The sounds of your voice and laughs, the smell of ready hot coffee in the kitchen ...... To simulate ocean acidification conditions, a pH-controller (AQUA1 MOD.
Volatile Organic Compounds from Marine Plants: Effects on the Behaviour of Benthic Invertebrates A thesis submitted to the Open University of London for the degree of
Doctor of Philosophy by
Emanuela Butera Master of Science in Marine Ecology, University of Palermo, Palermo-Italy
Stazione Zoologica Anton Dohrn, Naples, Italy The Open University, London, United Kingdom
Director of studies: Dr.Valerio Zupo External supervisor: Prof. Eric von Elert Co-supervisor: Dr. Elisabetta Tosti Advisor: Prof. Marco Vannini
September, 2015
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Ai miei nonni, For all of their sacrifices, patient and love
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ABSTRACT Marine organisms rely on chemical cues- most as scents- to obtain information (i.e. infochemicals) about their environment. Volatile organic compounds (VOCs) from plants represent one group of infochemicals that can shape ecological interactions and the structure of the ecosystems through the influence of behaviour of receivers. However, there is still little evidence about the ecological importance of VOCs on the structure and interactions of mesograzers community within ecosystems like the Mediterranean seagrass Posidonia oceanica. The studies presented in this thesis were designed, using behavioural choice assays, to investigate the effects of epiphytes-borne VOCs from Posidonia oceanica on the associated benthic invertebrates and further clarify how their responses to these putative infochemicals could affect the spatial organization of this stable community. The objectives of this thesis were: 1) to standardize a behavioural choice method for benthic invertebrates (e.g. static chambers vs. flumes and the determination of the species-specific minimum number of replicates); 2) to define the existence of associative behavioural patterns of invertebrates (i.e. mollusc and decapods) to epiphyte-borne VOCs at the community level; 3) to identify the roles of VOCs as infochemicals (e.g. food or toxins cues); and 4) to investigate the behavioural responses to infochemicals within the range of pH levels predicted for the end of this century. Species-specific behaviours of mesograzers to VOCs depended on the method of investigation, the concentration of VOCs tested, body constraints and animal ecology. Behaviours of mesograzers appeared more coherent with the concentrations of VOCs within the static chambers compared with those recorded in flumes. When sympatric from Posidonia oceanica and generalist epiphytes were considered, mesograzers showed to fine-tune their behaviours to VOCs according to their ecology responding to infochemicals faced in their own microhabitat like the leaf axis of P. oceanica. However, at lower pH the invertebrates switched their behavioural patterns across the VOC bouquets replacing their ―natural ecological preferences‖ with ―taxonomic preferences‖, leading to a simplification of chemical relationships within P. oceanica.ecosystem. Overall, the contribution of this thesis to knowledge is that, within ecosystems like the seagrass P. oceanica, co-evolutionary patterns of infochemicals create microhabitats and the coexistence of herbivores on a single plant leaf. These findings support a better understanding of the entire ecosystem for further coastal management also in prevision of near-future climate changes. iii
ACKNOWLEDGEMENTS I would like to thank the Open University and Stazione Zoologica Anton Dohrn for giving me the opportunity and fellowship to pursue my Ph.D. In particular, I would like to thank my thesis defense committee members, Dr. Mark Briffa, Dr. Paolo Sordino, and Dr. Christophe Brunet for their brilliant comments, questions and suggestions, which made my defense one of the best moment of my Ph.D. I would like to thank my supervisors for their advice and support during this project. Thanks Valerio, for conceding me the great freedom in carrying out the research and to have shared throughout these years your knowledge on the maintenance of animals and algae under laboratory conditions. Thanks Eric, although the kilometers of distance, you have been always present with your constructive criticism and comments, which helped me remaining focused during the writing-up of this thesis. I will never forget the way that you listened to me, and your pat on my shoulder. Thanks Elisabetta for your kind encouragements over these years. I am thankful for your way of appreciating the smallest of my results. Special thanks go to all the scientists and students with whom I had the luck to collaborate with during my studies: Dr. Patrick Fink (University of Cologne) because his expertise on volatiles, their extraction, his constant and kind presence and his precious suggestions, as only a supervisor does, have been much appreciated. Dr. Maria Cristina Buia (SZN) for her invaluable suggestions on epiphytes and Posidonia oceanica; Lucia Porzio (Università ‗Federico II‘ di Napoli) for all the times I asked her to help with the identification of epiphytes. She always helped me with a smile! Dr. Francesco Paolo Patti (SZN) and Antonia Chiarore (Università ‗Federico II‘ di Napoli) for their help in the identification of Molluscs and their suggestions; to Dr. Maria Cristina iv
Gambi (SZN) and Alexia Massa Gallucci (SZN) for the taxonomy identification and help with Polychaetes; to Dr. Giovanna Romano (SZN) for her kindness of human being and precious help with the decadienal and sea urchins experiments; to Prof. Michele Scardi (Università ‗Tor Vergata‘ di Roma) for the long afternoon giving me suggestions on statistics and graphs. Very special thanks go to my previous supervisor, Sam Dupont (The Sven Lovén Centre for Marine Sciences, University of Gothenburg) and my best lab mates of ever and now friends, Narimane Dorey (The Sven Lovén Centre for Marine Sciences, University of Gothenburg), Triranta Sircar (University of Gothenburg), Macarena S. Valiñas (Universidad Nacional de Mar del Plata, Argentina), Isabel Casties, Meike Stumpp and Marian Yong-An Hu (IFM-GEOMAR, Kiel, Germany), who taught me all the tips on how to work with pH and larvae. I will never forget the long months of the experiment and our funny talks on The Walking Dead and of course, sometimes science, mackerel season and barbecue nights in Kristineberg. Next, it will be a long list! I would like to thank all the members of Stazione Zoologica of Ischia! Coffee breaks (those rare times- that all of you know - I came for cakes) and lunchtime were the most important parts of my long days in lab. The most special thank goes to Rosanna Messina. You gave me ―life‖ advice, reminding me to eat and drink and to take care of my health. I miss already the internal calls for ―coffee‖ breaks: , or the glass of water filled up by you…What else I could say. You have been my adoptive mom throughout these years in Ischia-thank you! Thanks Amit Kumar, for our long days spent in lab and our way walking back home, talking about science problems and solutions and giving each other support. Only a good friend does that, so thank you! Thanks to all my other lab mates: Antonia Chiarore, Giulia Valvassori, Sara Fioretti, Emanuela Di Meglio, Nuria Teixido for our girls‘ chatting; Martina Mulas for our long v
talks about everything and especially on rock music bands. Our first concert together was awesome! To Daniele Grech for the wild spring fennel gift harvested on mount Epomeo during his Sunday walks. To Maria Cristina Buia for her amazing way to say always what she thinks. I liked her from the first welcoming moment! To Maurizio Lorenti for his constant presence and immediate help when exhausted autoclave and pHmeter fought against me. To Anna Rando for her way to remind at eleven o‘clock: and to see life like a mom; To ―The Captain‖ Vincenzo Rando for his funny whistling way and, of course, for his tasty Tarallini pugliesi always ready on the boat after sampling. To Bruno Iacono for his very analytical way to describe people. The sounds of your voice and laughs, the smell of ready hot coffee in the kitchen every day, made me feeling like home and you as my adoptive family. So, just thank you! Last but not least, I would thank my family and the two Francesco making me being who I am. To my dad and mom, because they thought me what Freedom is. I will always say what I think and I will always follow my heart. To my tall brother to be my anchor in life shaking my nerd brain in his rude way every time I need, and to my short sister in law for being the sister I have never had. To my first Francesco, my almost four years old nephew who every time reminds me what the unconditional love is! And thanks to my second unexpected love, Francesco, for his patience, for all the times I said: . He never went. Thanks to him, that makes me smiling to life every day, reminding me that something beautiful always might be there. Right there, in the front spot during a very annoying seminar.
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TABLE OF CONTENTS ABSTRACT _________________________________________________________ iii ACKNOWLEDGEMENTS ____________________________________________ iv TABLE OF CONTENTS _____________________________________________ vii LIST OF FIGURES __________________________________________________ xi LIST OF TABLES __________________________________________________ xix CHAPTER 1 _________________________________________________________1 Introduction __________________________________________________________1 1.1 Overview________________________________________________________2 1.2 The general context of marine chemical ecology _______________________2 1.3 The current knowledge on infochemicals _____________________________6 1.4 Volatile Organic Compounds from marine plants and their functions as infochemicals __________________________________________________________9 1.5 The method: behavioural choices in the investigation of infochemicals ___17 1.6 Behavioural traits and mechanisms of chemoreception in marine invertebrates _________________________________________________________18 1.7 The seagrass Posidonia oceanica, its epiphytes and associated invertebrates _____________________________________________________________________21 1.8 Epiphytes-herbivores interactions: the role of infochemicals within Posidonia oceanica ecosystem____________________________________________27 1.9 Research aims and objectives ______________________________________30
CHAPTER 2 ________________________________________________________33 VOCs of epiphytes from Posidonia oceanica trigger infochemicals based communication for benthic inverterbrates ___________________________________33 2.1 INTRODUCTION _______________________________________________34 2.2 MATERIALS AND METHODS ___________________________________37 2.2.1 Isolation and culture of benthic epiphytes from the seagrass Posidonia oceanica ____________________________________________________________37 vii
2.2.2 Extraction of algal odours: VOCs _________________________________38 2.2.3 Study organisms: animal sampling and biology ______________________38 2.2.4 Experiment 1: choice assay in static chambers _______________________41 2.2.5 Experiment 2: choice assay in straight flow-through flume _____________43 2.2.6 Baseline choice assays _________________________________________47 2.2.7 Distribution of VOCs in static chambers ___________________________47 2.2.8 Distribution of VOCs in straight flow-through flume _________________48 2.3 STATISTICAL ANALYSIS _______________________________________49 2.3.1 Standardization of behavioural data _______________________________49 2.3.2 Effect of time in behavioural responses of invertebrates _______________50 2.3.3 Recognition of algal odours and associative patterns __________________51 2.3.4 Distribution of odours in static chambers and in straight flow-through flumes ___________________________________________________________________52 2.4 RESULTS ______________________________________________________53 2.4.1 Kriging analysis for the diffusion in static chambers __________________53 2.4.2 Kriging analysis for the flow in straight flow-through flume____________54 2.4.3 Behavioural responses of invertebrates to VOCs in static chambers ______55 2.4.4 Behavioural responses of invertebrates to VOCs in flumes _____________58 2.4.5 Behavioural associative patterns to VOCs __________________________61 2.5 DISCUSSION___________________________________________________65 2.6 CONCLUSION _________________________________________________69
CHAPTER 3 ________________________________________________________71 Epiphyte-borne infochemicals rule the community structure of mesograzers in seagrasses ____________________________________________________________71 3.1 INTRODUCTION _______________________________________________72 3.2 MATERIALS AND METHODS ___________________________________76 3.2.1 Study organisms: animal sampling and biology ______________________76 3.2.2 Production of epiphytes: seaweeds and diatoms______________________78 3.2.3 VOCs extraction ______________________________________________81 3.2.4 Gel preparation and concentrations _______________________________81 3.2.5 Behavioural choice tests ________________________________________82 3.2.6 Standardization of behavioural choice test: controls __________________84 viii
3.3 STATISTICAL ANALYSIS _______________________________________85 3.3.1 Standardization of controls ______________________________________85 3.3.2 Behavioural choices to VOCs, effect of concentration and time of exposure ___________________________________________________________________87 3.3.3 Multivariate analysis: taxonomy vs. environmental hypothesis __________88 3.4 RESULTS ______________________________________________________89 3.4.1 Control experiments: the need for standardization of behavioural data ____89 3.4.2 Responses to VOCs, concentrations and time of exposure for each mesograzer __________________________________________________________93 3.4.3 Responses of the community of mesograzers to infochemicals _________101 3.5 DISCUSSION__________________________________________________105 3.5.1 The mesograzers‘ standardized behaviour _________________________105 3.5.2 Volatile epiphyte-borne infochemicals structure mesograzers‘ microhabitat selection upon the leaves of Posidonia oceanica ____________________________107 3.6 CONCLUSION ________________________________________________112
CHAPTER 4 _______________________________________________________114 Decoding the messages beyond epiphyte-borne volatiles: food kairomones, alarm and habitat cues ________________________________________________________114 4.1 INTRODUCTION ______________________________________________115 4.2 MATERIALS AND METHODS __________________________________118 4.2.1 Animal collection ____________________________________________118 4.2.3 Preparation of artificial foods ___________________________________118 4.2.4 Feeding test and survival ______________________________________120 4.2.5 Statistical analysis ____________________________________________121 4.3 RESULTS _____________________________________________________122 4.3.1 Putative toxicity _____________________________________________122 4.3.2 Feeding preferences __________________________________________125 4.4 DISCUSSION__________________________________________________129 4.5 CONCLUSION ________________________________________________133
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CHAPTER 5 _______________________________________________________135 Ocean acidification effects on the behaviour of mesograzers to epiphyte-borne infochemicals __________________________________________________________135 5.1 INTRODUCTION ______________________________________________136 5.2 MATERIALS AND METHODS __________________________________139 5.2.3 Animal collection and epiphyte cultures __________________________139 5.2.4 Simulated ocean acidification treatment ___________________________141 5.2.5 Experimental design and behavioural choice tests ___________________142 5.2.6 VOCs extractions and gels preparations ___________________________144 5.2.7 Statistical analysis ____________________________________________144 5.3 RESULTS _____________________________________________________146 5.3.1 Responses of molluscs to volatile infochemicals at different pH ________146 5.3.2 Responses of decapods to volatile infochemicals at different pH _______149 5.3.3 Reactions of mesograzers‘ community to infochemicals at different pH __152 5.4 DISCUSSION__________________________________________________157 5.5 CONCLUSION ________________________________________________161
CHAPTER 6 _______________________________________________________163 Discussion and Conclusion ____________________________________________163 6.1 General discussion ______________________________________________164 6.2 Conclusions and future directions _________________________________171 6.2.1 Epiphyte-odours are infochemicals for benthic invertebrates __________171 6.2.2 Epiphyte-borne infochemicals structure the benthic community within the Posidonia oceanica ecosystem __________________________________________172 6.2.3 OA disrupts behaviour to infochemicals and indirectly the structure of ecosystems _________________________________________________________173 Appendix 1_Results on Full Factorial ANOVA for Chapter 2 _______________176 Appendix 2_Table of pH measurements within Petri-dishes for Chapter 5 ____179 REFERENCES _____________________________________________________181
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LIST OF FIGURES CHAPTER 1 Figure 1.1 The Venn diagram illustrates chemical ecology as an integrative science at the intersection of the four main approaches of chemistry, ethology, neuroscience, and ecology. Figure from Derby and Sorensen, (2008). Figure 1.2 Chemical-ecological interactions driven by chemical defences and infochemicals produced by higher animals and primary producers in freshwater and marine ecosystems. Figure from Brӧnmark and Hansson, (2012). Figure 1.3 The terminology of infochemicals given according to Dicke and Sabelis (1988). E is the Emitter and R is the Receiver, the symbol + indicates which one between emitter and/or receiver benefits from the chemical message. Figure 1.4 The planktonic crustacean Daphnia sp. shows several defence strategies against predators‘ chemical cues. (A) Scanning electron micrographs showing morphological defences against several predators in various Daphnia species (Photograph by C. Laforsch, from Chemical ecology in aquatic systems. Brӧnmark & Hansson eds. 2012). (B) Reproduction of Daphnia showed in the absence of predators (middle panel) and defence strategies with predators. In the left panel, behavioural (i.e. diel vertical migrations) and physiological changes (e.g. earlier sexual maturity at a smaller size with increased number of offspring of reduced size) are present in response to fish predation. In the right panel are showed morphological changes (e.g. spines in the neck) and increased body size but decreased number of offspring in the presence of the Chaoborus larvae as predators (Illustration by L. Weiss, from Chemical ecology in aquatic systems. Brӧnmark & Hansson eds. 2012).
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Figure 1.5 (a) Dynamic web of interactions and constraints driven by infochemicals (Figure modified from Klaschka, 2008); (b) examples of infochemical relationships within terrestrial ecosystem with specific insects-plant interactions and (c) with a generalist herbivore (Figure modified from Ueda et al. 2012). Figure 1.6 (a) Schematic description of the planktonic use of dimethyl sulphide (DMS) that is a foraging kairomone attracting birds to patches of food (Figure modified from Steinke et al. 2002). (b) Schematic description about the tri-trophic interaction described in Coleman et al. (2007a). Point 1 indicates the herbivore Littorina obtusata feeding on the brown algae Ascophyllum nodosum in a rocky pool. Point 2 indicates the release of VOCs from grazed A. nodosum, acting as SOS signal that attracts as foraging infochemical the predator green crab Carcinus maenas that in point 3 ―rescues‖ the algae by eating the herbivore. Figure 1.7 Photo of a meadow of Posidonia oceanica (L.) Delile with visible epiphytes on the leaves (left) and on the right (d) an image of a shoot of P.oceanica with the rhizome covered by scales (remaining of the old leaf bases) showing also (a) adult leaf, (b) intermediate leaf and (c) juvenile leaf. (Image modified from Buia et al. 2004). Figure 1.8 Illustration of the spatial succession of algal epiphytes over a leaf axis of Posidonia oceanica that shows the increase in species and abundance from the basal towards the apical zone. (Figure modified from Mazzella et al. 1994 (eds.), Le praterie sommerse del Mediterraneo). Figure 1.9 The diagram shows the complex trophic relationships within a meadow of Posidonia oceanica against the ‗seagrass grazing paradigm‘ (Figure from Mazzella and Zupo, 1995).
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Figure 1.10 Example of some mesograzers associated to Posidonia oceanica: (left) the shrimp Hippolyte inermis, (centre) the hermit crab Cestopagurus timidus, and (right) the mollusc Bittium latreillii. CHAPTER 2 Figure 2.1 The sampling site in Lacco Ameno (40°45‘ N/13°50‘ E) at the north east of Ischia Island (Tyrrhenian Sea, Italy) is indicated by a grey square. Figure 2.2 Experimental arena used in static choice experiments indicating (a): different sectors (from Jüttner et al. 2010); (b) Photo of the experimental setup (April, 2013).where each arena was positioned with the (+) targets opposite, to randomize directional effects. Figure 2.3 Straight flow-through flume system designed according to Atema et al. (2002): (A) project in 3D (gently courtesy from M. Mutalipassi). Overall, the chamber size was 21 cm x 14 cm x 10 cm (length x width x height) in overshadowing glass, 4 mm thick. The arrows indicate the test area and the two inflow compartments. (B) The scheme shows the choice flume system from the upside view with: c) water inflow compartments (with arrows); d) packed drink straws to reduce the turbulence; e) barrier-separated channels to further laminate flow; f) upstream fine mesh (0.5 mm) net to contain animals; g) test area; h) starting point for the acclimation; i) downstream containment net and j) drain area with the central drain opening. Figure 2.4 Flumes are visualized here in the (a) upside and (b) lateral view for photographic purposes using 0.25 g L-1 of Methylen blu dye dissolved in ambient seawater. Figure 2.5 Kriging reconstruction of the distribution of the 2-trans-4-trans-decadienal volatile compound at (a): 5; (b): 10; (c): 15; and (d): 20 min after the start of the experiment. Isolines indicate areas with identical concentrations of this volatile organic
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compound according to the intensity scale on the right (µg mL-1) while the scales on the left and at the bottom indicate the diameter of the static choice arena (cm). Figure 2.6 Kriging reconstruction of the distribution of Methylen blue dye at (a): 3; (b): 4; (c): 5 min after the start of the experiment. Isolines indicate areas with identical concentrations of the dye according to the intensity scale on the right (µg mL-1) while the scales on the left and at the bottom indicate the dimension of the flume chambers 21 cm x 14 cm (length x width). Figure 2.7 Mean (± SE) of the score weight preferences for the 9 species of invertebrates at the different concentrations of VOC bouquets of (a) Enteromorpha prolifera, (b) Colaconema daviesii and (c) Cyanobacteria in static chambers. Only the significant choices from one sample t-test analysis (p
