reflect the phosphoarginine content, the intensity of phosphoarginine use during phasic contractions and the efficacy of valve closure as a defence against predation.

Interspecific differences in the use of tonic contractions raise the question as to their role and importance. The best known roles of tonic contractions are in prolonged valve closure and maintenance of specific valve opening during routine ventilation. In both, the lower energetic cost of tonic contractions is beneficial. Force recordings revealed that P. magellanicus alternate between phasic and tonic contractions during escape responses (Fleury et al. 2005) leading to the suggestion that tonic contractions facilitate metabolic recovery of the phasic adductor muscle. In P. magellanicus long tonic contractions (1 min) do allow partial metabolic recovery of the phasic adductor muscle as the adenylate energy charge returns to control levels (Pérez et al. 2008b). On the other hand, replenishment of phosphoarginine takes longer (Grieshaber 1978, Livingstone et al. 1981, Chih and Ellington 1983, Bailey et al. 2003, Pérez et al. 2008b). Comparison of force recordings of electrically stimulated versus sea star-stimulated P. magellanicus (Fig. 4 in Pérez et al. 2008b) shows that short (<5 s) tonic contractions are absent in electrically stimulated scallops, although prolonged tonic contractions begin at a similar time in scallops stimulated electrically and by sea stars. It is believed that improvement of the energetic status of the phasic adductor is unlikely to occur during short duration tonic contractions. Given the lack of a plausible metabolic role for short tonic contractions, interspecific differences in the use and the duration of tonic contractions may reflect functional attributes of the tonic adductor muscle. Scallop tonic adductor muscles use the catch mechanism typical of molluscan smooth muscle, which allows maintenance of tension at a relatively low cost of ATP (Lowy and Millman 1963). Tonic muscle contains a high quantity of paramyosin at the core of the myosin-containing thick filaments (Chantler 1983). In addition to its structural role, paramyosin may play an indirect role in the maintenance of catch (Chantler 2006). Most of our knowledge of molluscan catch muscle physiology comes from studies of the anterior byssus retractor muscle of Mytilus edulis. It is unknown whether these characteristics apply to scallops and if they vary between species. Interestingly, Bayliss and colleagues observed that in vitro “contracture” (tonic contraction) of the tonic adductor muscle diminished in an hour in P. maximus, while that of P. magellanicus fell to low values much more quickly (Bayliss et al. 1930). Possibly, interspecific differences among scallops in the catch mechanism and in tension

development during tonic contractions underlie the differences in the use of tonic contractions during escape responses.

Habitat temperature and escape response patterns In designing this study, each species was characterised at habitat temperatures at the time of sampling to have similar measurement temperatures for these species. Thus C. gigantea, M. asperrima and P. fumatus were measured at 12.5°C, P. magellanicus at 14°C and A. balloti at 18.5°C. Many aspects intervene to establish the impact of temperature upon performance. Each species has its thermal optimum for performance and individual thermal history can modify the position of this optimum (Guderley et al. 2009). Intraspecific thermal sensitivities provide a guide to the potential impact of the differences in measurement temperature in our study. In P. magellanicus, total number, overall rate and minimum interval between phasic contractions differed little between 12 and 18°C but clapping rate in the first series increased (Guderley et al. 2009). This suggests that although A. balloti was measured at the highest temperature, its performance was not markedly enhanced by this thermal difference. Furthermore, as 18.5°C is a cool temperature for A. balloti, we may be underestimating its performance. Thus, it can be concluded that the interspecific performance differences observed are not a reflection of the differences in measurement temperature.

Potential influence of ontogenetic changes on escape responses This study of interspecific differences in the use of the phasic and tonic adductor muscles during scallop escape responses aimed to interpret these patterns in the context of morphology and life style (habitat and activity levels). Nonetheless, morphology and life style change during ontogeny and these changes may affect escape response performance. Although scallops within a similar size range were compared to reduce the influence of size, as the species studied reach different maximal sizes (A. balloti 110 mm, P. magellanicus 150 mm and sometime up to 200 mm, P. fumatus 145 mm, M. asperrima 110 mm and C. gigantea 250 mm) [Lauzier and Bourne (Lauzier and Bourne 2006) and references therein) (see also Edgar 2001, Dredge 2006, Naidu and Robert 2006), the 67 potential influence of ontogenetic state must be considered in interpreting interspecific differences in adductor muscle use.

For any shell shape, basic allometric considerations predict certain aspects of swimming performance. As body mass increases in proportion to the cube of shell length (L3) and lift requirements increase in proportion to L2 (Gould 1971), larger scallops need to swim more rapidly (employing more phasic contractions) to produce enough lift to compensate for their increased mass (Gould 1971, Gruffydd 1976). The size dependence of swimming is best known in P. magellanicus (Dadswell and Weihs 1990, Manuel and Dadswell 1991, Manuel and Dadswell 1993). Hydrodynamic efficiency and swimming performance peak between 40 and 80 mm shell height, after which both decrease (Dadswell and Weihs 1990). Properties of the adductor muscle in P. magellanicus show similar size dependence; enzymatic activities and phosphoarginine content peak at a shell height of 60 mm and decline at greater sizes (Labrecque and Guderley 2011). Parameters related to escape response endurance, the number of phasic contractions and force production peak at 70 mm shell height and then decrease (Labrecque and Guderley 2011). In many species, small scallops are more active than larger ones (Yonge 1936, Olsen 1955, Baird and Gibson 1956, Caddy 1968, Gruffydd 1976, Tremblay et al. 2006, Labrecque and Guderley 2011). The pattern of movement (swimming versus jumping) may also vary with size/age, with small Aequipecten opercularis swimming more than large individuals (Schmidt et al. 2008). Ontogenetic changes in behaviour can occur in response to the predators in an environment as greater size can provide a refuge from predation, allowing larger scallops to reduce their response to predators (Barbeau and Scheibling 1994a, Wong and Barbeau 2003). Ontogenetic variation in performance also reflects the increasing influence of reproductive investment as scallops become larger and favour reproductive investment over escape response capacities (Brokordt et al. 2000a, Brokordt et al. 2000b, Kraffe et al. 2008). All the species we studied were near their spawning season; only A. balloti had finished spawning at the time of the experiment (Table 2.1). Even though spawning can reduce swimming performance and recovery (Brokordt et al. 2000a, Brokordt et al. 2000b, Kraffe et al. 2008), A. balloti was the most active species tested confirming that, if anything, we underestimated its capacities relative to the other species.

Links between shell morphology, lifestyle and escape response behaviour Interpretation of the interspecific differences in muscle use during escape responses must take shell morphology, lifestyle and potential predators into account. Of the species we studied, the shell morphology of A. balloti has the most attributes favouring swimming (Gould 1971). The hydrodynamic shape and light mass of A. balloti allow it to climb easily in the water column and glide efficiently (Gould 1971). Nonetheless, A. balloti makes an intense series of phasic contractions at the beginning of its escape response. Amusium balloti is unusual, compared to other scallops, as it reacts very strongly to crustaceans. Of a suite of potential predators including crustaceans and a sea star, Thenus orientalis elicited the strongest escape response from A. balloti (Himmelman et al. 2009). Thenus orientalis can suddenly extend its tail to lunge forward to seize its prey at a distance of 30-50 cm (Jones 2007). Once caught, A. balloti is vulnerable because the gaps between the valves provide openings through which T. orientalis can insert its appendages. Himmelman and colleagues suggested that the most critical step in A. balloti’s escape from T. orientalis is the time to get off the sea bottom (Himmelman et al. 2009). To escape this very active predator, A. balloti needs to produce significant thrust rapidly. The high rate of phasic contraction produced by A. balloti during the first 30s of the escape response, as well as its highly hydrodynamic shell should allow it to escape a predator with good swimming performance.

Placopecten magellanicus has an excellent shell shape for lift production (Stanley 1970, Gruffydd 1976, Thorburn and Gruffydd 1979); however, its shell mass is relatively high compared with that of A. balloti (Table 2.1). Both juvenile and adult P. magellanicus responded to stimulation by crabs, but the most vigorous escape response was obtained with predatory sea stars (H.E.G., unpublished); (Wong and Barbeau 2003). Placopecten magellanicus, particularly those of smaller sizes, are preyed upon by numerous predators (Elner and Jamieson 1979, Naidu et al. 1986, Barbeau and Scheibling 1994a). Sea stars (A. vulgaris) prefer small individuals, presumably because of their greater vulnerability (Barbeau and Scheibling 1994a), and rock crabs (Cancer irroratus) only feed on small scallops (≤72 mm shell height); (Elner and Jamieson 1979). Nonetheless, small juvenile scallops are less responsive to crabs than to sea stars. It is possible that, even with their

69 good swimming capacities, juvenile P. magellanicus cannot escape fast moving crabs. The simultaneous decline in hydrodynamic efficiency, adductor muscle physiological capacities and swimming behaviour above a shell height of 65 mm (Labrecque and Guderley 2011) may indicate that P. magellanicus no longer “need” to swim once they have reached a certain size. This may explain the lower swimming performance of large P. magellanicus despite their apparently advantageous shell shape.

The flat upper valve and the convex lower valve of P. fumatus are not considered advantageous for lift generation or swimming (Verrill 1897, Baird 1958, Stanley 1970). Like P. magellanicus, P. fumatus shells are heavy compared with those of other scallops of a similar size (Table 2.1). At the beginning of its escape response, P. fumatus carries out an intense burst of phasic contractions. This reaction indicates that P. fumatus uses contractile activity to overcome the constraints of its shape and mass. The intense burst of phasic contractions of P. fumatus, in response to its predator, presumably allows it to attain enough speed to rise into the water column and swim away from potential predators.

For the less active M. asperrima and C. gigantea, predators may be deterred by valve closure and shell characteristics. Mimachlamys asperrima retains the capacity for byssal attachment throughout its life. Its shell is frequently covered by sponges, which is known to inhibit predation by sea stars (Pitcher and Butler 1987). Crassadoma gigantea are free living as juveniles but cement to rocks as adults, presumably once they are sufficiently large and heavy. The shells of cemented C. gigantea are irregularly shaped, their outer surface is spiked and the shell is encrusted with many organisms, providing camouflage. They can also avoid predators by cementing into places that are difficult to reach. Although C. gigantea loses its swimming ability once cemented (Yonge 1951, Lauzier and Bourne 2006), phasic contractions may by retained as a means of cleansing the mantle cavity, reflecting the ancestral condition from which scallop swimming may have arisen (Yonge 1936).

This study is the first to examine interspecific differences in the use of phasic and tonic contractions during escape responses by scallops. The observations revealed a variety of patterns of phasic contractions as well as marked differences in the types and deployment of tonic contractions. Short tonic contractions are widely used by more active scallop species, but their functional significance remains to be elucidated. To swim, a scallop can adjust some of its behaviour to compensate for its morphology. The use of phasic contractions to swim is energetically expensive, particularly for species with a disadvantageous shell shape. Keeping the valves shut for prolonged periods, using tonic contractions, is energetically cheap but requires tight junctions between the valves to prevent predation. Although all scallops have a simple locomotor system, our comparisons indicate that many factors intervene to determine muscle use during escape responses. Further, the position and proportions of the phasic and tonic adductor muscles, as well as the metabolic capacity of the muscles, will influence escape response performance. The properties of the hinge ligament will set an upper limit to the frequency of phasic contractions. These physiological and biomechanical properties have presumably co- evolved to facilitate escape response performance. This performance, in turn, must be adjusted to the ecological context within which each species exists. Alternatively, behavioural flexibility may allow scallops to overcome morphological constraints and to adjust to changing conditions. The marked interspecific variation in muscle use during escape responses clearly reflects such behavioural flexibility. Scallops respond to contact with a predator either by swimming away or by prolonged valve closure. Selection for these behaviours should favour different patterns of co-evolution of the underlying traits. The theory of symmorphosis postulates that the structural design of living systems is quantitatively matched to functional demands (Weibel et al. 1996). This implies a co- evolution of capacity at different levels of organisation and stipulates that no excess structure is maintained. Overall, the ‘design’ of oxygen and substrate use pathways in dogs and goats conforms to these principles, although pulmonary capacity exceeds that at other levels (Weibel et al. 1996). If symmorphosis also applies to scallops, the physiological and biomechanical properties favouring swimming should decrease in scallops that close their valves in response to their predator. Our analysis of the patterns of use of the adductor

71 muscle indicates such changes of muscle use and predicts that the biochemical, biomechanical and morphological properties should show similar interspecific differences.

Acknowledgements We are extremely grateful to the staff of the different institutes where this research was conducted. In Queensland at the BIARC this included Paul Palmer, Tim Lucas, Satoshi Mikami and Sizhong (Joe) Wang as well as Peter F. Duncan at the University of the Sunshine Coast. In Tasmania at TAFI we received invaluable assistance from Julian Harrington, Craig Mundy and all the technical staff. In Québec at the MAPAQ, Bruno Myrand, Madeleine Nadeau and the technical staff as well as Mélanie Bourgeois from CultiMer greatly facilitated our work. Finally in British Columbia, Brian Kingzett and the technical staff at the Centre for Shellfish Research made it possible for us to work with C. gigantea. We thank the anonymous reviewers for their constructive comments on our manuscript.

Funding This research was supported by funds from Natural Sciences and Engineering Research Council of Canada to H.E.G and J.H.H. and from Réseau Aquaculture Québec to H.E.G. IT was the recipient of a PhD scholarship from Fonds de recherche sur la nature et les technologies du Québec and program FONCER (Programme de formation orientée vers la nouveauté, la collaboration et l’expérience en recherche) from Natural Sciences and Engineering Research Council of Canada.

CHAPITRE 3

Scallops show that muscle metabolic capacities reflect locomotor style and morphology

ISABELLE TREMBLAY et HELGA E. GUDERLEY

Publié dans « Physiological and Biochemical Zoology, 2014, 87 :231-244»

Résumé Toutes les espèces de pétoncles utilisent leur muscle adducteur pour nager. Par contre, chaque espèce diffère au niveau du patron d’utilisation de leur muscle adducteur qui varie en parallèle avec les différences interspécifiques marquées au niveau de la morphologie de la coquille. Ceci fournit donc une excellente opportunité pour étudier les liens entre les capacités métaboliques du muscle et la performance locomotrice. Nous avons trouvé que les capacités aérobie et de la glycolytique anaérobique, ainsi que les niveaux de phosphoarginine dans le muscle adducteur phasique, diffèrent avec le type de réponse de fuite. Le contenu en phosphoarginine est plus élevé chez les espèces qui s’appuient principalement sur les contractions phasiques (Amusium balloti, Placopecten magellanicus and Pecten fumatus). L’activité de l’arginine kinase reflète la dépendance aux contractions phasiques rapides en début de réponse de fuite. Les espèces de pétoncles qui maintiennent leurs valves fermées pour des périodes prolongées (P. fumatus, Mimachlamys asperrima and Crassadoma gigantea) ont un muscle adducteur phasique avec des activités élevées au niveau des enzymes de la voie de la glycolyse anaérobique. L’activité de la myosine ATPase est plus faible chez l’espèce de pétoncle qui ne nage pas, C. gigantea, comparativement aux pétoncles qui nagent. Les différents rôles et patrons de nage observés chez les pétoncles, se reflètent au niveau de différences interspécifiques des attributs biochimiques du muscle adducteur phasique. Ces patrons suggèrent une co- évolution entre les capacités métaboliques du muscle adducteur, les patrons d’utilisation du muscle adducteur et la morphologie de la coquille chez les pétoncles.

Abstract Although all scallops swim using their adductor muscle to close their valves, scallop species differ considerably in how they use their muscle during escape responses, in parallel with the striking interspecific differences in shell morphology. This provides an excellent opportunity to study links between muscle metabolic capacities and animal performance. The capacity for anaerobic glycolysis and aerobic metabolism, as well as phosphoarginine levels in the phasic adductor muscle, differed with escape response strategy. Phosphoarginine contents were high in species that rely on phasic contractions (Amusium balloti, Placopecten magellanicus and Pecten fumatus). Arginine kinase activities reflect reliance on rapid initial bursts of phasic contractions. Scallops that maintain their valves closed for prolonged periods (Pecten fumatus, Mimachlamys asperrima and Crassadoma gigantea) have high activities of enzymes of anaerobic glycolysis in their phasic adductor muscle. Myosin ATPase activity was lower in the non- swimming scallop, C. gigantea, than in swimming scallops. The different patterns and roles of swimming are reflected in interspecific differences in the biochemical attributes of the phasic adductor muscle. These patterns suggest co-evolution of muscle metabolic capacities, patterns of adductor muscle use and shell morphology in scallops.

Introduction The link between morphology and physiology is fundamental in biological systems. Form may dictate function or evolve with it since major functions, such as locomotion or reproduction, can select for physiological mechanisms and favourable morphologies, within the constraints of phylogeny. As locomotor performance is critical for activities that determine survival, growth and reproductive success, such as escaping predators, prey capture, foraging and migrating, the underlying physiological mechanisms should be subject to selection. Effectively, comparisons of vertebrates with markedly differing locomotor styles, such as birds with different flight strategies (pheasants vs pigeons) or mammals with different capacities for long-distance running (dogs vs goats) show that cardiovascular performance and muscle aerobic capacity parallel the capacity for sustained activity (Davis and Guderley 1987, Weibel et al. 1996); as these interspecific differences in performance are accompanied by distinct musculoskeletal dynamics, it is difficult to link muscle metabolic capacities directly with the use of muscle during activity and whole animal performance. A locomotor system with fewer components, such as that of scallops, should be ideal for revealing links between muscle physiology and animal performance.

Scallops are exceptional among bivalve molluscs in their ability to swim considerable distances using jet propulsion. Although all scallops (Pectinidae) live in the subtidal, their life habits vary considerably ranging from byssal attachment with sporadic swimming, to free-living, gliding, recessing, and cementing. Byssal attachment with sporadic swimming is the most likely ancestral state in Pectinidae (Alejandrino et al. 2011). Convergent and parallel evolution are thought to have generated the other life habits (Alejandrino et al. 2011). The cementing life habit only occurs as a derived state, whereas the other life habits can be ancestral states or transitional outcomes (Alejandrino et al. 2011). Valve characteristics and patterns of muscle use vary with the life habits of Pectinidae (Tremblay et al. 2012). Differences in patterns of muscle use require distinct muscle metabolic capacities and neural activation. Here, we focus on links between muscle metabolic capacities and patterns of muscle use in scallop species with differing shell morphologies and life habits.

Scallop swimming relies on a single adductor muscle inserted between two shells linked by a hinge ligament. Swimming results from jet propulsion produced by a rapid succession of contractions where valve adduction (closure) and abduction (opening) alternate (Drew 1906, Dakin 1909, Bruddenbrock 1911). This mode of swimming is shared by scallops exhibiting a wide range of shell morphologies and life habits. The adductor muscle is responsible for valve closure while the uncalcified hinge ligament, with its rubber like properties (Alexander 1966, Marsh et al. 1976), acts as a spring to open the valves when the adductor muscle relaxes. The larger phasic adductor muscle is composed of cross-striated muscle fibres that contract rapidly during swimming or jumping movements (Lowy 1953, Millman 1967). The smaller tonic adductor muscle is composed of slow contracting smooth muscle fibres that allow prolonged valve closure or low-energy maintenance of a constant valve opening during filter feeding (Lowy 1953, Chantler 2006). Force recordings during escape responses show that the relationship between phasic and tonic contractions differs considerably among scallop species (Tremblay et al. 2012), with some species making many short tonic contractions between phasic contractions, while others primarily make long tonic contractions at the end of their escape response.

Scallop swimming depends upon anaerobic metabolism; scallops lack respiratory pigments and their muscle is poorly vascularised (Thompson et al. 1980, de Zwaan et al. 1980). The phasic adductor muscle uses ATP at a high rate for its rapid contractions, while the tonic adductor muscle uses the energetically efficient catch mechanism to maintain tension over prolonged periods. Most of the ATP used by phasic contractions (~ 70% in Placopecten magellanicus and Argopecten irradians concentricus) is generated from phosphoarginine by arginine kinase (Grieshaber and Gäde 1977, Gäde et al. 1978, de Zwaan et al. 1980, Livingstone et al. 1981, Chih and Ellington 1983, Chih and Ellington 1986). Hence, phosphoarginine concentrations in the phasic muscle decrease as a linear function of the number of phasic contractions (Livingstone et al. 1981, Bailey et al. 2003). The depletion of phosphoarginine, as well as a decreased free energy of ATP hydrolysis in the phasic adductor, likely bring on the prolonged tonic contractions that indicate exhaustion (Bailey et al. 2003, Pérez et al. 2008a). While vertebrate phasic muscle uses glycogen to fuel anaerobic exercise, in scallops only the final (~30%) phasic contractions

79 rely on anaerobic glycolysis (Grieshaber and Gäde 1977, Gäde et al. 1978). The scallop phasic adductor muscle uses anaerobic glycolysis primarily during the tonic contraction that initiates recovery from exhaustive exercise (Grieshaber and Gäde 1977, Gäde et al. 1978, de Zwaan et al. 1980, Livingstone et al. 1981). Adenylate energy charge recuperates partially during valve closure (Pérez et al. 2008b). To recover completely from exhaustion, scallops open their valves and markedly increase rates of oxygen consumption (Thompson et al. 1980, Tremblay et al. 2006). Rapid ATP production by mitochondrial oxidative phosphorylation allows replenishment of phosphoarginine and glycogen in the phasic adductor muscle (Grieshaber 1978, Livingstone et al. 1981, Guderley et al. 1995).

In this study, the phasic adductor muscles of scallops with distinct escape response strategies and shell morphologies (Fig. 3.1) were examined to determine if they differ in their muscle metabolic capacities. These Pectinidae use their adductor muscle differently during escape responses (Tremblay et al. 2012). The scallop A. balloti and P. magellanicus have shells with a hydrodynamic cross-sectional profile and perform many phasic contractions interspersed with short tonic contractions during prolonged escape responses (Fig. 3.1). Pecten fumatus and Mimachlamys asperrima do not have shells with a hydrodynamic cross-sectional profile and differ in how they deal with their disadvantageous shell morphology (Fig. 3.1). The scallop P. fumatus carries out an intense burst of phasic contractions, presumably to overcome the constraints imposed by its shape and mass (Tremblay et al. 2012). The scallop M. asperrima makes very short bursts of phasic contractions at the beginning of the response, but generally keeps its valves shut (Tremblay et al. 2012), possibly as its tightly closed shells are covered by sponges known to inhibit predation by sea stars (Pitcher and Butler 1987). Although the shells of Equichlamys bifrons have a similar cross-sectional profile and mass to those of P. magellanicus, adult E. bifrons are more sedentary than M. asperrima and P. fumatus (Olsen 1955). Adult Crassadoma gigantea are cemented to the substrate and generally keep their valves shut in response to a predator, only occasionally deigning to make phasic contractions (Fig. 3.1). Finally, a non-swimming monomyarian bivalve, the Pacific oyster Crassostrea gigas, was sampled to see whether the biochemical attributes of C. gigantea reflect the swimming condition in scallops or the ancestral condition of non-swimming

Amusium balloti Total number of phasic 40.9±1.37 Min. interval between two phasics, s 0.38±0.036 Phasic contraction rate 1st series, nb∙s-1 0.92±0.07 Time to fatigue, s 229±12.5 Total number of tonic 12.5±0.76 Mean duration of tonic, s 20.9±1.71

Placopecten magellanicus Total number of phasic 27.5±1.65 Min. interval between two phasics, s 1.12±0.144 Phasic contraction rate 1st series, nb∙s-1 0.72±0.07 Time to fatigue, s 217±15.3 Total number of tonic 20.9±1.67 Mean duration of tonic, s 16.2±2.17

Pecten fumatus Total number of phasic 32.7±2.86 Min. interval between two phasics, s 0.32±0.042 Phasic contraction rate 1st series, nb∙s-1 2.39±0.15 Time to fatigue, s 76±14.3 Total number of tonic 8.0±1.12 Mean duration of tonic, s 46.4±10.95

Mimachlamys asperrima Total number of phasic 18.0±1.61 Min. interval between two phasics, s 0.65±0.123 Phasic contraction rate 1st series, nb∙s-1 1.25±0.14 Time to fatigue, s 111±13.8 Total number of tonic 9.9±0.88 Mean duration of tonic, s 33.2±2.94

Crassadoma gigantea Total number of phasic 1.8±0.84 Min. interval between two phasics, s - Phasic contraction rate 1st series, nb∙s-1 - Time to fatigue, s 45±21.1 Total number of tonic 2.3±0.86 Mean duration of tonic, s 276±31.4

81 monomyarian bivalves. Scallops and oysters both belong to the Subclass Heterodonta (Plazzi et al. 2011).

Our underlying premise was that the biochemical attributes of the phasic adductor muscle reflect the swimming behaviour of each species. Thus, scallop species performing many phasic contractions (A. balloti, P. magellanicus and P. fumatus) would have higher phosphoarginine contents than more sedentary species. Scallop species with high phosphoarginine contents should take longer to fatigue. Scallop species capable of high rates of phasic contractions (P. fumatus, A. balloti) would have high arginine kinase activities. On the other hand, scallops that keep their valves closed for prolonged periods (P. fumatus, M. asperrima, C. gigantea) would have higher activities of glycolytic enzymes in the phasic adductor muscle. Myofibrillar ATPase activity should be higher in scallops that perform phasic contractions at a high rate compared to those performing them at a low rate. As Olsen (1955) indicates that adult E. bifrons are more sedentary than P. fumatus and M. asperrima, we predicted that the biochemical signature of E. bifrons would be closer to that of the less active scallops. As the oysters tolerate prolonged periods of anaerobiosis, it is predicted that their muscles would have higher glycolytic capacities and lower levels of arginine kinase and phosphoarginine than those of the scallops.

Materials and methods

Sampling and maintenance of the scallops

Amusium balloti Mature A. balloti (92 to 103 mm shell height) were sampled in August 2007 near Gladstone (Queensland, Australia) and kept them in holding tanks with running seawater (18.5°C, salinity 35 ppt) at the Bribie Island Aquaculture Centre (Woorim, Queensland, Australia).

Placopecten magellanicus Mature P. magellanicus (85 to 103 mm shell height) were obtained from Culti-mer (Cap-Aux-Meules, Îles-de-la-Madeleine, Québec, Canada) in September 2008. Scallops were transferred to holding tanks with flow-through seawater (17.5°C, salinity 30 ppt) in the laboratory of Ministère des Pêcheries et de l’Alimentation du Québec (Cap-Aux- Meules, Îles-de-la-Madeleine, Québec, Canada) and were left undisturbed in the tanks. After a week, the water temperature dropped overnight to 14°C and remained constant. Scallops were left another week to habituate to this temperature prior to sampling.

Equichlamys bifrons, Pecten fumatus, and Mimachlamys asperrima These species were collected by SCUBA near Satellite Island (43° 32’491”S and 147°23’297”E, Channel d’Entrecastreux, Tasmania, Australia) in September 2007. We Animals of a similar size range were selected: E. bifrons (73 to 114 mm shell height), P. fumatus (93 to 108 mm shell height) and M. asperrima (83 to 97 mm shell height). Scallops were transferred to the Tasmanian Aquaculture and Fisheries Institute (Taroona, Tasmania, Australia) where they were kept undisturbed in tanks with running seawater (12.5°C, 34 ppt) for three weeks prior to sampling.

Crassadoma gigantea Mature C. gigantea were collected in May 2010 from oyster rearing systems that had been in the water for the past 4-5 years near Espinosa Inlet (Vancouver Island, British Columbia, Canada). After collection, scallops (71 to 120 mm shell height) were transferred to the Centre for Shellfish Research (Vancouver Island University, Nanaimo, British Columbia, Canada) where they remained in running seawater tanks (12.5°C, salinity 28 ppt) for 10 days prior to their sampling.

Crassostrea gigas Pacific oysters, C. gigas (85 to 133 mm shell height), were collected at low tide on the beach at Deception Bay (Vancouver Island, British Columbia, Canada) and were immediately transferred to the Centre for Shellfish Research (Vancouver Island University,

Nanaimo, British Columbia, Canada) where they remained in running seawater tanks (12.5°C, salinity 28 ppt) for two weeks prior to their sampling.

Muscle sampling Phasic adductor muscle was removed and cut in half. Each half was quickly frozen in liquid nitrogen (less than 30 s) using a clamping press (Gagnon et al. 1998) and stored at -80°C until use for biochemical analysis.

Anatomic and morphological measurements Shell height and mass of the experimental scallops were measured. As the phasic adductor muscle had to be frozen quickly for the biochemical assays, it was impossible to determine its mass. As a second group of scallops was kept under the same conditions for behavioural tests (Tremblay et al. 2012), the phasic adductor muscle mass of the scallops used for biochemical assays was estimated from the regression between shell height and phasic adductor mass of the scallops used for the behavioural tests (see appendix 3.1 for regression equations). Further, the water content, condition index (see Tremblay et al. 2012 for details), and protein concentration of the scallops used for behavioural tests (Table 3.1) are presented, as for all the species, both groups came from the same population, were collected at the same time and were kept in the same conditions. All the species had mature gonads; only A. balloti had finished spawning at the time of the sampling (Table 3.1). The mass, water content, condition index and protein concentration data for E. bifrons were obtained from a separate group used for visual examination of behaviour while data for C. gigas were obtained from a group kept at rest in running seawater tanks.

Muscle protein fractions The phasic adductor muscle protein was separated into soluble and insoluble fractions using the method of Bates and Millwad (1983) as modified by Somero and Childress (1990). Protein concentration was determined by bicinchoninic acid method (Smith et al. 1985) with bovine serum albumin as the standard.

Phosphoarginine concentrations Frozen phasic adductor muscles were ground in liquid nitrogen in a mortar and pestle, homogenised with ice-cold 6% PCA (w/v) (200mg∙2ml-1) using a Polytron (Brinkmann Instruments) and centrifuged for 15 min at 12 000 g at 4°C (Sorvall® RC-5B).

Supernatants were neutralised with 5 M K2CO3 and centrifuged at 12 000 g at 4°C for 5 min. The resulting supernatants were filtered through a nylon filter (0.45µm) and stored at -80°C till their use for phosphoarginine determination. Phosphoarginine concentration was determined using high-performance liquid chromatography following the mothod used by Viant et al. (2001) as described by Pérez et al. (2008a). The peak for phosphoarginine was identified by comparison with a standard (Sigma Biochemical). In all scallops the phosphoarginine peak was clear and easy to identify. For oysters, no peak was found at the expected location, only a broad shoulder, out of the elution zone. Phosphoarginine could not be identified with certainty in the oyster adductor muscle, therefore it was not considered in our analysis.

Enzyme assays Samples of phasic adductor muscle were homogenised on ice, using a minimasher

(Tremblay et al. 2006), in 10 vol of 50 mM imidazole-HCl, 2 mM EDTA-Na2, 5 mM EGTA, 1 mM dithiothreiol and 1% Triton X-100 (20 mM NaF for phosphofructokinase assay only) at pH 6.6. Homogenates were centrifuged (IEC MicroMax) for 10 min at 2462 g at 4°C. Enzymatic activities were measured at 18°C using a microplate spectrophotometer (SPECTRAmax 190, Molecular Devices). Enzyme activity was examined by following the absorbance changes of NAD(P)H at 340 nm, with the exception of CS, which was monitored at 412 nm to detect the transfer of sulphydryl groups from CoASH to DNTB. Extinction coefficients for NAD(P)H and DTNB were 6.22 and 13.6 cm-1 µmol-1, respectively. All assays were run in triplicate and the specific activities are expressed in international units (µmol substrate converted to product per min) per g of phasic adductor muscle wet mass.

The assay conditions followed those given by Brokordt et al. (2000a) except for the following modifications (all concentrations in mM l-1). In all cases the substrate concentrations shown give the maximal activities in the species in question.

Arginine kinase (AK; EC 2.7.3.3): Phosphoarginine (omitted for the control) concentrations were 2.5 (E. bifrons), 5 (A. balloti, P. magellanicus, P. fumatus, M. asperrima and C. gigas) and 10 (C. gigantea), pH 6.6.

Octopine dehydrogenase (ODH; EC 1.5.1.11): Arginine (omitted for the control) concentrations were 1.5 (P. magellanicus), 3 (A. balloti and E. bifrons) and 6 (P. fumatus, M. asperrima and C. gigantea), pH 6.6.

Phosphofructokinase (PFK; EC 2.7.1.11): ATP (omitted for the control) concentrations were 1 (A. balloti), 2 (P. magellanicus, P. fumatus, M. asperrima, E. bifrons, and C. gigas) and 4 (C. gigantea). No fructose 2,6 biphosphate was used for these assays, pH 7.5.

Citrate synthase (CS; EC 4.1.3.7): Oxaloacetate (omitted for the control) concentrations were 0.2 (A. balloti, P. magellanicus, P. fumatus, M. asperrima, C. gigantea and C. gigas) and 0.4 (E. bifrons), pH 8.0.

Alanopine dehydrogenase and strombine dehydrogenase were only measured in the oyster, C. gigas. Assay conditions (all concentrations in mmol ∙ l-1) were adapted from Fields and Hochachka (1981):

Alanopine dehydrogenase (ADH; EC 1.5.1.17): Imidazole-HCl 100, sodium pyruvate 3, NADH 0.2 and alanine 400 (omitted for the control), pH 7.0.

Strombine dehydrogenase (SDH; EC 1.5.1.22): Imidazole-HCl 100, sodium pyruvate 3, NADH 0.2 and glycine 200 (omitted for the control), pH 7.0.

Myosin ATPase Samples of phasic adductor muscle (~0.2 g) were homogenised on ice, using a minimasher, in 500µl of 20 mM imidazole-HCl, 100 mM KCl, 1 mM EDTA-Na2, and 1% Triton X-100. Homogenates were centrifuged (IEC MicroMax) at 3000 g for 5 min at 4°C. Supernatants were removed and pellets were homogenised in 500µl of suspension buffer (20 mM imidazole-HCl and 100 mM KCl) and centrifuged at 3000 g for 5 min at 4°C. Samples were submitted to three cycles of homogenisation-centrifugation after which supernatants were removed and pellets were suspended in 1 ml of suspension buffer and centrifuged at 400 g for 2 min at 4°C. Final pellets were suspended in 150µl of suspension buffer and assayed for myosin ATPase activity. Aliquots were frozen at -80°C for determination of protein concentration. To measure myosin ATPase activity, we used a coupled enzyme assay that followed the disappearance of NADH at 340 nm (Glyn and Sleep 1985, Guegen et al. 2005). Assay conditions were from Seebacher and James (2008) except that NADH was 0.6 mM. Assays were run in triplicate at 18°C using a microplate spectrophotometer (SPECTRAmax 190, Molecular Devices). Protein concentration of the myofibrillar solution was determined by the bicinchoninic acid method (Smith et al. 1985) with bovine serum albumin as the standard.

Chemicals Biochemicals were obtained from Sigma Chemical (St. Louis). All other biochemicals were of analytical grade.

Statistical analysis The normality of the residuals was tested using a Shapiro-Wilks test and the homogeneity of variances was analyzed visually by plotting residuals relative to predicted values. Since residuals were not normal and the variances were not homogeneous, non- parametric tests were used. Comparisons between the scallop species used Kruskall-Wallis tests. All analyses were done using SAS 9.2 (SAS Institute) and significance was accepted at P ≤ 0.05.

Results

Anatomical characteristics of the experimental scallops The initial step was to examine the size and condition indices of the scallops, as these factors can affect muscle metabolic capacities (Labrecque and Guderley 2011, Brokordt et al. 2000a). Shell heights of the experimental scallops overlapped, with mean shell heights between 90 and 100 mm. The scallop P. magellanicus and M. asperrima were the smallest while E. bifrons was the largest (Table 3.1). The oyster, C. gigas, had a slightly greater shell height than the scallops (Table 3.1). As the muscles of our experimental scallops were snap frozen, the anatomic characteristics of the scallops were assessed on individuals of similar size sampled at the same time (see Material and Methods). The scallop E. bifrons and C. gigantea had the biggest phasic adductor muscle (mean values 18.0 and 16.8 g respectively) while A. balloti had the smallest (7.9 ± 0.35 g); (Table 3.1). The scallop P. magellanicus, P. fumatus and M. asperrima had intermediate phasic adductor muscles weighing approximately 12 g (Table. 3.1). The phasic adductor muscle of the oyster was much smaller than that of any scallop (1.5 ± 0.06 g); (Table 3.1). Water content in the soft tissues was highest in A. balloti (85.5 ± 0.2 %) and lowest in C.

Table 3.1. Morphological characteristics, water content and condition index of experimental scallops.

Amusium Placopecten Equichlamys Pecten Mimachlamys Crassadoma Crassostrea balloti magellanicus bifrons fumatus asperrima gigantea gigas Biochemical studies Shell height, mm 97.0±0.75a 90.6±0.67b 100.1±2.25a 96.4±1.57a 90.9±1.14b 96.8±3.74a 111.9±3.27c Phasic muscle wet mass, g 7.9±0.35a 12.2±0.27b 18.0±1.09c 12.4±0.60b 11.2±0.46b 16.8±1.43c 1.5±0.06d N 23 16 20 10 12 18 20 Behavioural studies Shell height, mm 96.3±1.10a 90.4±0.85b,c,d 96.2±2.53a 94.4±1.71a,d 84.2±2.04c 97.0±4.54a,b 105.9±3.27e Water content Soft tissues, % 85.5±0.2a 80.2±0.3b 82.1±0.2c 84.1±0.4d 84.6±0.3d 79.2±0.3e 76.8±0.5f Phasic muscle, % 80.7±0.3a 74.3±0.3b 76.8±0.1c 79.4±0.6d 80.0±0.4a,d 74.6±0.3b 74.4±0.3b Condition index 0.48±0.01a 0.51±0.01a 0.71±0.06b 0.42±0.02c 0.41±0.01c 0.61±0.02b 0.41±0.01c Gonadosomatic index, % 5.4±0.3a 10.8±0.7b,e 15.4±1.0c 11.5±0.7b 17.8±0.9d 9.9±0.6e n.a. Total proteins concentration, mg∙g-1 69.0±1.6a 85.0±4.3b 94.7±2.3c 79.2±2.6b,d 77.6±1.6d 99.7±2.7c 111.5±2.9e N 18-30 14-15 20 15 16 19 18 Data are means ± S.E. Condition index: (scallop soft tissue wet mass/shell volume) (Tremblay et al. 2012) Soft tissues: all tissues including the adductor muscle. Gonadosomatic index: (gonad tissue wet mass/total animal wet mass)*100. Total proteins concentration in the phasic adductor muscle (mg∙g-1 muscle wet mass). In a given row, different letters indicate significant differences (Kruskall-Wallis and multiple comparisons, P<0.05).

89 gigantea (79.2 ± 0.3 %), but the values overlapped between the scallop species (Table 3.1). Water content in the soft tissues of the oyster was lower than in scallops (Table 3.1). The same pattern was observed for the water content in the phasic adductor muscle (Table 3.1). In contrast to the relative stability of the water content, the condition index (g soft tissue mass per ml volume between valves) varied considerably between species. The scallop E. bifrons had the highest condition index (0.71 ± 0.06 g∙ml-1), followed closely by C. gigantea (Table 3.1). The scallop P. magellanicus and A. balloti had similar condition indices and were one third less than E. bifrons (Table 3.1). The condition indices of P. fumatus and M. asperrima were approximately 0.41 g∙ml-1 (Table 3.1). The condition index of the oyster was similar to that of P. fumatus and M. asperrima (Table 3.1). Overall, these limited differences in size and condition were unlikely to cause the appreciable metabolic differences observed between the scallop species.

Phasic muscle protein concentrations The oyster, C. gigas, had the highest protein concentration (111.5 ± 2.9 mg∙g-1 wet muscle mass) in the phasic adductor muscle (Table 3.1). Among the scallops, C. gigantea and E. bifrons had the highest levels with 95-100 mg∙g-1 muscle wet mass (Table 3.1). P. magellanicus, P. fumatus and M. asperrima had slightly lower protein concentrations than C. gigantea and E. bifrons (Table 3.1). The protein concentration was lowest in A. balloti (69.0 ± 1.6 mg∙g-1 muscle wet mass); (Table 3.1).

The species examined differed little in the distribution of muscle proteins between the soluble and insoluble fractions (data not shown). The insoluble fraction represented 62 to 67 % of the total protein contents. The highest proportion of insoluble proteins (~67 %) was found in P. magellanicus, M. asperrima and E. bifrons. The scallop P. fumatus, C. gigantea and the oyster had intermediate proportions, ~64 %, while A. balloti had the lowest proportion of insoluble proteins: ~ 62%. Interspecific differences in soluble proteins followed the opposite order with A. balloti having the highest proportion and E. bifrons the lowest.

Phasic muscle phosphoarginine content The highest phosphoarginine contents was found in P. fumatus and E. bifrons with values near 40.0 µmol∙g-1 muscle wet mass (Fig. 3.2). The scallop A. balloti and P. magellanicus had approximately 30 µmol∙g-1 muscle wet mass, whereas M. asperrima had 22.4 ± 1.0 µmol∙g-1 muscle wet mass (Fig. 3.2). Among the scallops, C. gigantea had the lowest phosphoarginine levels (9.1 ± 0.8 µmol∙g-1 muscle wet mass); (Fig. 3.2). Phosphoarginine was not measurable with certainty in the phasic muscle of the oyster (see Material and Methods). As predicted, the reliance of A. balloti, P. magellanicus and P. fumatus on phasic contractions (Fig. 3.1) was reflected in high phosphoarginine contents (Fig. 3.2).

Phasic muscle enzyme activities Arginine kinase (AK) activity differed approximately two-fold among the scallop species. The scallop P. fumatus had the highest AK activity (1873 ± 247 U∙g-1 muscle wet mass) while M. asperrima and E. bifrons had slightly, but not significantly, lower levels (Fig. 3.3). The scallop A. balloti and C. gigantea and the oyster had intermediate activities, while P. magellanicus had the lowest activity (953 ± 53 U∙g-1 muscle wet mass); (Fig. 3.3). These results confirm the prediction that species making strong initial bursts of phasic contractions (P. fumatus and M. asperrima) have the highest AK activities (Fig. 3.1 and 3.3). Muscle activities of the glycolytic enzymes, octopine dehydrogenase (ODH) and phosphofructokinase (PFK) also differed at least two-fold among the scallop species. The scallop E. bifrons had the highest ODH activity (71 ± 4 U∙g-1 muscle wet mass) while P. fumatus and C. gigantea showed intermediate activities (Fig. 3.3) and A. balloti, P. magellanicus and M. asperrima had significantly lower ODH activities (approximately 30 U∙g-1 muscle wet mass) (Fig. 3.3). In the oyster, the activities of the terminal enzymes of anaerobic glycolysis, ADH and SDH, were 61.8 ± 5.2 and 66.6 ± 5.9 U∙g-1 muscle wet mass respectively. When combined, these activities exceeded the ODH activity of any scallop (Fig. 3.3).

Phosphoarginine content 50 b b 40 a a

phasic muscle wet mass wet muscle phasic 30

1 c - 20

d Arg ∙ g ∙ Arg

- 10

0 µmol

A. balloti E. bifrons P. fumatus C. gigantea M. asperrima P. magellanicus

Figure 3.2. Concentration of phosphoarginine (µmol P-Arg ∙ g-1 phasic muscle wet mass) in the phasic adductor muscle. Means ± S.E. (N=10-23). Means with different letters are significantly different (P<0.05), as indicated by Kruskall-Wallis and a posteriori multiple comparison tests.

Arginine kinase Octopine dehydrogenase

2500 80 b c c 2000 c 60 a,c c 1500 a a,b a,b a b 40 a 1000 a 20 500

0 0

Phosphofructokinase Citrate synthase

2.0 d 8 b 1.5 6 b e b b 1.0 4 d c a,d a

phasic muscle wet muscle phasic a a 0.5 c c

1 1

- 2

mass U ∙ g ∙ U 0 0.0

C. gigas A. balloti C. gigas A. balloti E. bifrons E. bifronsP. fumatus P. fumatus C. gigantea C. gigantea M. asperrima M. asperrima P. magellanicus P. magellanicus

Figure 3.3. Enzymatic activities (U ∙ g-1 phasic muscle wet mass) in the phasic adductor muscle measured at 18°C. Values represent means ± S.E. (N=7-10 for AK and N=14-22 for others). Means with different letters are significantly different (P<0.05), as indicated by Kruskall-Wallis and multiple comparison tests.

The oyster had the highest PFK activity (7.6 ± 0.4 U∙g-1 muscle wet mass); (Fig. 3.3). The scallop P. fumatus, E. bifrons, and C. gigantea had similar (approximately 4.0 U∙g-1 muscle wet mass) but significantly lower PFK activities than the oyster (Fig. 3.3). Phosphofructokinase activity in M. asperrima was intermediate while A. balloti and P. magellanicus had the lowest PFK activities (Fig. 3.3). The interspecific differences among these glycolytic enzymes partially follow our predictions, as two of the scallop species which use long tonic contractions (P. fumatus and C. gigantea; Fig. 3.1) had high glycolytic enzyme activities (Fig. 3.3). Citrate synthase activity was highest in P. magellanicus with 1.56 ± 0.07 U∙g-1 muscle wet mass (Fig. 3.3). The scallop A. balloti, M. asperrima and C. gigantea had intermediate CS activities while in P. fumatus and E. bifrons CS activities were only 0.3 U∙g-1 muscle wet mass (Fig. 3.3). Citrate synthase activity in oyster adductor muscle fell between that of P. magellanicus and the other scallops (Fig. 3.3). To examine whether the interspecific differences in enzyme activities were a reflection of differences in protein contents, these patterns were examined when enzyme activities were expressed relative to protein contents. The interspecific trends among scallops were retained when enzyme activities were expressed relative to muscle protein contents (data not shown). As muscle protein concentrations were considerably higher in oysters than in scallops (Table 3.1), expressing activities relative to protein contents changed the relative activities in oyster and scallops. Even so, when PFK activity was expressed relative to protein contents, oysters retained the highest activity.

Myosin ATPase Myosin ATPase activity was highest in P. magellanicus (0.58 ± 0.07 U∙mg-1 protein); (Fig. 3.4). The scallop A. balloti, P. fumatus, M. asperrima and E. bifrons had intermediate activities whereas C. gigantea and the oyster only had activities around 0.06 U∙mg-1 protein (Fig. 3.4). Given the extraction protocol, myosin ATPase activity was expressed relative to the protein content in the myofibrillar fraction and not relative to muscle mass. This provides information about the catalytic capacity of myofibrillar ATPase, but not of muscle capacities. Knowledge of muscle myosin ATPase activities

Myosin ATPase

0.6

0.4

protein

1 - a a,c a,c c 0.2

U ∙ mg U ∙ d d 0.0

C. gigas A. balloti E. bifrons P. fumatus C. gigantea M. asperrima P. magellanicus

95 would require separate quantification of myofibrillar abundance. The interspecific patterns of myofibrillar ATPase activities partially confirmed the prediction that less active species (C. gigantea) have lower activities.

Discussion Metabolic characteristics of the phasic adductor muscle differed considerably among the bivalves studied. The Pectinidae, while diversified in muscle enzyme activities, shared a pattern of metabolic organization distinct from that of oysters. Much as the morphological differences among scallop species are accompanied by marked differences in how the phasic and tonic adductor muscles are used during escape responses (Fig. 3.1) (Tremblay et al. 2012), the metabolic attributes of the adductor muscles differed among these species. Our salient findings are that muscle phosphoarginine content was higher in scallops that relied more on phasic than tonic contractions (A. balloti, P. magellanicus, and P. fumatus) and AK activity was highest in scallops that make their first phasic contractions in rapid bursts (P. fumatus and M. asperrima). Scallops that keep their valves closed for prolonged periods (P. fumatus, M. asperrima and C. gigantea) have higher glycolytic enzyme activities than those that primarily use short tonic contractions (A. balloti and P. magellanicus). Myosin ATPase activity was much higher in swimming scallops than the non-swimming scallop (C. gigantea) and the oyster. Overall, the biochemical attributes of the phasic adductor muscle reflected interspecific differences in muscle use during escape responses. On the other hand, some of our predicted relationships were not found, presumably as interspecific differences in behaviour and morphology obscured the postulated direct links. Shell morphology does not directly predict muscle metabolic capacities, since muscle use can overcome constraints imposed by shell morphologies (Tremblay et al. 2012). Differing life habits can favour specific patterns of muscle use which in turn require the appropriate metabolic machinery.

Interspecific differences in phosphoarginine levels and patterns of muscle use Neither the total number of phasic contractions nor the time to fatigue completely predicted interspecific differences in adductor muscle phosphoarginine contents. The scallop A. balloti, P. magellanicus, and P. fumatus performed more phasic contractions and had higher phosphoarginine contents than M. asperrima and C. gigantean; although P. fumatus had the highest phosphoarginine content (Fig. 3.2), it did not make the most phasic contractions (Fig. 3.1). Also, P. fumatus fatigued much more rapidly than A. balloti and P. magellanicus which had lower phosphoarginine contents (Fig. 3.1 and Fig. 3.2). On the other hand, phosphoarginine contents were even lower in scallops that make fewer phasic contractions. The combination of behavioural and biochemical data indicate potential interspecific differences in the cost of phasic contractions. Such differences are suggested by the fact that A. balloti was able to perform more phasic contractions with a smaller pool of phosphoarginine than P. fumatus. Scallops do not deplete their phosphoarginine pool completely when swimming until exhaustion; C. opercularis used 93% while P. magellanicus and P. maximus used 78% (Grieshaber 1978, Livingstone et al. 1981, Bailey et al. 2003). On average, the phosphoarginine pool drops to 5 µmol∙g-1 muscle wet mass (Grieshaber 1978, Livingstone et al. 1981, Bailey et al. 2003). If one assumes that the scallops we studied had reduced phosphoarginine levels to 5 µmol∙g-1 muscle wet mass when they stopped contracting, we can estimate how much phosphoarginine was used per phasic contraction in each of our species. The scallop A. balloti made the best use of its phosphoarginine, using only 4.86 µmol∙phasic contraction-1. Phasic contractions by P. fumatus, P. magellanicus and M. asperrima used much more phosphoarginine (13.77, 11.22 and 10.85 µmol∙phasic contraction-1 respectively). Combining visual estimates of escape response activity by E. bifrons (45.6 ± 10.2 phasic contractions before fatigue) with the phosphoarginine data suggests that E. bifrons used phosphoarginine at 13.50 µmole∙phasic contraction-1. It is more difficult to estimate how much phosphoarginine was used per phasic contraction for C. gigantea. We find it unlikely that C. gigantea is exhausted and has markedly depleted its phosphoarginine pool when it stops contracting. We deemed a scallop exhausted when it made no phasic contractions during 1 min of stimulation with its predator (Tremblay et al. 2012). The cemented scallop C. gigantea

97 usually keeps its valves shut and only occasionally makes phasic contractions in response to its predator. When C. gigantea made more than one phasic contraction, they were sometime separated by more than 1 min (I. Tremblay personal observations). Overall, our observations suggest that phasic contractions do not have the same energetic cost for all the species. The rate of recovery of the phosphoarginine pool differs between scallop species. Full recovery requires aerobic metabolism (Grieshaber 1978, Thompson et al. 1980, Bailey et al. 2003), as its time course follows the slow recovery of oxygenation of adductor sinus hemolymph (Grieshaber 1978). Complete recovery of phosphoarginine took 12 to 24 h in P. magellanicus (Livingstone et al. 1981, H. M. Pérez-Cortés personal communication), 2 h in Aequipecten opercularis (Grieshaber 1978), and 50% recuperation required 3–5 h in A. colbecki, A. opercularis and P. maximus (Bailey et al. 2003). Although A. opercularis uses its energy stores more quickly, its rate of phosphoarginine regeneration is similar to that of P. maximus and A. colbecki (Bailey et al. 2003). Interspecific variation in recovery time should reflect interspecific differences in initial phosphoarginine levels, the extent of phosphoarginine depletion, muscle aerobic capacities and probably environmental temperature. Phosphoarginine was present in C. gigantea although at low levels. Juveniles of C. gigantea are mobile but cement to rocky surfaces after reaching 30 mm (Yonge 1951, Lauzier and Bourne 2006). At that point, C. gigantea may reduce its phosphoarginine pool from levels typical of swimming scallops and only maintain what is necessary for occasional phasic contractions to clean the mantle cavity. The regulation of interspecific differences in muscle phosphoarginine contents merits more study.

Interspecific differences in enzyme activities and patterns of muscle use The available data for the metabolic capacities of scallop phasic adductor muscle (summarized in Table 3.2) show that overall, the scallop phasic adductor muscle has high activities of AK, intermediate levels of ODH and low levels of PFK and CS (Table 3.2). Low levels of ADH and SDH occur in some scallops (Wongso et al. 1999), but do not exceed 20% of ODH activities. In keeping with its role as an enzyme setting the rates of

anaerobic glycolysis, PFK levels were consistently much lower than those of AK or ODH. On the other hand, oyster muscle had a distinct metabolic profile, replacing ODH with ADH and SDH and possessing higher PFK activities (Table 3.2). The high protein content and low water content (Table 3.1) in the oyster adductor muscle only partly explain these high glycolytic capacities. These metabolic differences likely reflect both the acquisition of the swimming condition in scallops and the capacity of oysters to exploit oxygen-limiting habitats. Our data showing the use of muscle during escape responses (Fig. 3.1) allowed us to examine whether fine-scale metabolic differences among scallops reflect patterns of muscle use.

The activity of AK was highest in scallop species making rapid initial bursts of phasic contractions and fatiguing rapidly. Phasic contraction rate during the first series was highest in P. fumatus followed by M. asperrima and finally by A. balloti and P. magellanicus (Fig. 3.1). Time to fatigue followed the opposite pattern (Fig. 3.1). The scallop P. fumatus had high AK activity, high phosphoarginine contents and fatigued quickly. The scallop M. asperrima also fatigued quickly, but had low phosphoarginine contents. On the other hand, A. balloti and P. magellanicus had high phosphoarginine contents, but much lower AK activity and a longer time to fatigue than P. fumatus. Together AK activity and phosphoarginine contents should predict scallop time to fatigue, if the energetic cost of phasic contractions is similar. In the cemented scallop, C. gigantea, these relationships cannot apply, as phasic contractions were rare.

As expected, glycolytic enzyme activities were high in scallops that keep their valves closed for prolonged periods. In keeping with their reliance on short tonic contractions, A. balloti and P. magellanicus had low activities of glycolytic enzymes, whereas the scallop species (P. fumatus, M. asperrima, C. gigantea) that make fewer, but longer, tonic contractions (total number of tonic and tonic duration, Fig. 3.1) had higher activities. The metabolic capacities of C. gigantea differed from those of the oyster even though both are cemented as adults. The anaerobic capacities of C. gigantea were similar to those of scallop species that made prolonged tonics (P. fumatus and M. asperrima).

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Table 3.2. Phasic adductor muscle enzyme activities in different scallop and oyster species.

AK ODH PFK CS ADH SDH T Shell height References Species (U∙g-1) (U∙g-1) (U∙g-1) (U∙g-1) (U∙g-1) (U∙g-1) (°C) (mm) Amusium balloti 1245±74 27.8±1.7 1.77±0.10 0.51±0.05 - - 18 97.0±0.8 This study Placopecten magellanicus 953±53 29.9±3.1 1.92±0.17 1.56±0.07 - 18 90.6±0.7 This study P. magellanicus - - 2.93±0.37 1.93±0.53 - - 15 550-800g a Stewart et al. 1992 P. magellanicusb 414±27 40.5±1.6 0.92±0.03 2.40±0.05 - - 12.5 35-45 Lafrance et al. 2003 P. magellanicus c 372±19 29.9±1.2 0.73±0.04 2.43±0.07 - - 12.5 35-45 Lafrance et al. 2003 P. magellanicus 200±7 31.1±0.8 2.30±0.07 - - - 25 100-140 deZwaan et al. 1980 Equichlamys bifrons 1494±61 70.7±4.3 3.80±0.16 0.31±0.03 - - 18 100.1±2.3 This study Aequipecten opercularis - - - 1.58±0.11 - - - 68.0±1.9 Philipp et al. 2008 A. opercularis - 27.0 - - - - 25 40-50 Grieshaber 1978 Argopecten purpuratus - 300 - 0.48 - - 16 70-90 Martinez et al. 2000 A.irradians concentricus 317±14 97.6±15.6 2.9±0.6 - - - 25 - Chih & Ellington 1986 Mizuhopecten yessoensis - 50.8±3.0 12.37±2.54 - 6.10±0.08 6.23±0.94 25 260±20gd Wongso et al. 1999 Pecten fumatus 1873±247 57.7±5.6 4.12±0.34 0.33±0.04 - - 18 96.4±1.6 This study P. fumatus - 58.4±2.6 6.0±2.2 0.5±0.2 - - 25 - Baldwin & Opie 1978 P. maximus 930 29.5 9.4 - - - 25 - Zammit & Newsholme 1976 P. jacobaeus - 52.0 - - - - 25 - Gäde 1979 P. albicans - 64.1±3.4 9.03±0.96 - 7.21±0.65 6.75±0.52 25 80±5gd Wongso et al. 1999 Euvola ziczac e 4500 175 4 1.35 - - 21 70-80 Brokordt et al. 2000b E. ziczac - 21.8±7.9f - - - - 25 73.7±4.6 Alfonsi et al. 1995 Mimachlamys asperrima 1641±67 34.5±4.3 2.89±0.19 0.57±0.04 - - 18 90.9±1.1 This study M. crassicostata - 30.1±2.3 15.99±2.29 - 6.29±0.74 6.76±0.50 25 100±10gd Wongso et al. 1999 M. varia 900 30.2 3.2 - - - 25 - Zammit & Newsholme 1976 Chlamys islandica 842 7.4 - 2.8 - - 27 24-36 Tremblay et al. 2006 C. islandica g 400 14 0.9 1.05 - - 6 80-95 Brokordt et al. 2000a Crassadoma gigantea 1155±54 53.2±2.2 4.08±0.18 0.70±0.04 - - 18 95.3±3.8 This study Crassostrea gigas 1100±69 - 7.64±0.43 0.92±0.08 61.8±5.2 66.6±5.9 18 111.9±3.3 This study C. gigash - 2.0 - 0.77 21 39.5 20 60.2±16.3 Dunfy et al. 2006 C. gigas - - - - 40.5 - 25 - Fields & Hochachka 1981 C. virginica - <0.20 1.12±0.20 - - - 25 - deZwaan et al. 1980 Ostrea edulis 140 <0.1 2.8 - - - 25 - Zammit & Newsholme 1976 O. chilensish - 0.2 - 0.28 3.5 4.2 20 49.9±8.8 Dunfy et al. 2006 Note: Enzyme activities (U∙g-1 phasic muscle wet mass). Mean±S.E. a Shell mass (g), b Cultured scallops, c Wild scallops, d Animal total wet mass including shell (g), eMature 1st reproduction, f U/g muscle dry mass, g Mature, hWild at 15ºC, i Summer.

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Citrate synthase activity differed among the scallops examined, with P. magellanicus having the highest activity. As CS activity decreases with size in P. magellanicus (between 30 to 100 mm shell height); (Labrecque and Guderley 2011), the CS activity of P. magellanicus would decline with further growth. Citrate synthase activity is also high in scallop species from a range of thermal habitats, including A. opercularis, Euvola ziczac and C. islandica (Table 3.2). Thus, the cold habitat of P. magellanicus cannot explain the high CS activity. As an indicator of mitochondrial abundance, CS activity should reflect the scallop capacity to replenish phosphoarginine and glycogen levels in the phasic adductor muscle. The time for the recovery of phosphoarginine differs among scallop species (Grieshaber 1978, Livingstone et al. 1981, Bailey et al. 2003), but its correlation with the aerobic capacities of scallop adductor muscle remains to be demonstrated.

For E. bifrons, only visual observations of escape response behaviour were available. A high number of phasic contractions and an extended time to fatigue (191.7 ± 19.0 s) are the hallmarks of a strong swimmer, as predicted by shell morphology, but not by in situ observations (Olsen 1955). Virtually all of the muscle metabolic characteristics of E. bifrons were similar to those of the strong swimmer, P. fumatus. Visual observations may enhance estimates of performance, as attached P. magellanicus perform approximately 10 % fewer phasic contractions than scallops that are free to swim (X. Janssoone and H. Guderley personal observations). With these caveats, we conclude that the strong escape response of E. bifrons was appropriately accompanied by high levels of phosphoarginine, AK, PFK and ODH.

Interspecific difference of myosin ATPase activity and patterns of muscle use Given its central role in muscle contraction, myosin ATPase activity was expected to reflect the rapidity of muscle contraction. Contrary to our prediction, P. fumatus did not have higher myosin ATPase activity than other species. One way for P. fumatus to accelerate fibre shortening, without changing myosin ATPase activity per mg protein, is to increase muscle fibre length. The convex lower valve of P. fumatus might provide space to

101 increase muscle length. On the other hand, myosin ATPase activity was higher in swimming scallops than in the non-swimming C. gigantea and the oyster C. gigas (Fig. 3.4). We suspect that the activity of the myosin ATPase in C. gigantea decreases after the animals cement. Myosin ATPase activity was much higher in P. magellanicus than in the other species studied. Myofibrillar ATPase activity increases with cold acclimation in many fish species (Johnston and Temple 2002) therefore the high levels in P. magellanicus might be linked to the species cold habitat.

Potential effects of ontogenetic change on metabolic capacities Our study of interspecific differences in the metabolic capacities of scallop adductor muscles aimed to interpret them in the context of the scallops’ swimming behaviour and life style. As biochemical properties, shell morphology and life style change during ontogeny, we compared scallops of a similar size with a shell height close to their maximal size. Nevertheless, as the species we studied reach different maximal sizes (A. balloti 110 mm, P. magellanicus approximately 150 mm, E. bifrons 110 mm, P. fumatus 145 mm, M. asperrima 110 mm, C. gigantea 250 mm and C. gigas 300 mm) [Lauzier and Bourne (Lauzier and Bourne 2006) and references therein] (see also Abbott 1974, Edgar 2001, Dredge 2006, Naidu and Robert 2006), we must consider the potential influence of ontogenic state in our interpretations.

Biochemical properties of the phasic adductor change with size. Indeed, in P. magellanicus enzymatic activities and phosphoarginine content peak at a shell height of 60 mm and decline at greater sizes (Labrecque and Guderley 2011). In A. opercularis, larger (65-75 mm shell height) individuals have lower aerobic capacities (citrate synthase activity) and anaerobic capacity, as deduced from the amount of glycogen stored in the adductor muscle, than smaller ones (40-55 mm shell height); (Philipp et al. 2008). In C. islandica, anaerobic capacity increases with increasing size while aerobic capacity and AK activity both decrease (Tremblay et al. 2006). Scallop metabolic capacities seem to peak at a certain stage and decrease with increasing size. As we chose individuals with a shell height close their maximal size, we can assume that interspecific differences in the muscle

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metabolic capacities are not the result of the species being in different portions of their allometric curves.

Biochemical attributes, behaviour and morphology Scallops respond to contact with a predator either by swimming away or by prolonged valve closure. The use of phasic contractions is energetically expensive, particularly for species with a disadvantageous shell shape. Using tonic contractions to keep the valves shut for prolonged periods is energetically cheap but requires tight junctions between the valves to prevent predation. Selection for these contrasting behaviours should favour different patterns of co-evolution of the metabolic capacity of the adductor muscle and the morphology of the animal.

The scallop Amusium balloti, P. magellanicus and E. bifrons can sustain phasic contractions for long periods although their phosphagen contents and muscle metabolic capacities are not greater than those of the other species. This may be due to the hydrodynamic shell cross-section that facilitates lift and gliding in the water. On the other hand, the shell shape of P. fumatus suggests that it is a poor swimmer. Nonetheless, a high phosphoarginine content, high AK activity in the phasic muscle, paired with an intense use of phasic contractions, allow P. fumatus to rise in the water column and swim considerable distances. The shell shape of M. asperrima is not hydrodynamic and the tight junctions between the valves compromise the ability to eject water and swim, but may hinder predator access. On the other hand, byssally attached M. asperrima can detach from the substrate and make short bursts of phasic contractions using their limited phosphoarginine pool when necessary. Muscle metabolic capacities are one of a series of integrated traits that have evolved with behaviour, muscle anatomy and shell morphology to establish scallop escape response performance. These multifaceted relationships condition the relationships between muscle metabolic capacities and muscle use.

Biochemical attributes of the adductor muscle in adult cemented C. gigantea lay somewhere between scallops and oysters. The capacities of C. gigantea muscle for

103 anaerobic glycolysis were similar to those of scallops performing long tonic contractions, while phosphoarginine content was much lower than in other scallop species and myosin ATPase activity was similar to that observed in the oyster. These physiological characteristics of adult C. gigantea, coupled with the dramatic change in shell morphology, suggest that after cementing to the substrate its metabolism reverts to the ancestral condition of non-swimming monomyarian bivalves where closed shells were used to protect from predation and phasic contractions simply cleaned the mantle cavity.

While sharing an underlying functional design, the scallop escape response varies considerably within and among species. Morphological parameters such as shell shape and mass can facilitate or hinder scallop swimming. Patterns of muscle use can overcome the constraints imposed by shell shape and mass in scallops (Tremblay et al. 2012). Our study showed that the biochemical attributes of the phasic adductor muscle reflect patterns of muscle use during escape responses, as well as the scallop life habits. Byssally attached species (M. asperrima) with their short sporadic bursts of phasic contractions differ in their metabolic profile from gliding species (A. balloti and P. magellanicus) with their sustained phasic contractions; both in turn differ from the cementing species (C. gigantea). Muscle use seems a major determinant of muscle metabolic capacities. Other characteristics of the adductor muscle, such as the proportion and insertion of the phasic and tonic muscle could influence muscle performance. Even if scallops have few components in their locomotor system, there are multifaceted interactions among them. Understanding shell morphology, muscle use, muscle metabolic capacities, muscle anatomy and ligament characteristics in scallops with markedly different swimming behaviours should reveal their interactions and co-evolution, thereby demonstrating the links between form and function.

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Acknowledgements

This research was supported by funds from the RAQ (Ressources Aquatiques Québec) and NSERC to HEG. IT was recipient of a scholarship from FQRNT and RAQ. The authors are extremely grateful to the staff of the different institutes where this research was conducted. In Queensland at the BIARC: Paul Palmer, Tim Lucas, Satoshi Mikami and Sizhong (Joe) Wang. At the University of the Sunshine Coast: Peter F. Duncan. In Tasmania at TAFI: Julian Harrington, Craig Mundy and all the technical staff. In Québec at the MAPAQ in Îles-de-la-Madeleine: Bruno Myrand, Madeleine Nadeau and all the technical staff. Mélanie Bourgeois from Culti-mer in Îles-de-la-Madeleine. Finally in British Columbia at the Centre for Shellfish Research: Brian Kingzett and the technical staff. The assistance of Marie-Josée Martineau during the chromatographic determination of phosphoarginine levels was highly appreciated.

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Appendix 3.1. Regressions between shell height and phasic adductor mass of the scallops in the behavioural studies used to estimate the phasic adductor muscle mass of the scallops of the biochemical assays.

Species Regression equation R2 Amusium balloti y = 0.4671x – 37.433 0.7835 Placopecten magellanicus y = 0.4106x – 25.011 0.4455 Equichlamys bifrons y = 0.4823x – 30.292 0.8558 Pecten fumatus y = 0.3808x – 24.326 0.4643 Mimachlamys asperrima y = 3E-06x3.3535 0.9283 Crassadoma gigantea y = 0.0005x2.2717 0.7687 Crassostrea gigas y = 0.0023x1.3755 0.3482

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CHAPITRE 4

When behaviour and mechanics meet: Scallop swimming capacities and their hinge ligament

ISABELLE TREMBLAY, MYRIAM SAMSON-DÔ et HELGA E. GUDERLEY

Soumis dans « The Canadian Journal of Zoology 2014»

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Résumé Les pétoncles nagent à l’aide de jets expulsant l’eau contenue entre les valves lors de la contraction rapide du muscle adducteur. L’ouverture des valves, quant à elle, se fait grâce à un ligament qui agit tel un mécanisme à ressort. La résilience du ligament est plus élevée chez les pétoncles comparativement aux bivalves qui s’enfouissent ou qui sont sessiles. La résilience du ligament, la force déployée par le ligament pour ouvrir les valves ainsi que la force déployée par le muscle adducteur phasique et tonique ont été examinées chez six espèces de pétoncles (Amusium balloti, Placopecten magellanicus, Equichlamys bifrons, Pecten fumatus, Mimachlamys asperrima et Crassadoma gigantea) ayant des réponses de fuite et une morphologie de la coquille différentes. La force déployée par le ligament pour ouvrir les valves varie entre les espèces et est toujours égale ou inférieure à la force déployée par le muscle phasique et tonique. L’espèce de pétoncle ayant le taux de contraction phasique le plus élevé (P. fumatus) possède un ligament avec une plus grande résilience.

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Abstract Scallops swim using jet propulsion produced by expulsion of water from between the valves by rapid contraction of the adductor muscle. The valves are subsequently opened by a ligament that acts like a spring mechanism. The resilience of the ligament is higher in scallops than in burrowing or sessile bivalves. The ligament resilience, ligament opening force, and force deployed by the phasic and tonic adductor muscles were examined in six scallop species (Amusium balloti, Placopecten magellanicus, Equichlamys bifrons, Pecten fumatus, Mimachlamys asperrima and Crassadoma gigantea) with different escape response strategies and shell morphologies. The ligament opening force varied between species and was always equalled or exceeded by phasic and tonic closing forces. Scallop species that reached the highest frequencies of phasic contractions (P. fumatus) had the highest ligament resilience.

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Introduction The form and mechanical properties of the ligament vary among bivalves according to their lifestyle (Trueman 1953a, Kahler et al. 1976). The adductor muscle of bivalves acts against an elastic ligament rather than another muscle. When the adductor muscle contracts, the valves close and energy is stored in the ligament. When the muscle relaxes, the energy stored in the ligament is released opening the valves. Scallops are thought to have acquired the ability to swim by modifying their shell, mantle and adductor muscle, and by specializing their hinge ligament for efficient energy storage (Yonge 1936, Trueman 1953a, Trueman 1953b, DeMont 1990, Wilkens 2006).

The scallop ligament is situated dorsally between the valves, in front of and behind the umbo. The outer laminated layer connects the two valves at the dorsal margin and acts as a hinge (Fig. 4.1); (Trueman 1953b). The inner layer has a large non-calcified centre and two lateral calcified regions attaching the ligament to the valves (Trueman 1953b). Abductin composes the centre of the inner layer and acts as a compression spring having properties similar to those of elastin and resilin (Alexander 1966, Kelly and Rice 1967).

Inner layer

(cm)

umbo

Outer layer

Figure 4.1. Interior of the dorsal region of the left valve of Amusium balloti showing the ligament cut in longitudinal section. Scale is in cm.

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Scallops swim using water jets produced by successive contractions during which rapid closures alternate with valve openings (Drew 1906, Dakin 1909, Bruddenbrock 1911). The cycles of closing and opening require contraction and relaxation of the adductor muscle, as well as opening of the valves by the ligament. The striated phasic adductor muscle carries out rapid valve closures (Lowy 1953, Millman 1967) while the smooth tonic adductor muscle contracts slowly, maintaining constant valve positions (Lowy 1953, Chantler 2006). Extensive modelling by Cheng et al. (1996) shows that most of the mechanical energy produced by phasic muscle contraction is used to produce the jet that moves the scallop (Cheng et al. 1996). The ligament acts as a spring applying a force that pushes the valves apart. This force must counteract the inertia of the valves as well as that of the added mass of the water displaced by the moving shell. Flow-induced forces also help reopen the valves, but their role is small compared to that of the ligament (Vogel 1985, Cheng and DeMont 1996). The mechanical properties of scallop ligaments differ from those of ligaments from other bivalves (Trueman 1953a). The ligaments of Pecten maximus (Linnaeus 1758) and Chlamys opercularis (Linnaeus 1758) are more resilient and have a lower opening moment per gram of shell than those of non-swimming bivalves such as Cyprina islandica, Mytilus edulis, Mya arenaria, Spisula solidissima, Mercenaria mercenaria, Ensis directus, and Crassostrea virginica (Trueman 1953a, Kahler et al. 1976). Scallops exhibit a wide range of lifestyles, ranging from the highly active Amusium species to the more sedentary and byssally attached Chlamys species and differ in their use of phasic and tonic muscles during escape responses (Tremblay et al. 2012). We reasoned that ligament properties, in particular resilience, could reflect the escape response strategies of scallop. Specifically, we proposed that the ligament of scallop species that show a vigorous and intense escape response, as reflected by high rates of phasic contractions, should have higher resilience than those of species with a weaker escape response. Next, we reasoned that the force deployed by the ligament when opening the valves should vary with shell mass and not escape response strategy. Finally, although the force deployed by the phasic and tonic adductor muscles should be greater than that deployed by the ligament, scallops performing a vigorous escape response should have a greater divergence between the phasic closing force and the ligament opening force than more sedentary species. We reasoned that the

111 tonic closing force would remain similar to the ligament opening force, regardless of escape response strategy. To examine these predictions, the ligament resilience and opening force, as well as the force deployed by the phasic and tonic adductor muscle, were examined in scallop species with distinct escape responses and shell morphologies. The ligament opening force was assessed by measuring the mechanical force required to close the valves and measured phasic and tonic force production during escape responses (Tremblay et al. 2012). The scallop Amusium balloti (Bernardi 1861) and Placopecten magellanicus (Gmelin 1791) both perform phasic contractions throughout the escape response. The scallop Amusium balloti makes phasic contractions at a quicker pace than P. magellanicus, as shown by the short interval between phasic contractions and the high phasic contraction rate (Table 4.1). The scallop Pecten fumatus (Reeve 1852) performs intense bursts of phasic contractions at the beginning of its escape response (Table 4.1). The scallop Mimachlamys asperrima (Lamark 1819) makes short series of phasic contractions at the beginning of the response, with a slower overall phasic contraction rate (Table 4.1). Adult Equichlamys bifrons (Lamark 1819) are more sedentary than M. asperrima and P. fumatus (Olsen, 1955). Adult Crassadoma gigantea (Gray 1825) are cemented to the substrate (Yonge 1951) and only rarely make phasic contractions in response to predators (Tremblay et al. 2012).

Material and methods

Experimental scallops: shell and behavioural characteristics Shell characteristics and behavioural parameters of the experimental scallops are described in Tremblay et al. (2012) and summarized in Table 4.1. Mature Amusium balloti were collected near Gladstone (Australia). Mature Placopecten magellanicus were obtained from Culti-mer (Îles-de-la-Madeleine, Canada). Mature Pecten fumatus, E. bifrons and M. asperrima were collected by SCUBA in Channel d’Entrecastreux (Australia). Mature Crassadoma gigantea were collected near Espinosa Inlet (Vancouver Island, Canada). Holding conditions are described in Tremblay et al. (2012).

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Table 4.1. Shell and adductor muscle characteristics of the experimental scallops and parameters related to muscle contractile activity during escape responses.

Amusium Placopecten Equichlamys Pecten Mimachlamys Crassadoma balloti magellanicus bifrons fumatus asperrima gigantea Shell Height (mm) 96.3±1.1a 90.4±0.9b,c 96.2±2.5a 94.4±1.7a,c 84.2±2.0b 97.0±4.5a,c Mass (g) 34.6±1.6a 49.4±1.7b 61.6±3.6c 53.9±2.5b,c 27.3±2.0d 162.2±15.5e N 27 15 20 15 16 19 Dry mass at 90 mm (g) Phasic muscle 0.86±0.05a 3.08±0.13b 3.08±0.10b 2.05±0.16c 1.92±0.09c 3.60±0.24b Tonic muscle 0.05±0.004a 0.25±0.01b 0.51±0.01c 0.23±0.01b 0.17±0.01c 0.29±0.02d N 18 15 20 15 16 19 Phasic contractions Minimal interval between two phasics (s) 0.38±0.04a,c 1.12±0.14b - 0.32±0.04a 0.65±0.12c - Number during 1st series 9.4±1.3a 5.9±1.0b - 22.4±3.4c 3.0±0.4d 0.4±0.1e Rate during first 30 s (phasic s-1) 0.51±0.02a 0.38±0.03b - 0.45±0.11b 0.16±0.03c 0.01±0.01d Overall rate (phasic s-1) 0.12±0.004a 0.08±0.004b 0.28±0.02* 0.26±0.09a 0.05±0.01c 0.004±0.002d N 30 15 20 15 14-16 19 Data are means ± s.e.m. In a given row, different letters indicate significant differences (Kruskall-Wallis followed by multiple comparisons, P<0.05). *Overall rate was calculated relative to the time to fatigue for E. bifrons and relative to test duration of 355 s for other species (see Tremblay et al. 2012 for details). Note: Minimal interval between two consecutive phasic contractions. Number of phasics during the 1st series: We defined a series of phasic contractions as consecutive phasic contractions separated by <3 s. Contraction rate during the first 30 s of the escape response test.

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Animals were held at the temperature and salinity at which they were sampled for a maximum of two weeks before measurement of force production and ligament properties.

Muscle force measurements The force produced during escape responses was measured with a force gauge (AFG-50 N, Quantrol Advanced Force Gauge, Dillon, Fairmont, MN, USA) placed at the ventral edge of the upper shell giving a force/time curve for each individual (Tremblay et al. 2012). During phasic contractions, the upper shell edge hits the transducer while the lower shell is fixed. The impact of the edge of the upper shell on the transducer is influenced by inertial forces that cannot be estimated without measurements of the velocity of the shell mouvement. As these were not available, we could not estimate the force deployed by the phasic adductor muscle. Nonetheless, the impact of the upper shell on the transducer represents the force deployed by the moving valve, which ultimately produces jet propulsion. This measure was considered the best estimate of force produced by the phasic muscle for swimming and designated it as the phasic closing force. The force of all phasic contractions for each individual was averaged to assess the mean phasic closing force. This was 55-78% of maximal phasic force for the different species (Table 4.2). The sustained contraction of tonic muscle facilitates determination of its force production. The mean tonic force was estimated by dividing the area under the force/time curve by recording duration. This was feasible as phasic contractions represent a minimal proportion of the recordings. Mean tonic force was 38-43% of maximal tonic force. When the valves are prevented from moving, the force measured at the edge of the shell is lower than that measured near the muscle (Pérez et al. 2009b). Indeed, when the force measured was 7.55 N at the shell edge, it was 13.95 N near the muscle for a scallop with its muscle situated 4.5 cm from the edge (Pérez et al. 2009b). As our force measurements were made at the ventral edge of the shell and as the distance between the tonic muscle and the shell edge was approximately 4.5 cm in our species, we used the relationship from Pérez et al. (2009b) to estimate tonic force production in our species.

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Table 4.2. Forces measured in experimental scallops.

Amusium Placopecten Equichlamys Pecten Mimachlamys Crassadoma balloti magellanicus bifrons fumatus asperrima gigantea Ligament opening force (N) 4.2±0.1a 3.5±0.3b 4.6±0.3a,c 5.3±0.3c 2.8±0.2d 5.1±0.7a,c Phasic closing force (N) Mean force 10.10±0.25a 15.31±0.96b -* 8.16±0.90c 7.57±0.55c 14.21±3.42a,b Maximal force 14.57±0.24a 22.40±0.97b -* 14.55±1.01a 11.45±0.62c 17.09±4.22a Tonic closing force (N) Mean force 4.11±0.24a 8.01±0.91b,c -* 6.39±0.97c 6.83±0.70b,c 9.46±1.04b N 30 15 18 15 16 7-16 Data are mean ± S.E. In a given row, different letters indicate significant differences (Kruskall-Wallis followed by multiple comparisons, P<0.05). *As E. bifrons behaviour was visually assessed, we could not assess phasic or tonic closing forces.

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Experimental setup and measurement of ligament resilience Following behavioural tests, all tissues were removed from the valves without touching the ligament and ligament resilience was measured immediately. Emptied valves were placed on a base with an adjacent ruler as reference (Fig. 4.2). The force gauge, equipped with a disc shaped tip, was used to apply force at the centre of the phasic adductor muscle attachment area on the upper valve. The force gauge was attached to a manual stand, which allowed fine control of force application while closing the valves and while releasing the force to allow valves opening. The valves were closed and opened in steps to allow photographs of the experimental setup. The photographs were taken regularly with a digital camera during the closing and opening cycle giving approximately 30 photographs per individual. The time lapse between consecutive photographs did not exceed 5 s. In Placopecten magellanicus, the ligament resilience measured in two consecutive closing/opening cycles did not differ (Kruskall-Wallis test, P=0.98). Therefore, only one closing and opening cycle for each individual was measured. Each photograph (Fig. 4.2) was analysed using image analysis (ImageJ, ver. 1.42, National Institutes of Health) to determine the force applied and the distance between the valves during closing and opening. From these photographs, data points from the loading (valves closing) and unloading (valves opening) cycle were graphed and joined by straight lines (SigmaPlot 11.0), giving a hysteresis loop for each individual (Fig. 4.3). Using image analysis, the areas under the loading and unloading curves were measured and used to calculate ligament resilience (area under unloading curve / area under loading curve). The force required to mechanically close the valves was taken as a proxy for the ligament opening force.

Statistical analysis Normality was tested using Shapiro-Wilk test and homogeneity of variances was analyzed visually by plotting residuals relative to predicted values. Due to non-normality of residuals and non-homogeneity of variances, non-parametric statistics were used. Interspecific comparisons of ligament resilience were made using Kruskall-Wallis tests and multiple comparisons. Spearman’s correlations were used to assess whether the

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Force gauge

Tip of the force gauge

Scallop emptied shell on a base

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Amusium balloti Placopecten magellanicus 3.5 4 Loading curve 3.0 Unloading curve 3 2.5

2.0 2 Force (N) 1.5

1.0 1 0.5

0.0 0 0.0 0.5 1.0 1.5 2.0 2.5 3.0 0.0 0.5 1.0 1.5 2.0 2.5 3.0

Pecten fumatus 6 Equichlamys bifrons 6

5 5

4 4

3 3

Force (N) 2 2

1 1

0 0 0 1 2 3 4 5 6 0 1 2 3 4 5 6

Mimachlamys asperrima Crassadoma gigantea 4 10

8 3

6 2 4

Force (N)

1 2

0 0 0 1 2 3 4 5 0.0 0.5 1.0 1.5 2.0 2.5 Distance between the two valvesDistance (cm) between the two valves (cm)

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ligament opening force was correlated with shell mass and to assess whether ligament resilience was correlated with phasic contractile activity during escape responses. Spearman’s correlations were also used to assess whether the mean phasic force was correlated with shell mass and phasic muscle mass and to assess whether the mean tonic force was correlated with shell mass. These correlations were examined inter- and intra- specifically. Since the behaviour of E. bifrons was visually assessed, only the phasic contraction rate relative to time to fatigue could be estimated. Therefore, data for E. bifrons were not included in the interspecific correlations. All analyses were done using SAS 9.2 (SAS Institute). Significance was accepted at P<0.05.

Results

Shell characteristics Shell heights of the experimental scallop species overlapped, although M. asperrima (84.2±2.0 mm) was slightly smaller than the other species (Table 4.1). The scallop Crassadoma gigantea had the heaviest shell (162±16 g) and A. balloti and M. asperrima the lightest (35 and 27 g respectively) (Table 4.1). The biggest phasic muscle was found in Crassadoma gigantea while A. balloti had the smallest (Table 4.1). The scallop Amusium balloti had the smallest tonic muscle while P. fumatus had the largest (Table 4.1).

Hysteresis loops and muscle force production The ligament opening force showed considerable interspecific variation (Table 4.2 and Fig. 4.3), and tended to be correlated with shell mass (correlation coefficient=0.77, P=0.07, N=6). The scallop Mimachlamys asperrima and P. magellanicus had the lowest values with 2.8 and 3.5 N, A. balloti and E. bifrons had intermediate levels at 4.2 and 4.6 N while C. gigantea and P. fumatus had the highest forces at 5.1 and 5.3 N (Table 4.2). For all species, mean phasic closing force markedly exceeded the ligament opening force (from 1.5 to 5 times); (Table 4.2). Mean phasic closing force was higher in species that rely upon phasic contractions throughout their escape responses (A. balloti and P. magellanicus) as

119 well as in C. gigantea (Table 4.2). Mean phasic force was not correlated with shell or phasic muscle mass (correlation coefficient=0.50 and 0.60, P=0.39 and 0.28 respectively, N=5). Mean tonic closing force also exceeded the ligament opening force, but to a lesser degree than mean phasic force (Table 4.2). Mean tonic closing force was not correlated with shell mass (correlation coefficient=0.50, P=0.39, N=5) and was markedly lower in A. balloti than in the other species (Table 4.2).

Ligament resilience was highest in P. fumatus at 0.91±0.01 followed by A. balloti which had a slightly lower resilience (Fig. 4.4). The scallop Placopecten magellanicus and C. gigantea had similar ligament resilience, lower than that of A. balloti (Fig. 4.4). Finally, Equichlamys bifrons and M. asperrima had the lowest ligament resilience with 0.70-0.76 (Fig. 4.4).

When comparing mean values per species, ligament resilience was not correlated with parameters describing rates of phasic contraction during escape responses (Table 4.3); when the pattern produced by data points for all individuals was examined, individuals showing higher rates of phasic contraction (as shown by the number of phasic contractions during the first series and phasic contraction rate during the first 30 s of the escape response) showed significantly higher ligament resilience (Fig. 4.5a,b). There was a significant correlation between ligament resilience and phasic contraction rate during the first 30 s of the escape response (correlation coefficient=0.65, P=0.009, N=15) in Placopecten magellanicus.

Discussion The extensive morphological variation among scallop species is reflected in a wide range of escape response strategies (Tremblay et al. 2012) and metabolic attributes (Tremblay & Guderley 2014). It was shown here that the properties of the hinge ligament also differ between scallop species with distinct escape response behaviours. Ligament

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1.0 a d b b c 0.8 c

0.6

Resilience 0.4

0.2

0.0

A. balloti E. bifrons P. fumatus C. gigantea M. asperrima P. magellanicus

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Table 4.3. Correlations between ligament resilience and phasic contractile activity during escape responses by the experimental scallop species.

Minimal interval Nb of phasics in Contraction rate Contraction rate

between two phasics the 1st series during the first 30 s during 355 s Corr. coefficent -0.80 0.70 -0.20 0.70 Prob > |r| 0.20 0.19 0.75 0.19 N 4 5 5 5 *Spearman’s correlations were done on the mean for each species and used data from all the species with the exception of the minimal interval between two phasic contractions which could not be estimated for C. gigantea.

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A) 1.0

0.9

0.8 A. balloti M. asperrima P. fumatus Resilience 0.7 P. magellanicus

0.6

0.5 0 10 20 30 40 50

Number of phasic contractions during the 1st series (number of phasic) 1.0 B)

0.9

0.8 A. balloti M. asperrima P. fumatus P. magellanicus Resilience 0.7

0.6

0.5 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4

Phasic contraction rate during the first 30 s (phasic s-1)

Figure 4.5. The ligament resilience plotted against A) the number of phasic contractions during the first series (y=0.76+0.011x-0.0002x2, R2=0.27 and B) the phasic contraction rate during the first 30 s of the escape response for each species (y=0.75+0.23x-0.06x2, R2=0.25). Sample size is A. balloti N=30, P. magellanicus N=15, P. fumatus N=15, M. asperrima N=16.

123 hysteresis loops differed in terms of ligament opening force and in terms of resilience. As predicted, species with heavier shells tended to have greater ligament opening forces. Phasic closing force always exceeded ligament opening force and, as predicted, was highest in species relying on phasic contractions throughout the escape response. Mean tonic force exceeded ligament opening force to a lesser degree. Ligament opening force was not paralleled by phasic or tonic closing forces (correlation coefficient=0.10 and -0.10 respectively, P=0.87 for both, N=5). As the ligament opening force was measured in the air and phasic and tonic forces were measured in the water, it is difficult to quantify their respective roles during scallop swimming. Clearly, complex forces are involved in cycles of valve closing and opening in swimming scallops (Cheng and DeMont 1996) and our measurements were not made on free-swimming scallops. That being said, our study did demonstrate considerable variation in ligament resilience and opening force amongst scallop species.

Ligament resilience varied among species with various escape response strategies. As hypothesized, more active scallops tended to have a more resilient ligament with species that reach the highest frequencies of phasic contraction (P. fumatus and A. balloti) having the highest resilience. On the other hand, ligament resilience in Crassadoma gigantea was similar to that in Placopecten magellanicus and higher than that in Mimachlamys asperrima. As adults, the latter species perform phasic contractions at much higher rates than C. gigantea. During early life Crassadoma gigantea is free-living (<30 mm shell height) but then cements its lower valve to rocky surfaces (Yonge 1951, Lauzier and Bourne 2006). While new material is layered on the ligament as the animal grows, older material remains. The ligament of C. gigantea probably retains characteristics from its juvenile period when it manifests swimming escape responses. The resilience of the ligament measured in our scallop species is similar to that observed in other studies. Alexander (1966) estimated the ligament resilience in Pecten maximus and Chlamys opercularis to be ≥ 90% as estimated from graphs in Trueman (1953a). In his own experiments, Alexander (1966) measured a mean ligament resilience of 91% in C. opercularis. The resilience of the ligament was found to be 79% in P. magellanicus at 10ºC and at physiological swimming frequency (Bowie et al. 1993). In the

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studies noted previously (Trueman 1953a, Alexander 1966, Bowie et al. 1993), the resilience of the ligament was measured in the air and the ligament was maintained humid. It was shown that there is practically no variation in the unloading and loading cycles when repeated over relatively short period of time (Trueman 1953a). The ligament resilience in P. magellanicus measured at a physiological frequency of 3 Hz (Bowie et al. 1993) is similar to the resilience of the ligament measured in the same species in our study.

Ligament resilience is influenced by its arrangement in the shell and its biochemical composition (Trueman 1953b, Kahler et al. 1976) as well as by environmental temperature (Denny and Miller 2006). Our scallops came from, and were measured in, similar thermal conditions (12–18ºC); thus temperature is unlikely to have caused interspecific differences in ligament resilience. Although differences in ligament efficiency between non-swimming bivalves and scallops partly reflect the arrangement of the ligament in the shell (Trueman 1953b, Kahler et al. 1976), ligament anatomy is relatively constant among scallops (Trueman 1953b, Kahler et al. 1976). That being said, migration of the hinge line, and thus of the ligament, to a more ventral position occurs in C. gigantea when it cements in areas where spatial restriction prevents the growing valves from opening (Yonge 1951). Nevertheless, the general arrangement of C. gigantea ligament remains similar to that of other scallop species. Changes in ligament arrangement unlikely explain differences in ligament resilience among scallops.

The biochemical composition of the inner ligament differs between non-swimming bivalves and scallops (Kahler et al. 1976). The molluscan inner ligament protein contains a high percentage of glycine (Kahler et al. 1976). When compared to non-swimming bivalves, scallop abductin contains the most glycine and the least proline and cysteine (Kahler et al. 1976). The resilience of the bivalve ligament was inversely correlated with

CaCO3 and cysteine concentrations and directly correlated with glycine levels making the scallop ligament particularly well adapted for swimming (Kahler et al. 1976). While the differences in the biochemical composition of the ligament among bivalves are clear, differences among scallop species are less apparent (Denny and Miller 2006). Variation in

125 the biochemical composition of ligaments from different scallop species may underlie interspecific differences in resilience.

To close the valves, the force developed by the adductor muscle must exceed the ligament opening force. This excess was consistently greater for the phasic than the tonic muscle, although the extent of overshoot varied among species. Phasic muscle force production not only counteracts the ligament opening force, but also provides thrust to propel the scallops. As predicted, the divergence between ligament opening force and phasic closing force was greater in scallops that rely mainly on phasic contractions (i.e. A. balloti and P. magellanicus, Table 4.2). The high mean phasic closing force in C. gigantea reflected its paucity of phasic contractions that caused the mean phasic closing force to be much closer to the maximal phasic force than in the other species (Table 4.2). Tonic muscle force production maintains valve closure and resists predators trying to open the valves. The low mean tonic force in A. balloti paralleled the small size of the tonic muscle (Table 4.1) compared to other species. Valve closure via prolonged tonic contractions in A. balloti is of little use against its crustacean predators, given the large gaps between the closed valves. For example, if T. orientalis catches A. balloti, it inserts its appendages into the gaps and slices away at the flesh. Therefore, A. balloti mainly uses phasic contractions to swim away from its predators (Tremblay et al. 2012). On the other hand, scallop species, such as M. asperrima and C. gigantea, that use prolonged tonic contractions to close their valves firmly in response to predators, had a greater difference between tonic muscle force and ligament opening force (Table 4.2).

The hinge ligament is one component of a relatively simple, functionally elegant locomotor system. While we showed that ligament resilience reflects the frequency of phasic contractions in scallops, theory indicates that such changes in ligament resilience would have little impact on scallop swimming capacity. Indeed, Denny and Miller (2006) showed that even a drastic change in the resilience of the ligament would have only a small impact on the resonant period of the shell-hinge system and therefore on scallop locomotion. Nonetheless, our data and those of Denny and Miller (2006) show that scallops with different escape response strategies differ in ligament resilience. High 126

ligament resilience may facilitate extensive valve gape without modifying the resonant period of the shell hinge system. The adaptive value of modifications of ligament resilience in scallops is not clear and invites consideration of other aspects of scallop movement. Clearly, shell shape strongly influences swimming, and muscle use can partly overcome some of the attendant morphological constraints (Tremblay et al. 2012). Ligament properties could intervene in an analogous fashion. Muscle function is influenced by its metabolic capacities as well as by its size and position in the shell. Characterisation of these parameters in scallops with markedly different escape responses will help reveal links between, and co-evolution, of the underlying traits.

Acknowledgements This research was supported by funds from NSERC to H.E.G and Ressources Aquatiques Québec to H.E.G. I.T. was the recipient of a PhD scholarship from FQRNT and FONCER (NSERC). The authors are extremely grateful to the staff of the different institutes where this research was conducted. In Queensland at the BIARC this included Paul Palmer, Tim Lucas, Satoshi Mikami and Sizhong (Joe) Wang as well as Peter F. Duncan at the University of the Sunshine Coast. In Tasmania invaluable assistance was provided from Julian Harrington, Craig Mundy and the technical staff at TAFI. In Québec at the MAPAQ, Bruno Myrand, Madeleine Nadeau and the technical staff as well as Mélanie Bourgeois from Culti-mer greatly facilitated our work. Finally in British Columbia, we thank Brian Kingzett and the technical staff at the Centre for Shellfish Research. We are also grateful to Dr. Doug Syme for valuable discussions and comments on this paper.

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128

CHAPITRE 5

Can scallop swimming styles be predicted from shell and adductor muscle morphology?

ISABELLE TREMBLAY et HELGA E. GUDERLEY

Format prêt pour la soumission

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Résumé Les modifications au niveau de la structure de la coquille, du manteau et du muscle adducteur sont considérées des adaptations dérivées ayant permis aux pétoncles de nager. Ainsi, les propriétés morphologiques du muscle adducteur et de la coquille devraient être reliées à la performance de nage des pétoncles. Plusieurs charactéristiques de la coquille (masse, allongement et volume entre les valves) et du muscle adducteur (taille, position et arrangement dans le coquille) ont été mesurées chez 6 espèces de pétoncles (Amusium balloti, Placopecten magellanicus, Equichlamys bifrons, Pecten fumatus, Mimachlamys asperrima et Crassadoma gigantea) avec des réponses de fuite distinctes telles que documentées par les mesures d’utilisation du muscle adducteur pendant une réponse de fuite. Les charactéristiques morphologiques de la coquille et du muscle adducteur diffèrent de façon marquée entre les espèces, mais elles ne sont pas toujours le reflet de la stratégie de nage des pétoncles. Les analyses en composantes principales ont révélé que l’épaisseur de la coquille, la masse de la coquille et du muscle adducteur, ainsi que les attributs morphologiques apparentés, sont étroitement liés à l’endurance de la réponse de fuite. L’intensité de la réponse de fuite, est quant à elle principalement prédite par l’allongement de la coquille et l’oblicité du muscle adducteur.

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Abstract Modifications in shell structure, mantle and adductor muscle are considered to be derived adaptations that allowed scallops to swim. Thus, morphological properties of the adductor muscle and shell should relate to swimming performance in scallops. Various morphological characteristics of the shell (mass, aspect ratio and volume between the valves) and the adductor muscle (size, position and arrangement in the shell) were measured in 6 scallop species (Amusium balloti, Placopecten magellanicus, Equichlamys bifrons, Pecten fumatus, Mimachlamys asperrima, and Crassadoma gigantea) with distinct escape responses, as documented by measurements of muscle use during escape responses. Morphological characteristics of the shell and adductor muscle differed markedly between the species, but did not always follow their swimming strategies. Principal components analysis revealed that shell width, shell and muscle masses, and related morphological attributes were closely linked with swimming endurance. The intensity of the escape response was best predicted by the aspect ratio and the obliqueness of the adductor muscle.

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Introduction The ability to swim has been acquired by only a few bivalves that are almost exclusively monomyarian (i.e. only one adductor muscle) and is most predominant among the Pectinidae, or scallops. Diverse morphological, physiological and biochemical attributes underlie the wide range of locomotor styles developed by animals. Even within the generally sedentary bivalves, one finds a range of locomotor strategies accompanied by specific morphological attributes. For example, the soft-shell clam Mya arenaria uses its pedal foot and valves adductions to burrow in the substrate. In response to the contact with a predator, the cockle Cardium tuberculatum escapes via a series of jumps produced by the contractions of the foot muscle (Gäde 1980). Other bivalves, such as the file shell Lima hians can swim via two types of mouvements: 1) coordinated rowing mouvements of the pallial tentacles, and 2) rapid adductions of the valves to produce water jets that allow them to swim away or the jumping cockle (Gilmour 1967). Finally, scallops can swim with its ventral edge leading, seeming “to take a series of bites out of the water” (Dakin, 1909).

The scallop locomotor system is mainly composed of two valves, an adductor muscle and a ligament. In response to a disturbance, such as contact with a predator, scallops can swim away by expelling jets of water through lateral openings. Swimming is produced by rapid cycles of valve closures and openings (Drew 1906, Dakin 1909, Buddenbrock 1911). Contractions of the phasic adductor muscle rapidly close the valves (Lowy 1954, Millman 1967) whereas the hinge ligament (Alexander 1966, Marsh et al. 1976) acts as a spring to open the valves when the adductor muscle relaxes. The phasic adductor muscle is composed of cross-striated fibres while the smaller tonic adductor muscle is composed of slow contracting smooth fibres which allow prolonged valve closure (Lowy 1954, Chantler 2006). This mode of swimming is shared by scallops exhibiting a wide range of shell morphologies and life styles ranging from the highly active Amusium balloti to the byssally attached Mimachlamys asperrima.

Logically, the swimming ability of bivalves should be reflected in their morphological characteristics, including adductor muscle size and position and shell characteristics. Several studies have compared these properties in swimming and non- 132

swimming monomyarian bivalves, inferring swimming abilities from the literature, and have generated numerous predictions as to characteristics that should enhance swimming performance. Swimming monomyarians are thought to have smaller bodies, relative to the shell, than their non-swimming counterparts, presumably to increase the volume of water ejected during valve closure (Thayer 1972). The obliqueness of the phasic adductor muscle in a plane perpendicular to the hinge is greater in swimming monomyarians, presumably to increase the angular velocity of valve closure (Thayer 1972) and generate more powerful water jets (Yonge 1936, Thayer 1972, Soemodihardjo 1974). The ratio of phasic to tonic adductor muscle areas (Yonge 1936, Gould 1971, Soemodihardjo 1974) is greater in scallops thought to swim more, reflecting the central role of phasic contractions. A greater phasic moment would enhance force production. Scallops with good swimming abilities generally have light smooth shells, an upper valve that is slightly more convex than the lower one and a high aspect ratio (calculated either as valve length/valve height or valve length2/valve area) (Gould 1971, Soemodihardjo 1974). A greater aspect ratio increases the lift/drag ratio when movement is perpendicular to the anterior-posterior axis (Stanley 1970). Finally, the cemented scallop sinks rapidly (Gould 1971), but more active scallops have an approximately circular shape that slows sinking (Cheng and DeMont 1996). While these interspecific differences in shell and muscle morphology have been interpreted in light of swimming capacities, these studies did not actually measure swimming abilities, but inferred them from literature anecdotes. Two conclusions are clear from the existing literature: 1) non-swimming bivalves and scallops differ considerably in their muscle and shell morphologies, and 2) the smoothness and lightness of the shells of Amusium sp. favour its strong swimming (e.g. Gould 1971, Morton 1980). The literature does not, however, reveal whether differences in shell and muscle morphology are quantitatively linked with the wide range of scallop swimming strategies.

The objective of this study was to determine whether morphological characteristics of the shell and adductor muscle in a range of scallop species, reflect their escape response strategies and muscle use. Six scallop species (Amusium balloti, Placopecten magellanicus, Equichlamys bifrons, Pecten fumatus, Mimachlamys asperrima, and Crassadoma gigantean) with different shell shapes (Fig. 5.1) and distinct escape responses,

133 as documented by our measurements of muscle use during escape responses (Tremblay et al. 2012) were compared. The scallop Amusium balloti and Placopecten magellanicus are active swimmers and perform phasic contractions throughout the escape response, with A. balloti making phasic contractions at a quicker pace than P. magellanicus. Pecten fumatus performs intense bursts of phasic contractions at the beginning of its escape response, but fatigues quickly and then makes prolonged tonic contractions. The scallop Mimachlamys asperrima performs short series of phasic contractions at the beginning of the response, with a slower phasic contraction rate than the foregoing species. Adult Equichlamys bifrons are more sedentary than M. asperrima and P. fumatus (Olsen 1955). Finally, the purple-hinge scallop Crassadoma gigantea, is free-living in early life (<30 mm shell height) and then cements its lower, right valve, to rocky surfaces (Yonge 1951, Lauzier and Bourne 2006). Adult C. gigantea generally respond to predators by closing their valves, and only rarely make phasic contractions. The oyster, Crassostrea gigas served as a non- swimming monomyarian outlier.

For each species, the adductor muscle proportions, size, position, and obliqueness, as well as mass, were measured. The sinking rate and shell characteristics including aspect ratio and volume between the valves were measured. Principal component analysis (PCA) of these morphological characteristics was used to examine links among the morphological characteristics and how they differentiated our experimental species. Next, a PCA on our data for patterns of muscle use during escape responses (Tremblay et al. 2012) was carried out to evaluate links among the principal components describing morphology and those describing behaviour. The scallops shell and adductor muscle morphological characteristics should reflect their escape response strategies. Scallops that are active swimmers and mainly make phasic contractions during an escape response should have light shells, a high aspect ratio and sink slowly and these species should have a higher proportion of phasic muscle (relative to tonic muscle) than scallops using prolonged tonic contractions. Moreover, the phasic muscle in scallops performing mostly phasic contractions should have both a greater moment and obliqueness, in a plane perpendicular to the hinge, than that in species using prolonged tonics.

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Material and methods

Experimental scallops The experimental scallops were the individuals used in a complementory behavioural study (Tremblay et al. 2012). Adult Amusium balloti (90-113 mm shell height) were collected in August 2007 near Gladstone (Queensland, Australia). Adult Placopecten magellanicus (85-96 mm shell height) were obtained from Culti-mer (Québec, Canada) in September 2008. Adult Pecten fumatus (86-104 mm shell height), E. bifrons (74-121 mm shell height) and M. asperrima (71-95 mm shell height) were collected by SCUBA near Satellite Island (43º 32’ 491”S and 147º 23’ 297”E, Channel d’Entrecastreux, Tasmania, Australia) in September 2007. Adult Crassadoma gigantea (73-130 mm shell height) were collected in May 2010 from oyster rearing systems near Espinosa Inlet (British Columbia, Canada). The oyster Crassostrea gigas (85-133 mm shell height) were collected at low tide on the beach at Deception Bay (British Columbia, Canada) and were immediately transferred to the Centre for Shellfish Research (Nanaimo, British Columbia, Canada) where they remained in running seawater tanks for 2 weeks prior to their analysis. The experimental scallops had similar condition indices (Tremblay et al. 2012).

Sinking test The time taken by living scallops to sink in a 35 cm water column was measured. Scallops were disturbed slightly to make them shut their valves and then held in the tank, containing seawater, with the upper valve leveled with the water surface. Using a stopwatch, the time from when the scallop was released to when it touched the bottom was measured. Three trials were made with each individual and the average sinking time was calculated. Since our experimental P. fumatus and C. gigantea had a screw attached to their lower valve, another sample of scallops was used for this test. In both species, the individuals were a similar size as those used for measurements of swimming behaviour. As the shells of oysters, C. gigas, were encrusted with shells, barnacles, and rocks that could not be totally removed without damaging the oyster, sinking time was not measured for oysters.

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Given the slight size differences between the samples of our experimental species, the sinking time was adjusted to a shell height of 90 mm (a size present in our samples of each species). First, the regressions between the shell height and the the sinking time were done. These regressions were used to calculate T1 T2 and T3 where T1 was the sinking time expected for a 90 mm shell height scallop, A2 the sinking time expected for a scallop with the size of the experimental scallop and A3 the measured sinking time for the experimental scallop. Finally, the adjusted sinking time, at 90 mm shell height, was calculated using the following formula:

Adjusted sinking time = (T1/T2)×T3

Morphological measurements Shell height, length and width of each individual were measured using a digital caliper (±0.01 cm). The shell height corresponded to the maximum distance between the dorsal (hinge) and ventral margins, whereas length was the maximum distance between the anterior and posterior margins and was perpendicular to shell height (Fig. 5.1a). Shell width was measured at the point of maximum convexity with the two valves placed in their natural closed position (Fig. 5.1a). Due to the variable chord of the scallop shells, we chose to measure the aspect ratio as the square of the shell length divided by valve area (Dadswell and Weihs 1990). Digital photos were taken of the valves with an adjacent ruler as a reference (Fig. 5.2a). Valve area was determined from photographs of the shell using image analysis software (ImageJ, ver. 1.42, National Institutes of Health). Wet tissue mass was obtained after carefully removing soft tissues from the shell and placing them on absorbent paper to remove excess water. The phasic and tonic adductor muscles and remaining soft tissues were weighed separately (±0.01 g). Soft tissues were dried for 48 h at 60°C to assess dry mass and percentage water content. Once the soft tissues had been removed, shells were wiped dry and weighed. As the lower shell of P. fumatus and C. gigantea had a screw attached to it, other individuals were used to establish the relationship between shell dimensions and damp-dried shell mass and then the shell mass for individual scallops was calculated.

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A) height width

length

Placopecten magellanicus

Amusium balloti Equichlamys bifrons

Pecten fumatus Mimachlamys asperrima

Crassotrea gigas Crassadoma gigantea

137

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The volume between the valves was estimated by weighing the upper and lower valves filled with sifted sand (500 µm) levelled with the shell margin. Using the weight of given volume of sand, the volume in each valve was estimated. The sum gave the total volume. The shell and soft tissue dry masses were adjusted to a shell height of 90 mm using the same formula used for sinking time.

Adductor muscle measurements Following tissue removal, the shells were wiped dry before retracing the phasic and tonic muscle impressions with a permanent marker inside both valves (Fig. 5.2a). Digital photos of both valves with an adjacent ruler as a reference were taken (Fig. 5.2a). Photographs of the both shells with retraced muscle impressions were used to determine the area of the valves and adductor muscles using image analysis software (ImageJ). Muscle position on both valves was defined in terms of ratios as in Gould (1971) and Soemodihardjo (1974) (Fig. 5.2b). The dorso-ventral position of the phasic muscle corresponded to the ratio of line EF to line GH. Line EF was the shortest distance between the phasic muscle and the ventral most edge of the valve whereas line GH was the shortest distance between the phasic muscle and the hinge (Fig. 5.2b). Similarly, the ratio IJ/KL represented the dorso-ventral position of the tonic muscle. The antero-posterior position of the phasic and tonic muscles was defined by the ratios OP/MN and ST/QR respectively (Fig. 5.2b). The obliqueness of the adductor muscle, relative to the valve, was defined as the ratio between the dorso-ventral position of the muscle on the left valve over that on the right one. Accordingly, the obliqueness of the phasic adductor muscle was EF × G’H’/ E’F’ × GH, while that of the tonic muscle was IJ × K’L’/I’J’ × KL (Fig. 5.2b). The more the ratio departs from one, the greater the obliqueness. The moment of the phasic muscle was estimated as the product of the area of the muscle impression on the valve and the distance between its centre and the hinge. As some of the species in this study have valves with a strong curvature, it had to be demonstrated that measurements of muscle area and position on photographs of the valves were not biased. The muscle impression was retraced on a clear plastic paper that closely

139 fitted shell curvature. Muscle area was then measured from photographs of the flattened plastic paper using image analysis software (ImageJ). For muscle position, the measurements were made directly on the shell using a thin plastic ruler that could closely fit the shell. No significant differences were observed between measurements based on photographs of the valves and measurements that took shell curvature into account.

Statistical analysis Normality was tested using a Shapiro-Wilks test and the homogeneity of variances was analyzed visually by plotting residuals relative to predicted values. Due to the non normality of residuals and non homogeneity of variances, non parametric tests were used. Comparisons between the different scallop species were made using Kruskall-Wallis tests. The non-parametric Wilcoxon signed-rank test was used when data were expressed in terms of ratios to assess if these ratios differed from one. Intraspecific differences in muscle area and position on the left and right valves were assessed using paired T-tests. Spearman’s correlations were used to assess whether shell mass and aspect ratio were correlated with sinking time. The large number of morphological parameters measured increases the risk of erroneous identification of significant relationships between morphological and behavioural characteristics. Therefore, a principal component analysis (PCA) was performed to create new combined variates (principal components (PC), i.e. random variables; Legendre & Legendre 1998) of the morphological characteristics and a second PCA of the behavioural data measured on our experimental scallops (Tremblay et al. 2012). As only visual evaluations of behaviour for E. bifrons were available, the PCA for morphology and behaviour used data for A. balloti, P. magellanicus, P. fumatus, M. asperimma and C. gigantea. After running the PCA, an orthogonal rotation was applied to have uncorrelated PC that would be easier to interpret. Onle the PC that explained more than 10% of the overall variance were interpreted. Each variable was linked with the PC on which it had the greatest loading. The PC were named and interpreted according to the variables assigned to it.

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Principal component analysis omits observations that have missing values for any variable. Due to the presence of scattered missing values in the morphological data set, Monte Carlo multiple imputations (20 imputations) was performed to replace the missing values. The morphological PCA was performed using the 20 data sets that resulted from the multiple imputations. As fewer values were missing in the behavioural data set, such imputations were not needed. To examine interspecific differences for the PC for the morphological and behavioural data, a score was calculated for each individual for each PC. Then, an analysis of variance (ANOVA) was performed to see if these scores differed between the species. The links between morphological and behavioural characteristics were examined by performing a regression of the individual scores calculated for each morphological PC against the individual scores calculated for each behavioural PC. All analyses were done using SAS 9.2 (SAS Institute). Significance was accepted at P<0.05.

Results In the tables and figures, the species are placed in approximate order of their swimming endurance, starting with Amusium balloti and ending with Crassadoma gigantea and the oyster, Crassostrea gigas. It was initially evaluated whether interspecific differences in the parameters measured followed the hypotheses. Then PCA were used to examine how these parameters were related and how the resulting PC differentiated the species. Next, the behaviour during escape responses (Tremblay et al. 2012) was examined to see whether it was related to morphology, first using PCA to create behavioural variates. After examining how the behavioural variates differed between the species, regressions were used to evaluate how the PCs for morphology were linked to those for escape response behaviour.

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Shell characteristics The individuals studied had similar shell heights, although the sample of M. asperrima was slightly smaller than those of the other species (Table 5.1). After adjusting shell and tissue masses to a common shell height (90 mm), C. gigantea had the heaviest shell followed by P. fumatus and P. magellanicus (Table 5.1). The shells of M. asperrima and A. balloti were considerably lighter than the other species (Table 5.1). The expectation that scallops that are active swimmers have a lighter shell than less active and non- swimming species was only partially borne out. The aspect ratio was highest in P. fumatus, while it was lowest in M. asperrima and C. gigas (Table 5.1). The scallop A. balloti, P. magellanicus, E. bifrons, and C. gigantea had intermediate aspect ratios (Table 5.1). This pattern only partly conformed to the prediction that a high aspect ratio characterizes good swimmers.

Sinking test Amusium balloti took the most time to sink (Table 5.2). The scallop M. asperrima and P. magellanicus took slightly but significantly less time while E. bifrons and P. fumatus sank more quickly (Table 5.2). The scallop C. gigantea plummeted to the bottom in only 1.29±0.04 s (Table 5.2). Sinking time was correlated with shell mass (P=0.005), but not with the aspect ratio (P=0.872). The expectation that sinking time reflects swimming capacity was partly supported.

Muscle morphology The phasic adductor muscle (corrected to 90 mm shell height) was heaviest in C. gigantea, P. magellanicus and E. bifrons (Table 5.1). The scallop P. fumatus and M. asperrima had intermediate phasic muscle masses while A. balloti had the smallest phasic muscle among the scallops (Table 5.1). The oyster had a much smaller phasic muscle than all scallops (Table 5.1). The heaviest tonic muscle was found in E. bifrons while A. balloti had by far the smallest and the other species were intermediate (Table 5.1). The

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Table 5.1. Shell and morphological characteristics of experimental scallops.

Amusium Placopecten Equichlamys Pecten Mimachlamys Crassadoma Crassostrea balloti magellanicus bifrons fumatus asperrima gigantea gigas Shell Height, mm 96.3±1.1a 90.4±0.9b,c 96.2±2.5a 94.4±1.7a,b 84.2±2.0c 97.0±4.5a,b 105.9±3.3d Length, mm 95.5±1.4a 99.6±1.0b 100.8±2.9b,c 107.0±1.6c 82.4±2.1d 92.9±3.5a 70.2±2.2d Width, mm 19.8±0.4a 27.7±0.4b 29.6±0.6b.d 22.6±0.4c 31.3±1.1b,d 34.5±2.0d 36.6±1.6d N 27-30 15 20 15 16 16-19 18 Dry mass at 90 mm, g Soft tissue 2.00±0.08a 6.71±0.26b 7.16±0.14c 4.96±0.30d,e 5.45±0.17e 8.87±0.55c 4.61±0.23d Phasic muscle 0.86±0.05a 3.08±0.13b 3.08±0.10b 2.05±0.16c 1.92±0.09c 3.60±0.24b 0.31±0.02d Tonic muscle 0.05±0.004a 0.25±0.01b,d 0.51±0.01c 0.23±0.01d 0.17±0.01e 0.29±0.02b 0.19±0.01e Shell 26.5±0.4a 48.8±1.1b 54.9±0.8 c 45.9±1.4b 32.7±0.7d 136.4±9.9e 94.8±4.5f N 18-27 15 19-20 9-15 16 18-19 18 Ratio† Adductor muscle/total animal 0.37±0.01a,c 0.39±0.01b 0.38±0.01b,c 0.36±0.01a 0.30±0.01d 0.36±0.01a 0.09±0.005e Soft tissue/shell volume, g ml-1 0.48±0.01a 0.52±0.01b 0.63±0.01c 0.42±0.02d 0.41±0.01d 0.61±0.02c 0.41±0.01d N 18 13-15 18-20 15 16 19 18 Aspect ratio Shell length2/valve area 1.29±0.01a 1.21±0.01b 1.26±0.02a,b 1.52±0.02c 1.10±0.01d 1.24±0.05b 1.00±0.05d N 27 15 20 15 16 19 18 Muscle area/valve area Phasic Left valve 0.10±0.002a,d* 0.08±0.001b* 0.10±0.004a,c 0.11±0.01c* 0.09±0.003d 0.10±0.01c 0.03±0.002e* Right valve 0.08±0.002a* 0.06±0.002b* 0.10±0.002c 0.07±0.002b* 0.09±0.01a 0.09±0.005c 0.02±0.002d* Tonic Left valve 0.009±0.001a* 0.012±0.001a* 0.019±0.001b* 0.018±0.001b* 0.018±0.002b 0.021±0.006b 0.019±0.002b Right valve 0.006±0.0002a* 0.015±0.001b,d* 0.028±0.001c* 0.014±0.001d* 0.020±0.002e 0.023±0.005b,e 0.017±0.002b,e N 30 15 9-10 15 13-14 15-17 13-14 Moment phasic muscle Left valve 31.5±2.0a* 30.8±1.0a,b* 38.0±4.2b* 35.3±2.5b* 22.4±2.3c 40.4±6.2a,b* 6.5±0.6d Right valve 22.5±1.5a,d* 17.1±0.9b* 28.8±2.2c,d* 17.6±1.2a,b* 19.9±2.6a,b,d 34.4±5.5c,d* 6.5±0.8e N 30 15 10-20 15 14 17 13-15 Data are means ± S.E. Volume: refers to the volume between the two empty valves. Aspect ratio: left valve. Soft tissue dry mass was adjusted for 90 mm shell height. Soft tissue refers to all soft tissues including the adductor muscle. †Masses are the wet mass. Adductor muscle includes the phasic and tonic muscles. Moment phasic muscle: phasic muscle 3

143 area x distance between phasic muscle centre and hinge (cm ). In a given row, different letters indicate significant differences (Kruskall-Wallis and multiple comparisons, P<0.05). *An asterix indicates a difference between the left and right valves in a given species (paired T-test, P<0.05).

143

144

Table 5.2. Sinking time adjusted for 90 mm shell height*.

Amusium Placopecten Equichlamys Pecten Mimachlamys Crassadoma Crassostrea balloti magellanicus bifrons fumatus asperrima gigantea gigas Time (s) 2.68±0.03a 2.30±0.03b 2.07±0.05c 2.08±0.03c 2.36±0.04b 1.29±0.04d - N 22 15 20 15 16 19 - Mean ± S.E. Kruskall–Wallis and multiple comparisons, P<0.05. *See material and methods for detail about the adjustment to 90 mm shell height.

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ratio of adductor muscle to soft tissue wet mass was lowest in the oyster while scallops all had considerably higher ratios (Table 5.1). Muscle masses were not simply related to swimming ability. The ratio of the soft tissue wet mass to shell volume was highest in E. bifrons and C. gigantea followed by P. magellanicus with a slightly but significantly lower ratio (Table 5.1). The lowest ratios were found in A. balloti, P. fumatus, M. asperrima and C. gigaswith less than half of the shell volume filled with soft tissues (Table 5.1). The highly active Amusium, the byssally attached M. asperrima, and the oyster shared a reduced body volume, contradicting the prediction that this characteristic favours active swimming. The portion of the valves covered by the phasic muscle scar differed among species. Interspecific differences were greater for the ratio on the right valve (Table 5.1). Here E. bifrons, M. asperrima, C. gigantea, had the highest value, followed by A. balloti and then P. magellanicus and P. fumatus (Table 5.1). Phasic muscle size, as assessed by the muscle scar, was not a predictor of swimming capacity. The proportion of the valves covered by tonic muscle differed among scallops. On the left valve, 4 scallops (E. bifrons, P. fumatus, M. asperrima, C. gigantea) shared high values with the oyster while A. balloti and P. magellanicus had considerably lower values (Table 5.1). The proportion of the right valve covered by tonic muscle also differed among species, with A. balloti having the lowest value, E. bifrons the highest and the other species having intermediate values. Generally, tonic muscle area was lower in most active swimmers. The moment exerted by the phasic adductor muscle varied among species. When calculated for the left valve, A. balloti, P. magellanicus, E. bifrons, P. fumatus and C. gigantea had the highest moments while M. asperrima had an intermediate moment and the oyster had the lowest (Table 5.1). When the phasic moment was calculated for the right valve, interspecific differences among scallops changed. E. bifrons and C. gigantea had the highest moments while A. balloti, P. magellanicus, P. fumatus and M. asperrima had intermediate values (Table 5.1). The oyster had the lowest moment (Table 5.1). Again simple predictions based on swimming capacities do not explain these patterns.

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Proportions of tonic and phasic adductor muscle The impression of the phasic and tonic adductor muscles on the valves shows variability among the species (Fig. 5.3). When the percentage of tonic adductor muscle was calculated from the muscle impression on the right valve, A. balloti was shown to have very little tonic muscle compared to the other species (Fig. 5.4). The scallop P. magellanicus, P. fumatus, M. asperrima and C. gigantea had intermediate proportions of tonic muscle while the oyster had the highest proportion of tonic muscle (Fig. 5.4). The proportion of phasic and tonic adductor muscles was also calculated from the masses of each part of the muscle and interspecific differences in phasic and tonic muscle proportions were similar to those observed from muscle impressions on the shell (data not shown). While muscle proportions separated A. ballotti from the other scallops, these proportions did not follow differences in swimming strategy among the other scallop species.

Muscle obliqueness: adductor muscle impression on left versus right valves Having a larger area of muscle attachment on one valve than the other is one means by which muscle obliqueness can be increased. In agreement with a role of muscle obliqueness in increasing the angular velocity of valve closure, the size of the adductor muscle attachment on the two valves differed more in actively swimming scallops than in more sedentary species (Fig. 5.5). The area of the phasic adductor muscle was larger on the left than right valve in A. balloti, P. magellanicus, E. bifrons and P. fumatus whilst no significant differences were observed in M. asperrima, C. gigantea and C. gigas (Fig. 5.5). The ratio was approximately 1.50 in P. magellanicus and P. fumatus, significantly higher than in A. balloti and E. bifrons (Fig. 5.5). This mechanism of increasing phasic muscle obliqueness followed overall swimming strategy. The impressions of the tonic adductor muscle did not differ between the two valves in any species except E. bifrons in which the area of the tonic muscle was larger on the right than the left valve (Fig. 5.5).

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Amusium balloti Placopecten magellanicus

Equichalmys bifrons Pecten fumatus

Mimachalmys asperrima Crassadoma gigantea

20 mm L R Crassotrea gigas

Figure 5.3. Typical phasic (gray) and tonic (black) adductor muscle impressions on left (L) and right (R) valves in experimental scallops. Arrowed line corresponds to scale: 20 mm for all species.

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50 f

c b b,d 20 d e

Percentage (%)

10 a

C. gigas A. balloti E. bifrons P. fumatus C. gigantea M. asperrima P. magellanicus

148

2.0

Phasic * * Tonic b b 1.5 a,c *a *c A A c c A,B A,B 1.0 * A,B B ratio B

0.5

0.0

C. gigas A. balloti E. bifrons P. fumatus C. gigantea

Muscle impression area left valve/muscle impression area right valve right area impression valve/muscle left area impression Muscle M. asperrima P. magellanicus

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Muscle obliqueness: muscle position on right and left valves The relative positions of the adductor muscle attachment on the right and left valves also influence obliqueness. All the species, including the oyster, exhibited this type of obliqueness for the phasic adductor (Fig. 5.6). The insertion on the right valve was always closer to the hinge than on the left valve. Phasic muscle obliqueness was much more pronounced in P. fumatus than in the other species (Fig. 5.6). In most species, the tonic muscle was oblique in the opposite direction than the phasic muscle, with the insertion on the left valve being closer to the hinge (Fig. 5.6). In P. fumatus and the oyster, tonic and phasic adductor muscles showed obliqueness in the same direction (Fig. 5.6). Obliqueness of the muscles in P. fumatus was slightly overestimated due to the curvature of the lower valve. When this was taken into account, obliqueness of the phasic muscle remained much higher than in other scallops while obliqueness of the tonic muscle was similar to that of the other scallops.

Principal component analysis (PCA) of scallop morphology and behaviour The first three Principal components of the PCA for morphology accounted for 30, 19 and 12% of the variance respectively (Fig. 5.7). The first PC was influenced by sinking time, shell and tissue mass and various tissue proportions and was named “mass and proportions” (Fig. 5.7).

Sinking time and proportion of phasic muscle had negative loadings, whereas shell and muscle masses had positive loadings on this PC. The shared positive loadings of tonic muscle area and shell and muscle masses reflected the fact that poor swimmers have heavy shells and larger tonic muscles. The second PC was mainly influenced by the phasic muscle moment, relative size of the phasic muscle on the left valve and valve dimensions and was named “force and size” (Fig. 5.7). The last component, named “obliqueness and aspect ratio”, was influenced by parameters related to the obliqueness of the adductor muscle and the aspect ratio (Fig. 5.7).

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3.0 * d Phasic Tonic 2.5

2.0 * * b * b E 1.5 *a * * ratio c * a,c * c * * D A,B B A 1.0 * C * C

Adductor muscle obliqueness muscle Adductor 0.5

0.0

C. gigas A. balloti E. bifrons P. fumatus C. gigantea M. asperrima P. magellanicus

Figure 5.6. Scallop adductor muscle obliqueness. Obliqueness is the ratio of the dorso- ventral position of the adductor muscle on the left valve to that on the right valve Mean±S.E. Different letters indicate significant differences (Kruskall-Wallis and multiple comparisons, P<0.05). Lower case letters refers to phasic muscle while capital letters refer to tonic muscle. If the value is above the dashed line, the insertion on the right valve is closer to the hinge. Ratios significantly different than unity are identified by a star (Wilcoxon signed-rank test, P<0.05). Sample size A. balloti N=30, P. magellanicus N=15, E. bifrons N=9, P. fumatus N=15, M. asperrima N=14, C. gigantea N=14, C. gigas N=11.

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Factor 2 Factor 3 Mass andFactor proportions 1 Force and size Obliqueness and aspect ratio Sinking time (90 mm) 30% 19% 12% Proportion of phasic muscle Area tonic muscle left/area tonic muscle right Width Soft tissue mass/shell volume Area tonic muscle/ area valve (left valve) Area tonic muscle/ area valve (right valve) Shell mass (90 mm) Tonic muscle dry mass (90 mm) Phasic muscle dry mass (90 mm) Area phasic muscle/ area valve (right valve) Area phasic muscle left/area phasic muscle right Tonic muscle obliqueness Aspect ratio Phasic muscle obliqueness Muscle mass/soft tissue mass Area phasic muscle/ area valve (left valve) Length Height Phasic muscle moment (left valve)

-0.20 -0.15 -0.10 -0.05 0.00 0.05 0.10 0.15 0.20 -0.2 -0.1 0.0 0.1 0.2 0.3 0.4 -0.3 -0.2 -0.1 0.0 0.1 0.2 0.3 0.4

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The scores of the morphological PC differentiated the scallop species. For the first PC (mass and proportions), C. gigantea had the highest score and A. balloti the lowest (Fig. 5.8). The scallop P. magellanicus, P. fumatus and M. asperrima had intermediate scores (Fig. 5.8). For the second PC (force and size), M. asperrima had the lowest score while the other species were similar and slightly higher (Fig. 5.8), partly as the M. asperrima sampled were slightly smaller than the other species. For the third PC (obliqueness and aspect ratio), P. fumatus had the highest score, while M. asperrima and C. gigantea had the lowest (Fig. 5.8). The scallop A. balloti and P. magellanicus had intermediate scores, with P. magellanicus being slightly higher than A. balloti (Fig. 5.8).

The PCA on behavioural parameters, measured during escape response tests on these scallops (see Tremblay et al. 2012 for details), identified two PC that together explained 70% of the observed variability. The first PC grouped the parameters related to endurance (total number of phasics, time to fatigue, number of phasics before fatigue, mean duration of tonic contractions, number of tonic contractions >5 s) during an escape response and explained 47% of the variability (Fig. 5.9). As prolonged tonic contractions reduce the number of tonic contractions, a high number of tonic contractions was negatively related to the time spent in tonic contractions (Fig. 5.9). For this PC, A. balloti and P. magellanicus had the highest scores, P. fumatus and M. asperrima had intermediate scores and C. gigantea the lowest score (Fig. 5.10). The second PC included parameters related to the intensity of the escape response (number of phasics in the first series, number of phasic contractions before the first tonic, number of phasics over number of tonics, time at first tonic) and explained 23% of the variability (Fig. 5.9). Here P. fumatus had the highest score, A. balloti had an intermediate score while P. magellanicus, M. asperrima and C. gigantea had the lowest scores (Fig. 5.10).

Finally, links between morphological and behavioural parameters were examined by performing regressions between the three morphological PC and the two behavioural PC. The endurance of our scallops during an escape response was highly related to the PC “proportions and mass”. The intensity of escape responses was strongly related to

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154

Mass and proportions Force and size Obliqueness and aspect ratio

5 4 3 a c 4 3 2 a 3 d 2 b a 1 a,d a 2 1 a d b b b 0 1 c 0

Scores Scores -1 0 a -1 -1 -2 -2

-2 -3 -3

A. balloti A. balloti A. balloti P. fumatus P. fumatus C. gigantea C. gigantea P. fumatus C. gigantea M. asperrima M. asperrima M. asperrima P. magellanicus P. magellanicus P. magellanicus

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Endurance

Total number of phasic contractions during the first series relative to the total number of phasic contractions 47% Number of phasic contractions before the first tonic contraction Time at the first tonic contraction Total number of phasic contractions relative to the total number of tonic contractions Mean duration of tonic contractions Percentage of time spent in tonic contractions Phasic contraction rate during the first 30 s Percentage of time spent in phasic contractions Number of tonic contractions of a duration of 5 s or more Total number of tonic Number of phasics before fatigue Time to fatigue Total number of phasic

-0.2 -0.1 0.0 0.1 0.2 0.3

Intensity Total number of phasic contractions during the first series relative to the total number of phasic contractions 23% Number of phasic contractions before the first tonic contraction Time at the first tonic contraction Total number of phasic contractions relative to the total number of tonic contractions Mean duration of tonic contractions Percentage of time spent in tonic contractions Phasic contraction rate during the first 30 s Percentage of time spent in phasic contractions Number of tonic contractions of a duration of 5 s or more Total number of tonic Number of phasics before fatigue Time to fatigue Total number of phasic

-0.2 -0.1 0.0 0.1 0.2 0.3 Figure 5.9. Normalised scores of the behavioural variables associated with the first 2 principal components. Black bars identify

155 variables assigned to each component. The percentages indicate the proportion of variance explained by the principal component.

155

Endurance 2 a a

1 b b c

Scores -1

-2

-3

Intensity 5

4 c 3

2 a

Scores b,d d 0 b

-1

-2

A. balloti P. fumatus C. gigantea M. asperrima P. magellanicus

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the obliqueness of the adductor muscle and the aspect ratio, as well as the proportions and mass of tissues in the scallop (Table 5.3). Morphological parameters linked to force and size (PC2) were not significantly linked with either of the PC for behaviour.

Table 5.3. Links between the morphological and behavioural principal components.

Endurance Intensity t-value Pr > t t-value Pr > t Morphology Mass and proportions -7.28 <.0001 -2.51 0.012 Force and size -1.93 0.054 1.07 0.286 Obliqueness and aspect ratio 0.41 0.685 5.23 <.0001 Regression of the individual scores calculated for each morphological PC against the individual scores calculated for each behavioural PC (P<0.05).

Discussion Morphological characteristics of the shell and adductor muscle differed markedly between scallops and the oyster, but when examined individually, these morphological differences among scallop species did not always follow their swimming strategies. PCA revealed links among morphological parameters that nicely differentiated our experimental scallop species and revealed which morphological parameters were linked with specific escape response behaviours. The relationship between scallop behaviour and morphology is not simple, as it is the result of compromises imposed by the habitats, lifestyle and predators. To understand how the combinations of morphological parameters relate to the swimming behaviour of each species, it is important to interpret these results in the overall context of the habitats in which these scallops live.

Links between morphology and behaviour The PCA for the morphological characteristics allowed differentiating the species by groups of morphological parameters. The first PC, “proportions and mass”, separated

157 the most active swimmer (A. balloti) and the non-swimming scallop (C. gigantea) from the other species. The scallop A. balloti had the shortest sinking time, lowest width and lightest shell and muscle masses, whereas C. gigantea had the fastest sinking time, greatest width and heaviest shell and muscle masses. Major attributes of muscle morphology influencing this PC were the relative valve area occupied by tonic muscle and the proportion of phasic muscle. The adductor muscle contains virtually no tonic muscle in A. balloti but contains substantially more in heavy, sedentary species, particularly C. gigantea. The second PC, “force and size”, distinguished M. asperrima, the byssally attached species, from the others. Although the third PC, “obliqueness and aspect ratio”, only explained 12% of the variability, it separated the scallops into 3 groups: the swimmers A. balloti and P. magellanicus, the byssally attached M. asperrima and the cemented C. gigantea and finally P. fumatus alone with a much higher score than all the others. The scallop P. fumatus had the highest values for the aspect ratio and obliqueness of the phasic and tonic muscles while A. balloti and P. magellanicus had relatively high aspect ratios and values of muscle obliqueness. The PCA for behavioural parameters yielded two PC that explained much of the variability in the data. The first behavioural PC (endurance) nicely positioned the species in order of their swimming endurance: A. balloti, P. magellanicus followed by P. fumatus, M. asperrima and finally C. gigantea; a similar rank order as for PC1 (mass and proportions) of morphology. The swimming endurance in our scallops was best predicted by the slimness (width) and lightness of the shell and muscles and the ensuing changes in muscle morphology. The second PC (intensity) primarily highlighted P. fumatus. The swimming intensity of our scallops was best predicted by the obliqueness of the adductor muscle and aspect ratio, as well as the tissue mass and proportions. Neither behavioural PC was correlated with the second morphological PC, force and size, presumably as none of our behavioural data were expressed in terms of muscle force.

Obliqueness of the adductor muscle can be achieved by having the insertion of the muscle closer to the hinge on one valve than the other or by having the muscle impression larger on one valve than the other and has been observed for many years (Thayer 1972), but its role and adaptive significance remained unclear until now. Yonge (1936) suggested that

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the oblique arrangement increases the length of the muscle allowing the valves to open wider to take in more water and generate a more powerful water jet. Gould (1971) argued that increased length of the phasic muscle, due to its oblique arrangement, increases its capacity for work (cross-sectional area x length). Thayer (1972) demonstrated via calculations that the obliqueness of the phasic adductor muscle, in a plane perpendicular to the hinge, increases the angular velocity of valve closure. Finally, Soemodihardjo (1974) proposed that the increased diameter of the phasic muscle on the left valve relative to the right one, allows a pinnate arrangement of the muscle fibres which ultimately produces greater force. Our analysis showed that the obliqueness of the phasic muscle is linked to the intensity of escape response. As high velocity of valve closure and powerful water jet are both important determinants of the intensity (initial bursts of phasic contractions) of the escape response, our conclusions support the ideas proposed by the various authors (Yonge 1936, Gould 1971, Thayer 1972, Soemodihardjo 1974). Also, an increase of valve closure velocity requires the ligament to allow rapid opening of the valves. Indeed, the resilience of the ligament tended to be higher in scallops with more intense escape responses (Tremblay et al. unpublished).

Shell characteristics The experimental scallop species were chosen based upon visible differences in shell shape and texture, as well as on differences in known escape response strategies (Tremblay et al. 2012). Shell structure reflects a compromise between conflicting requirements in scallops. Light and smooth shells facilitate swimming by decreasing drag and gravity, but are more easily broken by predators than the thick heavy shells that restrict movement of more sedentary species. At one end of our range of species, the non- swimming scallop, C. gigantea, has a thick, heavy shell cemented to the substrate that gives efficient protection against predators. On the other extreme, A. balloti and M. asperrima have very light shells that facilitate swimming, but can easily be crushed or opened by a predator. In response to its crustacean predator, A. balloti strategy is to swim away while, in seeming contradiction to the fragility of its shell, M. asperrima tends to keep its valves closed for prolonged periods. The shell of M. asperrima is frequently covered by sponges

159 known to inhibit predation by sea stars (Pitcher and Butler 1987). Furthermore, the valves of M. asperrima fit closely together, hindering access by predators. Therefore, M. asperrima seems to have sufficient protection against predation without having a heavy shell or an extensive escape response.

Although the shell of P. fumatus is not hydrodynamic, its plano-convex shape facilitates recessing into the substrate and provides certain advantages for swimming. Plano-convex scallops produce significant lift when the commissural plane is in line with current flow (Gruffydd 1976, Millward and Whyte 1992). The high aspect ratio of P. fumatus shell means a high lift/drag ratio, helping it to rise in the water column and swim (Stanley 1970). The plano-convex shell is particularly important for recessing and camouflaging the scallop in soft substrate habitats. The compromises embodied in the shell shape of P. fumatus extend to its behaviour, muscle morphology and metabolic capacities. Effectively, the intensive burst of phasic contractions by P. fumatus at the start of its escape response (Tremblay et al. 2012), the obliqueness of its phasic muscle and its muscle metabolic capacities (Tremblay and Guderley 2014) can been seen as means of compensating for a shell shape that is unfavourable for swimming but useful for camouflage and feeding.

Muscle and body morphology In response to the presence of its predator, a scallop will either swim away or close its valves for prolonged periods. Swimming has been suggested to benefit from a reduction of body mass relative to shell volume, a bigger phasic adductor muscle or an oblique arrangement of the phasic adductor muscle (Thayer 1972). The volume of water expulsed per unit of time (V/t) and the force of the water jets could be increased by these mechanisms. While taken individually, muscle size and proportions did not differentiate escape response strategies in this study. However, these characteristics were linked on the first PC of the morphological PCA and in turn were strongly correlated with the PC for swimming endurance. Heavy shells require large muscles, increase the soft tissue volume

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and reduce sinking times and the proportion of phasic muscle. Thus, in combination, muscle size and proportions were strongly linked with swimming endurance and intensity. When a scallop claps its valves, the phasic muscle moment rotates the valves towards each other around the hinge and counteracts the opening moment of the ligament (Trueman 1953). This moment can be assessed as the cross-sectional area of the phasic muscle times its distance from the hinge. The moment exerted by the phasic muscle was a major determinant of the second morphological PC. As none of the escape response behaviours quantified in this study compared the force exerted by the scallop species, this property was not linked with escape response behaviours. Finally, the moment exerted by the phasic muscle was much higher in scallops than in the oyster. The behavioural PC did not yield a PC highlighting tonic contractions, rather the performance of the tonic and phasic contractions was closely intertwined in both PC. The frequent use of short tonic contractions in some actively swimming species (Tremblay et al. 2012) was apparent in the association of numbers of phasic and tonic contractions in the PC “endurance” of the behavioural data. Prolonged valve closure by tonic contractions provides time for phasic muscle recuperation (Pérez et al. 2008b) as well as protecting against predation in sedentary species. Scallop species that used mainly prolonged tonic contractions did not have a bigger proportion of tonic muscle than scallops performing mainly phasic contractions. Amusium balloti had a much smaller tonic muscle and developed less tonic force than the other species (Tremblay et al. unpublished). Amusium balloti cannot use tonic contractions in presence of predators as the large lateral openings of its shell give little protection against predators. It seems that the tonic muscle became accessory in Amusium species and resulted in its reduction. This study allowed the identification of parameters of swimming behaviour that are linked with specific aspects of muscle and shell morphology. Throughout evolution, scallops underwent various modifications in morphology and behaviour, with an ancestral byssally attached form giving rise to the various modern scallop species. While most of the life habits observed in scallops (gliding, free living, recession, byssally attached) can be either ancestral or transitory, the cemented form did not give rise to any other life-forms that scallops can assume (Alejandrino et al. 2011). This bias in life habit transitions may indicate constraints due to the complexity in the physiological and morphological changes

161 necessary for the transition from one life habit to another (Alejandrino et al. 2011). Alternately, cemented scallops could have physiological traits preventing the transition to another life habit (Alejandrino et al. 2011). Whereas this study examined how interspecific differences in shell and adductor muscle morphology are linked to escape response strategies, the underlying question is that of evolutionary plasticity in these attributes. Our data show that scallops can compensate for unfavourable shell morphology by changes in their behaviour (Tremblay et al. 2012), and that their muscle metabolic capacities follow these changes (Tremblay and Guderley 2014). While the behaviour and the metabolism of an organism are plastic on a relatively short time scale, morphological plasticity is manifested at a longer time scale. Morphological components, such as soft tissue mass and muscle arrangement within the shell, would change more rapidly than shell morphology; even shell morphology shows plasticity. Indeed, shell strength is influenced by the presence of predators with wild scallops having stronger shells than cultured ones (Lafrance et al. 2003, Grefsrud et al. 2006). The time scale required for changing shell shape (plano-convex to biconvex) or surface texture would be greater than the lifespan of individuals. Therefore, we view such aspects of shell morphology as constraints to which the morphology of soft tissues, behaviour and muscle metabolic capacities of scallops must adapt. Throughout it lifespan, scallops go through important changes in structure and activity. Indeed shell morphology, soft tissue and muscle arrangement, muscle metabolic capacities and swimming behaviour change as scallops grow (Gould 1971, Dadswell and Weihs 1990, Manuel and Dadswell 1993, Schmidt et al. 2008, Labrecque and Guderley 2011). Those modifications mostly take place during the juvenile stages and tend to stabilise when scallops reach maturity when more energy is put into reproduction than into growth. Thus, the links between shell and adductor muscle morphology and swimming strategy may change during ontogeny.

Overall, the differing escape response strategies of our scallop species are associated with specific morphological attributes without which these behavioural strategies would not work. Scallops have exploited different mechanisms to deal with the constraints of shell morphology, ranging from modifications of muscle size, position and

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proportions, to adjustments of muscle metabolic capacities and use during escape responses. The mechanisms that best enhance survival against predation reside on multiple levels.

Acknowledgements

This research was supported by funds from the RAQ (Ressources Aquatiques Québec) and NSERC to HEG. IT was recipient of a scholarship from FQRNT and RAQ. The authors are extremely grateful to the staff of the different institutes where this research was conducted. In Queensland at the BIARC (Paul Palmer, Tim Lucas, Satoshi Mikami and Sizhong (Joe) Wang and at the University of the Sunshine Coast (Peter F. Duncan). In Tasmania at TAFI (Julian Harrington, Craig Mundy and all the technical staff). In Québec at the MAPAQ (Bruno Myrand, Madeleine Nadeau and the technical staff) and Mélanie Bourgeois from Culti-mer. Finally in British Columbia at the Centre for Shellfish Research (Brian Kingzett and the technical staff).

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CHAPITRE 6

Discussion générale

Isabelle Tremblay

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La compréhension des liens structure/fonction est au cœur des buts de la physiologie évolutive. Bien que la performance locomotrice des vertébrés ait été beaucoup étudiée, la complexité de leur système musculo-squelettique rend difficile l’élucidation de tels liens. Grâce à la simplicité de leur système locomoteur et à la grande diversité au niveau de la morphologie de la coquille et du comportement de nage, les pectinidés étaient un groupe de premier choix pour notre étude des liens structure/fonction au niveau de la locomotion. Cette étude a comparé le comportement de nage, les capacités métaboliques du muscle adducteur, les propriétés du ligament et la morphologie de la coquille et du muscle adducteur chez 5 espèces de pétoncles présentant une morphologie de la coquille et un comportement de nage variés. Cette étude contribue à améliorer notre compréhension des liens fonctionnels et évolutifs entre la performance locomotrice et les différentes composantes du système locomoteur.

Rappel des grandes lignes de l’étude Dans un premier temps, il a fallu caractériser et quantifier la réponse de fuite des différentes espèces de pétoncles afin de les comparer de façon systématique en laboratoire. Nous avons opté pour les mesures de force, lors d’une réponse de fuite simulée, à l’aide d’un dynamomètre. Cette technique a permis de quantifier et comparer les patrons d’utilisation des deux parties du muscle adducteur lors d’une réponse de fuite chez les différentes espèces de pétoncles. Cette étude a révélé que l’utilisation des contractions phasiques et toniques variait de façon marquée entre les espèces. Il est donc maintenant possible de définir la réponse de fuite des pétoncles en termes de patron d’utilisation du muscle phasique et tonique et ainsi parler de stratégie de nage des pétoncles. La stratégie de nage des pétoncles varie avec la morphologie de leur coquille et leur mode de vie. Les pétoncles avec une coquille hydrodynamique ont tendance à utiliser principalement les contractions phasiques alors que les espèces dont la morphologie de la coquille est plutôt désavantageuse pour la nage vont s’appuyer davantage sur les contractions toniques. Aussi, le patron d’utilisation du muscle phasique et tonique peut être modifié afin de compenser pour une morphologie de la coquille désavantageuse pour la nage.

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En un deuxième temps, les capacités métaboliques du muscle phasique ont été étudiées afin de voir si elles différaient en fonction du patron d’utilisation du muscle adducteur. Les pétoncles qui font principalement des contractions phasiques ont des niveaux de phosphoarginine élevés, tandis que les pétoncles qui font des contractions toniques prolongées ont des capacités glycolytiques plus élevées que les autres. Ainsi, les capacités métaboliques du muscle phasique reflètent le patron d’utilisation du muscle adducteur des différentes espèces. L’activité de la myosine ATPase du muscle phasique est plus faible chez l’espèce de pétoncle qui ne nage pas, C. gigantea, comparativement aux espèces qui nagent. Les capacités métaboliques du muscle phasique des pétoncles sont très différentes de celles de l’huître, mais les capacités métaboliques du muscle phasique de C. gigantea sont similaires à celles des autres pétoncles malgré son mode de vie sédentaire au stade adulte.

À cause de son importance pour l’ouverture des valves, certaines propriétés du ligament des pétoncles ont été examinées afin de voir si ces propriétés varient selon la stratégie de nage des pétoncles. Ainsi, la résilience du ligament, la force déployée par le ligament pour ouvrir les valves, ainsi que la force déployée par le muscle adducteur phasique et tonique ont été caractérisées chez les espèces de pétoncles. La force déployée par le ligament pour ouvrir les valves varie entre les espèces et est toujours égale ou inférieure à la force déployée par le muscle phasique et tonique. De plus, la résilience du ligament des pétoncles varie entre les espèces avec P. fumatus ayant la résilience la plus élevée. Enfin, la résilience du ligament a tendance à être corrélée au taux de contraction phasique.

Finalement, les caractéristiques morphologiques de la coquille et du muscle adducteur des pétoncles ont été examinées afin de déterminer si ces propriétés reflètent l’utilisation des deux parties du muscle. Les caractéristiques morphologiques de la coquille et du muscle adducteur des pétoncles varient de façon marquée entre les espèces de pétoncles et aussi par rapport à l’huître. L’analyse en composantes principales (ACP) a montré comment les paramètres morphologiques sont reliés entre eux et comment ils permettent de différencier les espèces de pétoncles. Aussi, l’ACP a élucidé les liens entre 167 les paramètres morphologiques et les paramètres spécifiques du comportement. Ainsi, l’endurance de la réponse de fuite est principalement influencée par les différentes masses et proportions des tissus. L’intensité de la réponse de fuite, quant à elle, est fortement reliée à l’oblicité du muscle adducteur et l’allongement de la coquille, ainsi qu’aux différentes masses et proportions des tissus.

À chacun sa combinaison… Les liens entre la stratégie de nage, les capacités métaboliques du muscle phasique, ainsi que la morphologie du muscle et de la coquille, sont le résultat de compromis imposés par le style de vie, le type d’environnement et la présence de prédation dans l’environnement où évolue le pétoncle. Face à un prédateur, le pétoncle peut soit se déplacer ou tout simplement garder ses valves fermées pour une période de temps plus ou moins longue. Chacune des espèces de pétoncles opte pour la stratégie qui va lui procurer les meilleures chances de survie face aux prédateurs, et ce à l’intérieur des limites imposées par la morphologie de sa coquille. Ainsi, comme on le verra ci-dessous, nos résultats indiquent que chaque espèce de pétoncles a une combinaison morphologie-comportement qui lui est propre.

Face à ses principaux prédateurs, les crustacés, A. balloti est plutôt vulnérable avec sa coquille fragile et ses larges ouvertures sur les côtés où le crustacé peut insérer ses appendices pour déchirer la chair du pétoncle (Himmelman et al. 2009). Il est donc primordial pour A. balloti d’éviter de se faire capturer et la fuite par la nage est donc d’une haute importance pour sa survie. Nous avons montré qu’en réponse au contact avec T. orientalis, A. balloti va faire majoritairement des contractions phasiques tandis que les contractions toniques sont utilisées seulement à la fin de la réponse de fuite (Chap. 2, Fig. 2.2). Cette capacité d’A. balloti à maintenir les contractions phasiques tout au long de la réponse de fuite est possible grâce à plusieurs facteurs. Une coquille légère, lisse et de forme hydrodynamique, combinée à un ligament avec une résilience élevée (Chap. 4, Fig. 4.4) font en sorte que chaque contraction phasique se traduit en un déplacement aisé du pétoncle dans l’eau. De plus, les capacités métaboliques du muscle phasique indiquent que

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l’utilisation de la phosphoarginine chez A. balloti se fait à un rythme modéré et que le coût des contractions phasiques est moindre que chez les autres espèces (Chap. 3, Fig. 3.2 et 3.3). Ainsi, ce coût relativement faible chez A. balloti faciliterait la production des contractions phasiques tout au long de la réponse de fuite (Chap. 3, Fig. 3.2 et 3.3).

En ce qui concerne P. magellanicus, ses principaux prédateurs au stade adulte sont les étoiles de mer dont les déplacements sont plutôt lents (Elner & Jamieson 1979, Barbeau & Scheibling 1994b). Comme réponse de fuite, P. magellanicus fait majoritairement des contractions phasiques, et ce tout au long de la réponse de fuite comme c’est le cas pour A. balloti (Chap. 2, Fig. 2.2). Au niveau des capacités métaboliques du muscle phasique, P. magellanicus ressemble à A. balloti, mais semble utiliser la phosphoarginine à un rythme encore plus lent (Chap. 3, Fig. 3.2 et 3.3). Les résultats de l’ACP des paramètres comportementaux indiquent que P. magellanicus a un score élevé pour la composante « endurance » tout comme c’est le cas pour A. balloti (Chap. 5, Fig. 5.10). Par contre, les séries de contractions phasiques de P. magellanicus sont espacées par de courtes contractions toniques qui augmentent graduellement en durée et force au cours de la réponse de fuite (Chap. 2, Fig. 2.2). Ceci se reflète bien au niveau du faible score de P. magellanicus pour la composante « intensité » de l’ACP des paramètres comportementaux (Chap. 5, Fig. 5.10). Même si P. magellanicus a une coquille de forme relativement hydrodynamique, cette dernière a une masse relativement plus élevée ce qui augmente le poids que P. magellanicus doit déplacer afin de nager (Chap. 2, Tableau 2.1). Il est peu probable que les contractions toniques de courte durée (moins de 5 s) permettent une récupération métabolique du muscle phasique (Pérez et al. 2008b), ainsi il se pourrait qu’elles aient un rôle plutôt mécanique. Nous croyons que ces contractions toniques soient une façon pour P. magellanicus de profiter de l’effet de la poussée et de sa coquille de forme assez hydrodynamique. En utilisant des contractions toniques pour maintenir ses valves fermées, P. magellanicus pourrait prolonger la période de temps où il se laisse glisser dans l’eau avant d’ouvrir à nouveau les valves pour une autre contraction phasique. Ainsi, P. magellanicus augmenterait la distance parcourue par contraction phasique et ce sans augmenter le coût énergétique. Lors de la nage chez P. magellanicus et Adamussium colbecki, la phase de fermeture et d’ouverture des valves est de même durée alors que la

169 phase de glisse a une durée légèrement plus longue (Cheng et al. 1996, Bailey and Johnston 2005a). Contrairement à ce qui a été observé chez P. magellanicus et A. colbecki, la phase de glisse semble absente chez Chlamys hastata et Argopecten irradians (Marsh et al. 1992).

Le pétoncle Pecten fumatus est un cas intéressant à plusieurs niveaux. Avec sa coquille de forme peu hydrodynamique et lourde, P. fumatus ne semble pas très bien adapté pour la nage. Lorsqu’il n’est pas dérangé, P. fumatus s’enfouit dans le substrat avec la valve supérieure recouverte d’une fine couche de sédiments. Par contre, lorsque P. fumatus est en contact avec son prédateur, l’étoile de mer Coscinasterias muricata, il va faire une intense série de contractions phasique de courte durée suivie de contractions toniques prolongées (Chap. 2, Fig. 2.2). Ainsi, la fuite semble être la première option pour P. fumatus face à son prédateur. L’utilisation intense des contractions phasiques permet d’augmenter la force de poussée et de portance, ce qui rend possible la nage chez P. fumatus malgré les caractéristiques désavantageuses de sa coquille. Les capacités métaboliques du muscle phasique reflètent l’utilisation particulière des deux types de muscles chez P. fumatus. Le muscle phasique de P. fumatus a un contenu élevé de phosphoarginine qui serait utilisé à un rythme élevé, mais il a aussi des capacités glycolytiques élevées pour soutenir les périodes de fermeture prolongée des valves (Chap. 3, Fig. 3.2 et 3.3). Les résultats de l’ACP des paramètres comportementaux indiquent que P. fumatus se distingue des autres espèces au niveau de la composante « intensité » (Chap. 5, Fig. 5.10). Cette capacité de P. fumatus à faire des contractions phasiques à un rythme aussi élevé est possible grâce au positionnement très oblique du muscle phasique dans la coquille (Chap. 5, Fig. 5.6). En effet, l’intensité de la réponse de fuite est fortement corrélée à l’oblicité du muscle adducteur (Chap. 5, Tableau 5.3). Enfin, même si la résilience du ligament semble avoir peu d’influence sur l’efficacité de la locomotion des pétoncles (Denny & Miller 2006), la résilience du ligament de P. fumatus est nettement plus élevée que celle des autres espèces de pétoncles (Chap. 4, Fig. 4.4). Cette propriété particulière du ligament de P. fumatus joue certainement un rôle facilitant l’atteinte des taux élevés de contractions phasiques observés chez cette espèce, mais ce rôle reste encore à être élucidé.

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En milieu naturel, les coquilles de Mimachlamys asperrima sont recouvertes d’une éponge qui réduit la prédation par les étoiles de mer. En effet, la présence de l’éponge sur la coquille du pétoncle sert de camouflage en plus d’interférer au niveau de l’adhésion des podia de l’étoile de mer (Pitcher & Butler, 1987). Cette relation entre le pétoncle et l’éponge semble avoir joué un rôle au niveau de l’évolution de la réponse de fuite de M. asperrima face à un prédateur. En effet, la réponse première de M. asperrima est de fermer ses valves lorsqu’il est en contact avec son prédateur (Chap. 2, Fig. 2.2). Si le contact avec le prédateur persiste, M. asperrima va faire de courtes séries de contractions phasiques et va rapidement s’épuiser et garder ses valves fermées pour une période prolongée (Chap. 2, Fig. 2.2). La coquille de forme équiconvexe et légère pourrait faciliter la nage chez M. asperrima. D’un autre côté, la coquille est très étanche une fois les valves fermées ce qui permet une bonne protection contre la prédation, mais nuit à la propulsion par jet d’eau. De plus la coquille de M. asperrima est très rugueuse ce qui facilite fort probablement la colonisation par l’éponge. Ainsi, la meilleure stratégie pour M. asperrima est de garder ses valves fermées et d’attendre que le prédateur s’éloigne. Enfin, les capacités glycolytiques du muscle phasique de M. asperrima sont élevées, mais la capacité d’appuyer les contractions phasiques est plus faible (Chap. 3, Fig. 3.2 et 3.3).

Le pétoncle Crassadoma gigantea a pour principal prédateur les étoiles de mer. Puisqu’il est cimenté au stade adulte, C. gigantea ne peut évidemment pas nager et la seule option reste la fermeture des valves (Yonge 1951, Lauzier & Bourne 2006). La coquille de C. gigantea est lourde et de forme irrégulière avec une surface externe couverte de pics qui facilitent la colonisation de divers organismes, créant ainsi un camouflage pour le pétoncle. Lorsque les valves sont fermées, elles sont presque totalement étanches ce qui procure une bonne protection au pétoncle (Fig. 5.1). En réponse à son prédateur, C. gigantea va garder ses valves fermées et de temps à autres il peut faire quelques rares contractions phasiques (Chap. 2, Fig. 2.2). Crassadoma gigantea est semblable aux autres espèces de pétoncles, en termes de proportion muscle phasique et tonique (Chap. 5, Fig. 5.3 et 5.4). Les capacités glycolytiques du muscle phasique de C. gigantea sont élevées pour un pétoncle, mais restent beaucoup plus faibles que chez l’huître qui peut maintenir une fermeture pendant des périodes beaucoup plus longues (Chap. 3, Fig. 3.3). Les niveaux de

171 phosphoarginine sont plus bas chez C. gigantea comparativement à toutes les autres espèces de pétoncles à l’étude, ce qui reflète sa faible capacité pour les contractions phasiques (Chap. 3, Fig. 3.2). Une observation intéressante, c’est que l’activité de la myosine ATPase du muscle phasique de C. gigantea est beaucoup plus faible que celle des autres espèces de pétoncles, mais similaire à celle de l’huître (Chap. 3, Fig. 3.4).

Considérations ontogéniques Il est important de rappeler que cette étude s’est faite avec des pétoncles au stade adulte. Chez toutes les espèces, plusieurs aspects de la morphologie, la fonction et le mode de vie changent durant l’ontogénie. Ainsi les liens entre la stratégie de nage, les capacités métaboliques du muscle phasique, les propriétés du ligament et la morphologie du muscle et de la coquille, ne peuvent être généralisés au cours de l’ontogénie de nos espèces.

Les pétoncles changent typiquement de mode de vie au cours de leur développement. En effet, la plupart des espèces de pétoncles vont avoir un mode de vie attaché via un byssus tôt au cours de leur développement (Beninger & Le Pennec, 2006). Certaines espèces de pétoncles vont garder ce mode de vie au stade adulte, alors que d’autres espèces vont perdre définitivement leur byssus une fois qu’ils ont atteint la maturité (Beninger & Le Pennec, 2006). Certaines espèces, telle C. gigantea, vont avoir un changement drastique passant d’un mode de vie libre capable de nager à un mode de vie cimenté au substrat (Yonge 1951, Lauzier & Bourne 2006).

Au cours de l’ontogénie, il y a aussi des changements au niveau de la morphologie de la coquille et du muscle adducteur (Merrill 1961, Gould 1971, Dadswell & Weihs 1990). Ces changements affecteraient la capacité de nage et la vulnérabilité du pétoncle face aux prédateurs (Dadswell & Weihs 1990, Manuel & Dadswell 1991, Manuel & Dadswell 1993, Barbeau & Scheibling 1994b, Schmidt et al. 2008, Labrecque & Guderley 2011). De la même façon, l’environnement où se trouve le pétoncle peut changer au cours de l’ontogénie. En effet certaines espèces, telle que C. islandica, montrent un changement

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d’habitat et de type de prédateur au cours de leur ontogénie (Arsenault & Himmelman 1996a, Arsenault & Himmelman 1996b). Ainsi les compromis imposés par le style de vie, le type d’environnement et de prédateurs vont changer au cours du développement du pétoncle. De la même façon, les liens entre la stratégie de nage, les capacités métaboliques du muscle phasique, ainsi que la morphologie du muscle et de la coquille risquent de varier selon le stade de vie du pétoncle.

Même morphologie de la coquille, mêmes adaptations? Cette étude comparative a contribué à améliorer la compréhension des liens entre la performance locomotrice et les composantes du système locomoteur chez 5 espèces de pétoncles. Par contre, il est difficile d’extrapoler les observations à toutes les espèces de pétoncles. En effet, plusieurs espèces de pétoncles présentent une même morphologie de la coquille, mais vivent dans des environnements complètement différents. Par exemple, P. fumatus au large de la Tasmanie, P. maximus au large de l’Angleterre et E. ziczac au large du Vénézuela ont tous une coquille plano-convexe. Or, le type d’environnement et de prédateur ne sont pas nécessairement les mêmes. Pour une même morphologie de la coquille, est-ce que ces trois espèces de pétoncles ont les mêmes combinaisons de stratégie de nage, capacités métaboliques du muscle et morphologie du muscle? Est-ce qu’il existe plusieurs solutions possibles pour compenser pour une morphologie coquillaire désavantageuse pour la nage ou est-ce que c’est toujours la même combinaison qui est utilisée? Mimachlamys asperrima et Chlamys islandica sont très semblables au niveau de la morphologie de la coquille, mais M. asperrima est la seule des deux espèces dont la coquille est recouverte d’une éponge inhibant la prédation par les étoiles de mer. Qu’en est-il de la réponse de C. islandica face à un prédateur? Puisqu’il n’y a par d’éponge sur sa coquille pour le camoufler, quelle stratégie sera utilisée par C. islandica face à un prédateur en fonction des contraintes imposées par la morphologie de sa coquille?

L’évolution convergente existerait au niveau de la forme de la coquille des pétoncles. En effet, deux lignées de pétoncles reconnus pour leur capacité à nager sur de longues distances, Amusium balloti et Amusium pleuronectes, auraient évolué à partir de

173 deux ancêtres séparés ayant des modes de vie différents (Serb et al. 2011). Ces deux espèces de pétoncles, ayant le même mode de vie, auraient évolué vers une seule et même solution anatomique pour le même problème écologique (Serb et al. 2011). Serb et collaborateurs (2011) suggèrent que la sélection pour la forme de la coquille et le comportement serait importante pour la diversification des différents groupes de pectinidés. Par contre, le comportement de nage des pétoncles à l’étude a été inféré à partir d’anecdotes provenant de la littérature et l’étude s’est limitée à la morphologie de la coquille des pétoncles. Est-ce que l’évolution convergente observée au niveau de la coquille s’étend aussi au niveau de la morphologie du muscle adducteur ainsi qu’au patron d’utilisation des deux parties du muscle adducteur? Placopecten magellanicus et Adamussium colbecki seraient, tout comme A. balloti et A. pleuronectes, des pétoncles reconnus pour leur capacité à nager sur de longues distances (voir Fig. 2 dans Serb et al. 2011). Or, nos travaux ont montré des différences entre A. balloti et P. magellanicus au niveau du parton d’utilisation des deux parties du muscle adducteur, ainsi qu’au niveau de la morphologie de la coquille et du muscle adducteur. Ainsi, il semble que deux espèces de pétoncles, partageant le même mode de vie, présentent des différences au niveau de l’utilisation du muscle et de la morphologie de ce dernier.

Les liens fonctionnels et évolutifs entre la performance locomotrice et les différentes composantes du système locomoteur sont complexes. Ces liens sont le résultat de compromis imposés par le style de vie, le type d’environnement et de prédateurs où évolue le pétoncle. Les pétoncles sont un bon modèle animal afin d’essayer de mieux comprendre ces liens fonctionnels et évolutifs. Mon étude montre qu’il est important d’intégrer les différents niveaux d’organisation de l’animal car bien souvent la forme ne révèle pas tout.

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