Cues and consequences Copepodamides as mediators of defence induction in marine phytoplankton Milad Pourdanandeh Doctoral Thesis Department of Marine Sciences, Faculty of Science and Technology 2025 Dissertation for the degree of Doctor of Philosophy, Ph.D., in Natural Sciences, specialising in Marine Sciences, University of Gothenburg, 2025 Cues and consequences: Copepodamides as mediators of defence induction in marine phytoplankton Milad Pourdanandeh Except for the appended articles and figures, this thesis is licenced under a Creative Commons Attribution-ShareAlike 4.0 International Licence (CC BY-SA 4.0) Cover illustration: Ante Wiklund, Otyp form Back cover photo: Milad Pourdanandeh (Photo credit: Miranda Nichols) This book was typeset by the author. ISBN: 978-91-8115-449-8 (PRINT) ISBN: 9978-91-8115-450-4 (PDF) Electronic version available at http://hdl.handle.net/2077/89618 Printed by Stema Specialtryck AB, Borås, Sweden, 2025 Keywords: Bioluminescence, Chain-suppression, Chemical cues, Chemical ecology, NENMÄRVA KE Copepod, Copepodamides, Diatom, Dinoflagellate, Ecophysiology, Harmful algal blooms, Inducible defences, Phycotoxin, Phytoplankton, Plant defence, Plankton, Predator-prey Trycksak3041 0234 interactions i S T To my māmān, without whom I would not be here, and to Queen for an equally obvious reason. ii If you need a distraction during my defence, please enjoy this grid. It’s for playing Dots and Boxes/Squares, but feel free to use it as you wish. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . iii Table of contents Abstract ...................................................................................................................................... 1 Populärvetenskaplig sammanfattning ........................................................................................ 2 Acknowledgements ............................................................................................................... 3 List of papers ............................................................................................................................. 6 Funding statement ...................................................................................................................... 7 Preface ....................................................................................................................................... 8 Chapter 1. Background .............................................................................................................. 9 1.1 A pale blue dot ............................................................................................................. 10 1.2 Plankton ...................................................................................................................... 11 1.3 Chemically mediated interactions and plant defences ................................................. 12 1.4 Inducible defences ....................................................................................................... 15 1.5 Defences against smelly copepods: Copepodamide-mediated responses .................... 17 Chapter 2. Aims of thesis ......................................................................................................... 24 Chapter 3. Organisms and methods .......................................................................................... 26 3.1 Organisms ................................................................................................................... 27 3.2 Methods ....................................................................................................................... 27 Chapter 4. Summary of papers ................................................................................................. 31 Paper I: First description of copepodamides in freshwater copepods ................................ 32 Paper II: Chain suppression of new diatom species .......................................................... 35 Paper III: Simultaneous induction of multiple defence traits ............................................ 38 Paper IV: Inducing Dinophysis .......................................................................................... 41 Paper V: Bottom-up versus top-down induction of phycotoxins: A meta-analysis ............ 43 Chapter 5. Conclusions and implications ................................................................................. 47 References ........................................................................................................................... 52 iv Abstract Populärvetenskaplig sammanfattning Marine phytoplankton account for less than one percent of Earth’s primary-producer biomass, yet Marina växtplankton utgör mindre än en procent av jordens fotosyntetiska biomassa, men står ändå they sustain almost half of global primary production. These unicellular organisms face more för nästan hälften av den globala primärproduktionen. Dessa encelliga organismer utsätts för ett intense predation pressure than terrestrial plants, primarily from microzooplankton but also from starkt betningstryck, särskilt från mikrodjurplankton men även från hoppkräftor (copepods på copepods, which are among the most abundant animals on Earth. Consequently, phytoplankton have engelska), kanske jordens mest talrika djur. Växtplankton har därför utvecklat flera evolved a suite of defence traits to reduce grazing losses. Because plankton generally lack complex försvarsmekanismer mot sina betare. Eftersom plankton generellt saknar avancerade syn- och visual and auditory senses, they rely mainly on chemical and hydromechanical signals and cues in hörselsinnen förlitar de sig i hög grad på kemiska signaler i omgivningen för att navigera livets their environment to navigate the main Darwinian missions of life. Marine copepods exude a group utmaningar. Marina hoppkräftor utsöndrar en grupp kemiska ämnen som kallas copepodamider. of polar lipids, copepodamides, that have been co-opted by taxonomically diverse prey organisms, Dessa ämnen uppfattas av växtplankton – främst kiselalger och dinoflagellater – som signaler att mainly diatoms and dinoflagellates, to act as general alarm cues that copepods are nearby. This hoppkräftor finns i närheten. När de känner denna “lukt” av sina betare inducerar de leads prey to induce morphological, behavioural, and biochemical defences that help them evade försvarsmekanismer som minskar risken att upptäckas och ätas, till exempel kortare kedjelängd, and reduce predation. Examples of these defence traits include colony-size reduction, which lowers ökad produktion av alggifter, eller starkare bioluminiscens (mareld). encounter rates with predators, and elevated toxin production or bioluminescence, which increase rejection by copepods and redirects grazing towards less-defended prey. Denna avhandling undersöker hur utbredd förekomsten av copepodamider är bland hoppkräftor, vilka organismer som kan upptäcka och svara på dem, samt vilka konsekvenser sådana svar får på This thesis investigates how widespread copepodamides are among copepods, which organisms can individ-, populations-, och ekosystemnivå. Jag visar, för första gången, att sötvattenhoppkräftor detect and respond to them, and the consequences of those responses at individual, population, and innehåller copepodamider i mängder jämförbara med marina arter men att sammansättningen av ecosystem levels. Analysing individual copepods and bulk zooplankton samples, I show for the first copepodamiderna skiljer sig åt mellan hoppkräftor beroende på miljö. Med hjälp av renframställda time that freshwater copepods contain copepodamides in amounts comparable to marine copepods, copepodamider kan vi simulera närvaron av hoppkräftor i kontrollerade experiment, utan att that the composition differs markedly between the two groups, and that freshwater copepods samtidigt introducera störande faktorer från betande hoppkräftor. På detta sätt bekräftar jag tidigare putatively possess four unique copepodamide congeners. By using purified copepodamides to fynd att den vanligt förekommande kedjebildande kiselalgen Skeletonema marinoi förkortar sina simulate copepod presence in controlled exposure experiments I corroborate previous evidence that kedjor när de exponeras för copepodamider. Tre andra, tidigare otestade arter av kiselalger svarade the commonly occurring chain-forming diatom Skeletonema marinoi reduces chain length in däremot inte med att förkorta sina kedjor. Copepodamid-inducerad kedjeförkortning bör därför inte response to copepodamides, that three species not previously tested do not respond similarly, and antas vara en generell försvarsstrategi mot hoppkräftor bland kedjebildande kiselalger. Jag lägger that chain-suppression should thus not be assumed to be a general anti-copepod defence strategy. I också till ytterligare arter av dinoflagellater som bildar skadliga algblomningar (HAB, från also expand the catalogue of dinoflagellate species associated with harmful algal blooms (HABs) engelskans harmful algal blooms) till listan över organismer som svarar på copepodamider. that respond to copepodamides: Gymnodinium catenatum, Alexandrium catenella, and Dinophysis Gymnodinium catenatum, Alexandrium catenella och Dinophysis sacculus ökar i giftighet, medan sacculus increase phycotoxin production, while Alexandrium catenella and Protoceratium A. catenella och Protoceratium reticulatum ökar intensiteten av sin bioluminiscens. Induktionen av reticulatum increase bioluminescence intensity. Cue-mediated induction of such traits, combined sådana försvarsegenskaper, i kombination med att hoppkräftor ofta föredrar att äta mindre with selective grazing by copepods on less-defended cells, may facilitate HAB-formation and försvarade celler – vilket ökar den relativa andelen försvarade celler i närmiljön – kan underlätta should be incorporated into conceptual models of HAB drivers and HAB forecasting efforts. och driva på bildningen av skadliga algblomningar. Effekter från betare bör därför inkluderas i Finally, research into the drivers of phycotoxin induction in marine HAB species has long been modelleringen av, samt prognoser för, giftiga algblomningar. focused on bottom-up, nutrient availability effects. Accumulating evidence over the last 25 years, Forskning om vad som styr produktionen av alggifter i marina HAB-arter har länge fokuserat på however, shows that top-down grazer-induced effects are also important. However, bottom-up and botten-upp-effekter från näringstillgång. Sedan millennieskiftet har det dock kommit allt fler belägg top-down drivers have rarely been directly compared in primary studies. Using a meta-analytical för att topp-ned-effekten från djurplankton också är en viktig faktor. Trots detta finns mycket få framework, I synthesise and statistically compare published experimental studies on toxin induction primärstudier, och inga tidigare metastudier, som formellt jämför dessa huvuddrivkrafter. Jag caused by relative nitrogen enrichment (nitrogen enrichment or phosphorus limitation) or by presenterar en metaanalys som jämför experimentella studier där giftinduktion orsakats av antingen exposure to grazers or their chemical cues for the two most-studied marine HAB genera, relativ näringstillgång eller djurplanktonbetare (eller deras kemiska ämnen) för de två mest Alexandrium and Pseudo-nitzschia. I find that grazer-induced increases in phycotoxins rival—and studerade HAB-släktena, dinoflagellaten Alexandrium och kiselalgen Pseudo-nitzschia. Jag visar may even exceed—nutrient-driven effects. I discuss these findings in the context of well-developed att betare kan inducera giftighet som är jämförbar med, och ibland överstiger, de väletablerade frameworks of plant-defences in terrestrial plants, and suggest a path towards adopting a unified näringsdrivna effekterna. Jag diskuterar detta inom ramen för etablerade ekologiska ramverk om framework. kemiska växtförsvar utvecklade för landlevande växter, och föreslår en väg mot att anta ett enhetligt Copepodamides constitute a powerful experimental tool: by simulating grazer presence without ramverk som beskriver och förklarar giftproduktion hos växtplankton. direct predation, we can isolate non-consumptive inducible defences and test their mechanisms Copepodamider har visat sig vara ett kraftfullt verktyg inom experimentell planktonekologi. Genom under controlled, repeatable conditions. The use of copepodamides is improving our understanding att simulera hoppkräftors närvaro utan att införa faktisk predation från levande hoppkräftor kan vi of phytoplankton–zooplankton interactions and offers new leverage to test broader ideas in isolera kemiskt inducerade försvar hos deras byten. Upptäckten och användningen av predator–prey ecology and the evolution of anti-grazer defences. The detection of copepodamides copepodamider har redan fördjupat, och kommer att fortsätta fördjupa, vår förståelse av in freshwater copepods, in particular, opens an exciting new research frontier that warrants thorough interaktioner mellan växtplankton och djurplankton, samt bidra till att besvara centrala frågor inom investigation. rovdjur–bytesekologi och evolutionen av betningsförsvar. Särskilt öppnar fyndet av copepodamider hos sötvattenhoppkräftor för en ny och spännande forskningsfront som bör undersökas vidare. 1 2 Abstract Populärvetenskaplig sammanfattning Marine phytoplankton account for less than one percent of Earth’s primary-producer biomass, yet Marina växtplankton utgör mindre än en procent av jordens fotosyntetiska biomassa, men står ändå they sustain almost half of global primary production. These unicellular organisms face more för nästan hälften av den globala primärproduktionen. Dessa encelliga organismer utsätts för ett intense predation pressure than terrestrial plants, primarily from microzooplankton but also from starkt betningstryck, särskilt från mikrodjurplankton men även från hoppkräftor (copepods på copepods, which are among the most abundant animals on Earth. Consequently, phytoplankton have engelska), kanske jordens mest talrika djur. Växtplankton har därför utvecklat flera evolved a suite of defence traits to reduce grazing losses. Because plankton generally lack complex försvarsmekanismer mot sina betare. Eftersom plankton generellt saknar avancerade syn- och visual and auditory senses, they rely mainly on chemical and hydromechanical signals and cues in hörselsinnen förlitar de sig i hög grad på kemiska signaler i omgivningen för att navigera livets their environment to navigate the main Darwinian missions of life. Marine copepods exude a group utmaningar. Marina hoppkräftor utsöndrar en grupp kemiska ämnen som kallas copepodamider. of polar lipids, copepodamides, that have been co-opted by taxonomically diverse prey organisms, Dessa ämnen uppfattas av växtplankton – främst kiselalger och dinoflagellater – som signaler att mainly diatoms and dinoflagellates, to act as general alarm cues that copepods are nearby. This hoppkräftor finns i närheten. När de känner denna “lukt” av sina betare inducerar de leads prey to induce morphological, behavioural, and biochemical defences that help them evade försvarsmekanismer som minskar risken att upptäckas och ätas, till exempel kortare kedjelängd, and reduce predation. Examples of these defence traits include colony-size reduction, which lowers ökad produktion av alggifter, eller starkare bioluminiscens (mareld). encounter rates with predators, and elevated toxin production or bioluminescence, which increase rejection by copepods and redirects grazing towards less-defended prey. Denna avhandling undersöker hur utbredd förekomsten av copepodamider är bland hoppkräftor, vilka organismer som kan upptäcka och svara på dem, samt vilka konsekvenser sådana svar får på This thesis investigates how widespread copepodamides are among copepods, which organisms can individ-, populations-, och ekosystemnivå. Jag visar, för första gången, att sötvattenhoppkräftor detect and respond to them, and the consequences of those responses at individual, population, and innehåller copepodamider i mängder jämförbara med marina arter men att sammansättningen av ecosystem levels. Analysing individual copepods and bulk zooplankton samples, I show for the first copepodamiderna skiljer sig åt mellan hoppkräftor beroende på miljö. Med hjälp av renframställda time that freshwater copepods contain copepodamides in amounts comparable to marine copepods, copepodamider kan vi simulera närvaron av hoppkräftor i kontrollerade experiment, utan att that the composition differs markedly between the two groups, and that freshwater copepods samtidigt introducera störande faktorer från betande hoppkräftor. På detta sätt bekräftar jag tidigare putatively possess four unique copepodamide congeners. By using purified copepodamides to fynd att den vanligt förekommande kedjebildande kiselalgen Skeletonema marinoi förkortar sina simulate copepod presence in controlled exposure experiments I corroborate previous evidence that kedjor när de exponeras för copepodamider. Tre andra, tidigare otestade arter av kiselalger svarade the commonly occurring chain-forming diatom Skeletonema marinoi reduces chain length in däremot inte med att förkorta sina kedjor. Copepodamid-inducerad kedjeförkortning bör därför inte response to copepodamides, that three species not previously tested do not respond similarly, and antas vara en generell försvarsstrategi mot hoppkräftor bland kedjebildande kiselalger. Jag lägger that chain-suppression should thus not be assumed to be a general anti-copepod defence strategy. I också till ytterligare arter av dinoflagellater som bildar skadliga algblomningar (HAB, från also expand the catalogue of dinoflagellate species associated with harmful algal blooms (HABs) engelskans harmful algal blooms) till listan över organismer som svarar på copepodamider. that respond to copepodamides: Gymnodinium catenatum, Alexandrium catenella, and Dinophysis Gymnodinium catenatum, Alexandrium catenella och Dinophysis sacculus ökar i giftighet, medan sacculus increase phycotoxin production, while Alexandrium catenella and Protoceratium A. catenella och Protoceratium reticulatum ökar intensiteten av sin bioluminiscens. Induktionen av reticulatum increase bioluminescence intensity. Cue-mediated induction of such traits, combined sådana försvarsegenskaper, i kombination med att hoppkräftor ofta föredrar att äta mindre with selective grazing by copepods on less-defended cells, may facilitate HAB-formation and försvarade celler – vilket ökar den relativa andelen försvarade celler i närmiljön – kan underlätta should be incorporated into conceptual models of HAB drivers and HAB forecasting efforts. och driva på bildningen av skadliga algblomningar. Effekter från betare bör därför inkluderas i Finally, research into the drivers of phycotoxin induction in marine HAB species has long been modelleringen av, samt prognoser för, giftiga algblomningar. focused on bottom-up, nutrient availability effects. Accumulating evidence over the last 25 years, Forskning om vad som styr produktionen av alggifter i marina HAB-arter har länge fokuserat på however, shows that top-down grazer-induced effects are also important. However, bottom-up and botten-upp-effekter från näringstillgång. Sedan millennieskiftet har det dock kommit allt fler belägg top-down drivers have rarely been directly compared in primary studies. Using a meta-analytical för att topp-ned-effekten från djurplankton också är en viktig faktor. Trots detta finns mycket få framework, I synthesise and statistically compare published experimental studies on toxin induction primärstudier, och inga tidigare metastudier, som formellt jämför dessa huvuddrivkrafter. Jag caused by relative nitrogen enrichment (nitrogen enrichment or phosphorus limitation) or by presenterar en metaanalys som jämför experimentella studier där giftinduktion orsakats av antingen exposure to grazers or their chemical cues for the two most-studied marine HAB genera, relativ näringstillgång eller djurplanktonbetare (eller deras kemiska ämnen) för de två mest Alexandrium and Pseudo-nitzschia. I find that grazer-induced increases in phycotoxins rival—and studerade HAB-släktena, dinoflagellaten Alexandrium och kiselalgen Pseudo-nitzschia. Jag visar may even exceed—nutrient-driven effects. I discuss these findings in the context of well-developed att betare kan inducera giftighet som är jämförbar med, och ibland överstiger, de väletablerade frameworks of plant-defences in terrestrial plants, and suggest a path towards adopting a unified näringsdrivna effekterna. Jag diskuterar detta inom ramen för etablerade ekologiska ramverk om framework. kemiska växtförsvar utvecklade för landlevande växter, och föreslår en väg mot att anta ett enhetligt Copepodamides constitute a powerful experimental tool: by simulating grazer presence without ramverk som beskriver och förklarar giftproduktion hos växtplankton. direct predation, we can isolate non-consumptive inducible defences and test their mechanisms Copepodamider har visat sig vara ett kraftfullt verktyg inom experimentell planktonekologi. Genom under controlled, repeatable conditions. The use of copepodamides is improving our understanding att simulera hoppkräftors närvaro utan att införa faktisk predation från levande hoppkräftor kan vi of phytoplankton–zooplankton interactions and offers new leverage to test broader ideas in isolera kemiskt inducerade försvar hos deras byten. Upptäckten och användningen av predator–prey ecology and the evolution of anti-grazer defences. The detection of copepodamides copepodamider har redan fördjupat, och kommer att fortsätta fördjupa, vår förståelse av in freshwater copepods, in particular, opens an exciting new research frontier that warrants thorough interaktioner mellan växtplankton och djurplankton, samt bidra till att besvara centrala frågor inom investigation. rovdjur–bytesekologi och evolutionen av betningsförsvar. Särskilt öppnar fyndet av copepodamider hos sötvattenhoppkräftor för en ny och spännande forskningsfront som bör undersökas vidare. 1 2 Acknowledgements and Theo. I love you and will miss you both immensely. Thank you especially for these last few months as we’ve prepared our theses and defences. Good luck with your respective post-docs. If you know me, you might not be surprised by the fact that I began writing this section, perhaps Johan E—who would have thought that we would end up here, still together, after meeting while the first (and only) text that most of you will read in this thesis, with less than seven hours left until studying physics more than 11 years ago? Adele, Birte, Stina and Alex C, thanks for the intense the submission deadline. That’s not to say that I haven’t thought about what I would write here cruises, the conferences, and all the good times you always provided. almost every day for several weeks now. It’s hard not to reflect on one’s time and journey in the final weeks of writing one’s thesis, especially when life has changed so drastically in the last five A special thanks to all my officemates through the years, first and foremost to my Auntie Yvonne. years. When I started this PhD, I didn’t know what to expect. The world was still in the first year Thank you for putting up with me for so long, for indulging my R-OCD (at least we got rid of that of the COVID-19 pandemic, but Tiny-hands Donald lost the US presidential election on the second grey line eventually!), and for listening to my rants about the quality of student reports. Henry, I day of my first week, so things seemed to be looking up (but we all know how that’s gone). Looking hope you keep playing Hot Sake after I’m gone. Thanks for putting up with my bullshit! Teresa, back on that time feels strange; spending entire working days, weeks and months at home is almost thanks for being my tea person and for all the music. Apra, I’m sorry for being hard on you at times. a distant memory, obscured by a hazy fog of sourdough bread and Zoom interactions. It was, It comes from a place of love, but that is no excuse. Romain and Phoebe, thanks for the interesting however, also over Zoom that I first met the one who would arguably become the most important discussions we’ve had. I couldn’t think of two more different officemates, and I’m glad I got to person in my life for the entirety of my PhD. Lovisa, thank you for showing me that love is actually experience you both. Finally, to Solange: thank you for being my thesis-buddy in the last few weeks. worth it, even if ours didn’t quite make it. Thank you for always spreading positivity and happiness Your commitment to social issues is truly inspiring, and I wish I could be half the person you are in around you, for picking me up each time I fell, for filling my life with amazing dogs, for giving me that regard. some of the happiest memories of my life, and for being the best dog mum to our Leia. To the younger PhD generation in Natrium (including some of the ones already mentioned above): This thesis would not have been possible without my supervisor(s). Erik, you have been an amazing the twins Linnea & Wilma, our favourite baker Nicolai, Johan S, Doris, Vincent, Mauro, Alisa, mentor and colleague, and I am lucky to have had your support over these five years. Your almost Jakob, Justine, and Loraine. Take care of each other when we seniors are gone. You have some childlike curiosity about the natural world is inspiring, and I hope you never lose it. Thank you for big shoes to fill (specifically Estel’s), but I know you will do an amazing job. I’m always available always answering my barrage of questions, for teaching me so much, and for preaching the gospel for admin support if you need it. and inducting me into the copepodamide cult. To my co-supervisors Jon and Lars: you two sit at To all the people in Botan and Natrium (not already mentioned, and those too): Tobia, JC, Claudy, opposite ends of the spectrum in how you comment on and give feedback on (my) texts. Thank you Sina, Mariana, Izzy, Leo, Laura, Bastien, Seb, Marcel, Jenny, Heléne, Kim, Daniela, Salar, for straightening out my rambling; I’m sorry (or perhaps I should say, you’re welcome(?)) for not Niklas, Matilda, Maria, Grazzia, Joel, and many more. Thank you for all the lunches, fikas, AWs, giving you more to do as co-supervisors. Jon, thank you especially for all the rewarding discussions and other social activities that have made the last five years feel like having the biggest extended about stats and experimental design. You are a giant—both as a teacher and a scientist. By retiring, family imaginable at work every day. Extra love goes to Olga, Liz, and Carina. You have all been you have left an appropriately sized hole in this department; enjoy the quieter life on the Island. To rocks that I have leaned on time and time again. Thank you for all you do for your colleagues. my examiner Helle, thank you for always supporting and encouraging me. Through courses, teaching and other work, I have spent more than two years of my life at Tjärnö I owe many thanks to the copepodamide gang and adjacent people, especially my predecessor and Marine Laboratory without ever being officially stationed there. It truly feels like a second home. PhD big sister Kristie for always supporting and helping me when I felt out of my depth. Thank To the PhD students at Tjärnö, both current and former: Bella, Ben, Luisa, James R, Youk, Chloé, you also to Fredrik, Jenny, Aubrey, Harshith, Anna, Andy, and Josephine. Likewise, thank you Kristoffer, Simon, Steffi, Maru, Lara, Diego, Samuel, Cruise and others. We don’t get to see to all the students and interns who have passed through the group: Henke, Hope, Sina, Marc, each other that often, but I always love it when we do. Thank you for the fikas and defence parties; Paula, Ada, Andrra, Luísa, Anouschka, Hannah, Catherina; it has been a pleasure to help you keep taking care of each other. To the rest of the student and staff at Tjärnö: thank you for always with R over Zoom. To my current PhD sister Isa: we may not be doing similar research, but I have making me feel welcome when I visit. Extra special thanks to Ann L for being my BSc and MSc loved getting to know you over the last year and a half. I’ll make sure to stop by your 200 × 200 m supervisor and best teaching colleague in MAV109; to Gunilla T for always taking time for all who area in Möllan when I’m in Malmö. need it and for introducing me to the world of meta-analyses; and to Per J for always being available Many other collaborators, students, and colleagues need acknowledging. If you are reading this to talk stats, ecology, plankton, or anything else one might wonder about in marine research. right now, you are definitely one of them (even if I forget to mention you by name). To the PhD Another retired giant! students in the department as a whole and to the ECRs, I’ve said it before and I’ll say it again: we To everyone mentioned and not: It has truly been an honour and a privilege to share these five years (now you) are the single most cohesive and tight-knit group of people in this place. In a department (or parts of them) with you. that has at times experienced tensions and conflicts between factions, you have shown what true unity, collaboration, and collegial support look like. Never stop working to improve the PhD As some of you may know, I am of the opinion that free will is an illusion. Although one can make programme and your work environment, not just for yourselves but also for one another. philosophical arguments for why this is the case, as a biologist, I tend to prefer arguments based on empirical evidence and evolutionary perspectives. The more we learn about animal and human To the ones who came before and led the way: Björn, Rickard, Ella, James H, Alice, Matt, Astrid, behaviour and neuroscience, the more obvious it appears to me that we do what we do because of Johanna, Martins M & E. Thank you for putting up with all my many annoying questions in those who we are, and that we do not control who we are. Who we are is shaped by a complex interaction early years. To the ones who have been on this journey more or less with me from the start: Estel 3 4 Acknowledgements and Theo. I love you and will miss you both immensely. Thank you especially for these last few months as we’ve prepared our theses and defences. Good luck with your respective post-docs. If you know me, you might not be surprised by the fact that I began writing this section, perhaps Johan E—who would have thought that we would end up here, still together, after meeting while the first (and only) text that most of you will read in this thesis, with less than seven hours left until studying physics more than 11 years ago? Adele, Birte, Stina and Alex C, thanks for the intense the submission deadline. That’s not to say that I haven’t thought about what I would write here cruises, the conferences, and all the good times you always provided. almost every day for several weeks now. It’s hard not to reflect on one’s time and journey in the final weeks of writing one’s thesis, especially when life has changed so drastically in the last five A special thanks to all my officemates through the years, first and foremost to my Auntie Yvonne. years. When I started this PhD, I didn’t know what to expect. The world was still in the first year Thank you for putting up with me for so long, for indulging my R-OCD (at least we got rid of that of the COVID-19 pandemic, but Tiny-hands Donald lost the US presidential election on the second grey line eventually!), and for listening to my rants about the quality of student reports. Henry, I day of my first week, so things seemed to be looking up (but we all know how that’s gone). Looking hope you keep playing Hot Sake after I’m gone. Thanks for putting up with my bullshit! Teresa, back on that time feels strange; spending entire working days, weeks and months at home is almost thanks for being my tea person and for all the music. Apra, I’m sorry for being hard on you at times. a distant memory, obscured by a hazy fog of sourdough bread and Zoom interactions. It was, It comes from a place of love, but that is no excuse. Romain and Phoebe, thanks for the interesting however, also over Zoom that I first met the one who would arguably become the most important discussions we’ve had. I couldn’t think of two more different officemates, and I’m glad I got to person in my life for the entirety of my PhD. Lovisa, thank you for showing me that love is actually experience you both. Finally, to Solange: thank you for being my thesis-buddy in the last few weeks. worth it, even if ours didn’t quite make it. Thank you for always spreading positivity and happiness Your commitment to social issues is truly inspiring, and I wish I could be half the person you are in around you, for picking me up each time I fell, for filling my life with amazing dogs, for giving me that regard. some of the happiest memories of my life, and for being the best dog mum to our Leia. To the younger PhD generation in Natrium (including some of the ones already mentioned above): This thesis would not have been possible without my supervisor(s). Erik, you have been an amazing the twins Linnea & Wilma, our favourite baker Nicolai, Johan S, Doris, Vincent, Mauro, Alisa, mentor and colleague, and I am lucky to have had your support over these five years. Your almost Jakob, Justine, and Loraine. Take care of each other when we seniors are gone. You have some childlike curiosity about the natural world is inspiring, and I hope you never lose it. Thank you for big shoes to fill (specifically Estel’s), but I know you will do an amazing job. I’m always available always answering my barrage of questions, for teaching me so much, and for preaching the gospel for admin support if you need it. and inducting me into the copepodamide cult. To my co-supervisors Jon and Lars: you two sit at To all the people in Botan and Natrium (not already mentioned, and those too): Tobia, JC, Claudy, opposite ends of the spectrum in how you comment on and give feedback on (my) texts. Thank you Sina, Mariana, Izzy, Leo, Laura, Bastien, Seb, Marcel, Jenny, Heléne, Kim, Daniela, Salar, for straightening out my rambling; I’m sorry (or perhaps I should say, you’re welcome(?)) for not Niklas, Matilda, Maria, Grazzia, Joel, and many more. Thank you for all the lunches, fikas, AWs, giving you more to do as co-supervisors. Jon, thank you especially for all the rewarding discussions and other social activities that have made the last five years feel like having the biggest extended about stats and experimental design. You are a giant—both as a teacher and a scientist. By retiring, family imaginable at work every day. Extra love goes to Olga, Liz, and Carina. You have all been you have left an appropriately sized hole in this department; enjoy the quieter life on the Island. To rocks that I have leaned on time and time again. Thank you for all you do for your colleagues. my examiner Helle, thank you for always supporting and encouraging me. Through courses, teaching and other work, I have spent more than two years of my life at Tjärnö I owe many thanks to the copepodamide gang and adjacent people, especially my predecessor and Marine Laboratory without ever being officially stationed there. It truly feels like a second home. PhD big sister Kristie for always supporting and helping me when I felt out of my depth. Thank To the PhD students at Tjärnö, both current and former: Bella, Ben, Luisa, James R, Youk, Chloé, you also to Fredrik, Jenny, Aubrey, Harshith, Anna, Andy, and Josephine. Likewise, thank you Kristoffer, Simon, Steffi, Maru, Lara, Diego, Samuel, Cruise and others. We don’t get to see to all the students and interns who have passed through the group: Henke, Hope, Sina, Marc, each other that often, but I always love it when we do. Thank you for the fikas and defence parties; Paula, Ada, Andrra, Luísa, Anouschka, Hannah, Catherina; it has been a pleasure to help you keep taking care of each other. To the rest of the student and staff at Tjärnö: thank you for always with R over Zoom. To my current PhD sister Isa: we may not be doing similar research, but I have making me feel welcome when I visit. Extra special thanks to Ann L for being my BSc and MSc loved getting to know you over the last year and a half. I’ll make sure to stop by your 200 × 200 m supervisor and best teaching colleague in MAV109; to Gunilla T for always taking time for all who area in Möllan when I’m in Malmö. need it and for introducing me to the world of meta-analyses; and to Per J for always being available Many other collaborators, students, and colleagues need acknowledging. If you are reading this to talk stats, ecology, plankton, or anything else one might wonder about in marine research. right now, you are definitely one of them (even if I forget to mention you by name). To the PhD Another retired giant! students in the department as a whole and to the ECRs, I’ve said it before and I’ll say it again: we To everyone mentioned and not: It has truly been an honour and a privilege to share these five years (now you) are the single most cohesive and tight-knit group of people in this place. In a department (or parts of them) with you. that has at times experienced tensions and conflicts between factions, you have shown what true unity, collaboration, and collegial support look like. Never stop working to improve the PhD As some of you may know, I am of the opinion that free will is an illusion. Although one can make programme and your work environment, not just for yourselves but also for one another. philosophical arguments for why this is the case, as a biologist, I tend to prefer arguments based on empirical evidence and evolutionary perspectives. The more we learn about animal and human To the ones who came before and led the way: Björn, Rickard, Ella, James H, Alice, Matt, Astrid, behaviour and neuroscience, the more obvious it appears to me that we do what we do because of Johanna, Martins M & E. Thank you for putting up with all my many annoying questions in those who we are, and that we do not control who we are. Who we are is shaped by a complex interaction early years. To the ones who have been on this journey more or less with me from the start: Estel 3 4 between our biology (both genetic and otherwise) and the sum of our life experiences up to any given point, neither of which we fundamentally control. Robert Sapolsky describes this more List of papers eloquently than I can here (see e.g. Sapolsky 2024). Consequently, I do not think that I chose to do this PhD. I have been shaped by every person in my life, every experience, and every aspect of my This thesis is based on the following papers, which are referred to by their roman numerals: biology: every teacher who believed in me, every bully who harassed me, every colleague I’ve had a beer with, and every friend and family member. I should therefore also thank them and you all I: ↟Arnoldt, S., ↟Pourdanandeh, M., Spikkeland, I., Andersson, M. X., *Selander, E. (2024) for making me the person I am this very second. Some specific honourable mentions: I am deeply Mass spectroscopy reveals compositional differences in copepodamides from limnic and marine grateful to Sofia and Hanna, who got me through my BSc in biology and have been incredible copepods. Scientific Reports. https://doi.org/mf4s friends ever since. I also thank Alizz, Malin, and Jacob for making my MSc time much more II: *Pourdanandeh, M., Kourtchenko, O., Selander E. | Copepodamides do not universally enjoyable and manageable. I owe big thanks to Donald B and Peter T, who introduced me to the suppress chain formation in diatoms. Manuscript in preparation (intended submission to Journal world and power of biostatistics. of Plankton Research as a “Brief communications” article) Last but not least, I am eternally grateful to my families, both my biological family and my chosen III: Gonzalo-Valmala, P., Pourdanandeh, M., Lage, S., *Selander, E. | Grazer-induced family. To my mother and siblings: thank you for all you have done for me. Let’s try to get along bioluminescence and toxicity in marine dinoflagellates. Manuscript in revision (at Limnology and better, despite our passionate temperaments. To my chosen family: Alex, Carina, Sofia, Jakob, Oceanography). Earlier version available as preprint: https://doi.org/p6kg Elvira, Nico, Carl, and others. Thank you for always being there for me, and for tethering me so I don’t float away in my academia bubble. I look forward to being the cool uncle to all your kids, IV: *Pourdanandeh, M., Séchet, V., Carpentier, L., Réveillon, D., Hervé, F., Hubert, C., Hess, starting with Björn and Ingrid. P., Selander, E. (2025) Effects of copepod chemical cues on intra- and extracellular toxins in two species of Dinophysis. Harmful Algae. https://doi.org/nz3q Finally, to the light of my life, Leia, thank you for forcing me to go for walks outside and momentarily forget about some of life’s lesser issues. We could all learn a thing or two about living V: *Pourdanandeh, M., Selander, E. | Fear of grazing rivals the toxin induction effect of in the moment, the way dogs do. I love you. nitrogen enrichment in marine harmful algae –a meta-analysis. Manuscript in revision (at Biological Reviews). Earlier version available as preprint: https://doi.org/10/p6kf It can be hard to be optimistic about the future. A convicted sexual-assaulting con-man in control of the US nuclear codes, Putin’s war in Ukraine, several ongoing genocides, the clear re-emergence ↟ Shared first authorship of right-wing authoritarian and social-dominance-oriented ideologies across the world, and the * Corresponding author apparent abandonment of global efforts to mitigate climate change, biodiversity loss, and habitat exploitation make optimism difficult. It’s especially hard for me, as I’m from Örebro—the heart of the Swedish whining belt (gnällbältet)—where negativity tends to be the default. However, I believe My contributions to each paper, using the Contributor Role Taxonomy (CRediT) system: that science is our best tool for shaping a better tomorrow. It is a shining light of understanding and knowledge in the ruthless, suffering darkness of ignorance, hate, and dogma. I hope that this thesis Contribution I II III IV V contributes in some small way to the incrementally advancing scientific endeavour to better Conceptualisation x x x x understand the world and the universe that we have only a very limited and precious time to Methodology x x x x x experience. Validation x x x x x Formal analysis x x x x x Investigation x x x x Någonstans i Majorna, Göteborg Data Curation x x x x x 2025-10-02 Writing - Original Draft x x x x x Writing - Review & Editing x x x x x Visualisation x x x x x Milad Pourdanandeh 6 5 between our biology (both genetic and otherwise) and the sum of our life experiences up to any given point, neither of which we fundamentally control. Robert Sapolsky describes this more List of papers eloquently than I can here (see e.g. Sapolsky 2024). Consequently, I do not think that I chose to do this PhD. I have been shaped by every person in my life, every experience, and every aspect of my This thesis is based on the following papers, which are referred to by their roman numerals: biology: every teacher who believed in me, every bully who harassed me, every colleague I’ve had a beer with, and every friend and family member. I should therefore also thank them and you all I: ↟Arnoldt, S., ↟Pourdanandeh, M., Spikkeland, I., Andersson, M. X., *Selander, E. (2024) for making me the person I am this very second. Some specific honourable mentions: I am deeply Mass spectroscopy reveals compositional differences in copepodamides from limnic and marine grateful to Sofia and Hanna, who got me through my BSc in biology and have been incredible copepods. Scientific Reports. https://doi.org/mf4s friends ever since. I also thank Alizz, Malin, and Jacob for making my MSc time much more II: *Pourdanandeh, M., Kourtchenko, O., Selander E. | Copepodamides do not universally enjoyable and manageable. I owe big thanks to Donald B and Peter T, who introduced me to the suppress chain formation in diatoms. Manuscript in preparation (intended submission to Journal world and power of biostatistics. of Plankton Research as a “Brief communications” article) Last but not least, I am eternally grateful to my families, both my biological family and my chosen III: Gonzalo-Valmala, P., Pourdanandeh, M., Lage, S., *Selander, E. | Grazer-induced family. To my mother and siblings: thank you for all you have done for me. Let’s try to get along bioluminescence and toxicity in marine dinoflagellates. Manuscript in revision (at Limnology and better, despite our passionate temperaments. To my chosen family: Alex, Carina, Sofia, Jakob, Oceanography). Earlier version available as preprint: https://doi.org/p6kg Elvira, Nico, Carl, and others. Thank you for always being there for me, and for tethering me so I don’t float away in my academia bubble. I look forward to being the cool uncle to all your kids, IV: *Pourdanandeh, M., Séchet, V., Carpentier, L., Réveillon, D., Hervé, F., Hubert, C., Hess, starting with Björn and Ingrid. P., Selander, E. (2025) Effects of copepod chemical cues on intra- and extracellular toxins in two species of Dinophysis. Harmful Algae. https://doi.org/nz3q Finally, to the light of my life, Leia, thank you for forcing me to go for walks outside and momentarily forget about some of life’s lesser issues. We could all learn a thing or two about living V: *Pourdanandeh, M., Selander, E. | Fear of grazing rivals the toxin induction effect of in the moment, the way dogs do. I love you. nitrogen enrichment in marine harmful algae –a meta-analysis. Manuscript in revision (at Biological Reviews). Earlier version available as preprint: https://doi.org/10/p6kf It can be hard to be optimistic about the future. A convicted sexual-assaulting con-man in control of the US nuclear codes, Putin’s war in Ukraine, several ongoing genocides, the clear re-emergence ↟ Shared first authorship of right-wing authoritarian and social-dominance-oriented ideologies across the world, and the * Corresponding author apparent abandonment of global efforts to mitigate climate change, biodiversity loss, and habitat exploitation make optimism difficult. It’s especially hard for me, as I’m from Örebro—the heart of the Swedish whining belt (gnällbältet)—where negativity tends to be the default. However, I believe My contributions to each paper, using the Contributor Role Taxonomy (CRediT) system: that science is our best tool for shaping a better tomorrow. It is a shining light of understanding and knowledge in the ruthless, suffering darkness of ignorance, hate, and dogma. I hope that this thesis Contribution I II III IV V contributes in some small way to the incrementally advancing scientific endeavour to better Conceptualisation x x x x understand the world and the universe that we have only a very limited and precious time to Methodology x x x x x experience. Validation x x x x x Formal analysis x x x x x Investigation x x x x Någonstans i Majorna, Göteborg Data Curation x x x x x 2025-10-02 Writing - Original Draft x x x x x Writing - Review & Editing x x x x x Visualisation x x x x x Milad Pourdanandeh 6 5 Other publications not included in this thesis Preface Scientific publications A note to the reader Abrahamsson, K., Damm, E., Björk, G., Bunde, C., Sellmaier, A., Broström, G., Assmann, V., Dumitrascu, A., Maciute, A., Olofsson, N., Pourdanandeh, M. (2024) Methane plume detection I want to begin by briefly explaining why I have written this thesis the way I have. The kappa after the 2022 Nord Stream pipeline explosion in the Baltic Sea. Scientific Reports. presents a rare opportunity to set aside the strict conventions of scientific papers and to write more https://doi.org/m4c6 plainly about why this work matters and how its pieces fit together. Scientific articles rightly follow tight formats, and PhD projects often start with predefined aims. Here, however, I have room to tell Matteoni, H., Neun, S., Kourtchenko, O., Meirkhanova, A., Bezzubova, E., Siniakova, T., the story in my own voice. Bonaglia, S., Corcoll Cornet, N., Barteneva, N., Leder, E.H., Tronholm, A., Abrahamsson, K., Pourdanandeh, M., Bachimanchi, H., Damm, E., Laufer, N., Leray, L., Giebel, H., Flöder, S., The theme is straightforward: how chemical information shapes life in the plankton, and how Kellner, L., Kirsch, J., Kunze, C., Striebel, M., Bunse, C. | Effects of elevated methane grazers—especially copepods—influence phytoplankton via induced defences. The detailed concentration on zooplankton, phytoplankton and prokaryoplankton communities. Manuscript in methods, statistics and technical caveats live in the papers themselves. This kappa does not preparation. catalogue every model or test. Instead, it offers, I hope, a clearer, more accessible background so the rest of the thesis is easier to navigate. Think of it as a digestible guide to the ideas and Pourdanandeh, M. When billboards walk: The pros and cons of wearing your poster. Manuscript mechanisms that underpin the papers. in preparation (intended submission to Limnology and Oceanography Bulletin) My aim is to lead any scientifically literate reader—regardless of speciality—through the history and mechanisms that connect the studies into a coherent whole. For chemists: if I oversimplify, Repositories/data publications please remember that I am not a chemist and that my focus is not on pure chemistry. For Pourdanandeh, M., Arnoldt, S., Selander, E. (2023) Targeted and untargeted LC-MS planktologists and specialists seeking maximal technical depth, the primary papers await you. For copepodamide data for marine and freshwater copepods (Data and code repository). Zenodo. the intrigued but uninitiated, this is the place to start. https://doi.org/g6wpgz In other words, this thesis is intentionally focused on chemically mediated interaction, inducible Pourdanandeh, M., Séchet, V., Carpentier, L., Réveillon, D., Hervé, F., Hubert, C., Hess, P., and defences and copepodamide ecology, rather than being an exhaustive treatise on plankton ecology. Selander, E. (2024) Data and analysis code for a toxin induction study on two species of I hope it tells a cohesive story that frames the research questions and context leading up to the Dinophysis dinoflagellates (Data and code repository). Zenodo. https://doi.org/p62v discovery and study of copepodamides and chemically mediated predator–prey interactions in plankton. Pourdanandeh, M., Gonzalo-Valmala, P., Lage, S., Amorim, A., and Selander, E. (2025) Data repository for study on dinoflagellate bioluminescence and toxicity in response to copepodamides NB: because this thesis is based on five papers and manuscripts at various stages of publication, (Data and code repository). Zenodo. https://doi.org/p62x some passages in the kappa may resemble those papers, and some passages may be identical (e.g., figure captions). Pourdanandeh, M., Selander, E. (2025) Data and analysis code for a meta-analysis on relative phycotoxin induction due to elevated N:P ratio or elevated grazing risk in Alexandrium dinoflagellates and Pseudo-nitzschia diatoms (Data and code reposityory). Zenodo. https://doi.org/p622 Popular science Selander, E., Pourdanandeh, M. (2022) Doften av en hoppkräfta. Havsutsikt, https://www.havet.nu/havsutsikt/artikel/doften-av-en-hoppkrafta Funding statement This PhD thesis and related research was financed by the Swedish Research Council (VR 2019- 05238 to Erik Selander) and the Department of Marine Sciences of the University of Gothenburg. The Adlerbert Foundation supplemented these funds for travel to two of the four international conferences I attended. 7 8 Other publications not included in this thesis Preface Scientific publications A note to the reader Abrahamsson, K., Damm, E., Björk, G., Bunde, C., Sellmaier, A., Broström, G., Assmann, V., Dumitrascu, A., Maciute, A., Olofsson, N., Pourdanandeh, M. (2024) Methane plume detection I want to begin by briefly explaining why I have written this thesis the way I have. The kappa after the 2022 Nord Stream pipeline explosion in the Baltic Sea. Scientific Reports. presents a rare opportunity to set aside the strict conventions of scientific papers and to write more https://doi.org/m4c6 plainly about why this work matters and how its pieces fit together. Scientific articles rightly follow tight formats, and PhD projects often start with predefined aims. Here, however, I have room to tell Matteoni, H., Neun, S., Kourtchenko, O., Meirkhanova, A., Bezzubova, E., Siniakova, T., the story in my own voice. Bonaglia, S., Corcoll Cornet, N., Barteneva, N., Leder, E.H., Tronholm, A., Abrahamsson, K., Pourdanandeh, M., Bachimanchi, H., Damm, E., Laufer, N., Leray, L., Giebel, H., Flöder, S., The theme is straightforward: how chemical information shapes life in the plankton, and how Kellner, L., Kirsch, J., Kunze, C., Striebel, M., Bunse, C. | Effects of elevated methane grazers—especially copepods—influence phytoplankton via induced defences. The detailed concentration on zooplankton, phytoplankton and prokaryoplankton communities. Manuscript in methods, statistics and technical caveats live in the papers themselves. This kappa does not preparation. catalogue every model or test. Instead, it offers, I hope, a clearer, more accessible background so the rest of the thesis is easier to navigate. Think of it as a digestible guide to the ideas and Pourdanandeh, M. When billboards walk: The pros and cons of wearing your poster. Manuscript mechanisms that underpin the papers. in preparation (intended submission to Limnology and Oceanography Bulletin) My aim is to lead any scientifically literate reader—regardless of speciality—through the history and mechanisms that connect the studies into a coherent whole. For chemists: if I oversimplify, Repositories/data publications please remember that I am not a chemist and that my focus is not on pure chemistry. For Pourdanandeh, M., Arnoldt, S., Selander, E. (2023) Targeted and untargeted LC-MS planktologists and specialists seeking maximal technical depth, the primary papers await you. For copepodamide data for marine and freshwater copepods (Data and code repository). Zenodo. the intrigued but uninitiated, this is the place to start. https://doi.org/g6wpgz In other words, this thesis is intentionally focused on chemically mediated interaction, inducible Pourdanandeh, M., Séchet, V., Carpentier, L., Réveillon, D., Hervé, F., Hubert, C., Hess, P., and defences and copepodamide ecology, rather than being an exhaustive treatise on plankton ecology. Selander, E. (2024) Data and analysis code for a toxin induction study on two species of I hope it tells a cohesive story that frames the research questions and context leading up to the Dinophysis dinoflagellates (Data and code repository). Zenodo. https://doi.org/p62v discovery and study of copepodamides and chemically mediated predator–prey interactions in plankton. Pourdanandeh, M., Gonzalo-Valmala, P., Lage, S., Amorim, A., and Selander, E. (2025) Data repository for study on dinoflagellate bioluminescence and toxicity in response to copepodamides NB: because this thesis is based on five papers and manuscripts at various stages of publication, (Data and code repository). Zenodo. https://doi.org/p62x some passages in the kappa may resemble those papers, and some passages may be identical (e.g., figure captions). Pourdanandeh, M., Selander, E. (2025) Data and analysis code for a meta-analysis on relative phycotoxin induction due to elevated N:P ratio or elevated grazing risk in Alexandrium dinoflagellates and Pseudo-nitzschia diatoms (Data and code reposityory). Zenodo. https://doi.org/p622 Popular science Selander, E., Pourdanandeh, M. (2022) Doften av en hoppkräfta. Havsutsikt, https://www.havet.nu/havsutsikt/artikel/doften-av-en-hoppkrafta Funding statement This PhD thesis and related research was financed by the Swedish Research Council (VR 2019- 05238 to Erik Selander) and the Department of Marine Sciences of the University of Gothenburg. The Adlerbert Foundation supplemented these funds for travel to two of the four international conferences I attended. 7 8 Background 1.1 A pale blue dot The astronomer and celebrated science communicator Carl Sagan described Earth—as seen from about 6 billion (109) kilometres away in the photograph taken by the Voyager 1 space probe on 14 February 1990—as a pale blue dot1. All that blue is ocean. About 71% of Earth’s surface is water, most of it gathered into a single, connected sea that wraps the continents and links every coast. The ocean is our planet’s largest living space and its great moderator: it stores heat, drives weather, shapes climate, and shuttles water and energy around the globe. This is not a still reservoir, but a moving world. Winds push surface currents; tides breathe in and out; great overturning flows carry surface waters downward and return deep waters to the light. The ocean has layers: a sunlit skin where life can harvest light; a dim, quieter middle; and the vast dark below. In all of these layers, from the coral shallows to the trenches deeper than any mountain is Chapter 1. high, life finds a way to persist. Background Why care? Because the ocean does not only feed its permanent inhabitants, it also feeds billions of people, it shapes our coastlines, it influences storms and droughts, it absorbs heat and carbon, and it sustains economies, cultures, and livelihoods across the planet. What happens offshore does not stay offshore: warming seas, shifting currents, and changing ecosystems ripple outward to affect climate, fisheries, and communities far beyond the shoreline. It is interesting to contemplate an entangled bank, clothed with many plants of Within this vast moving system, most life is small and drifting. Plankton—the collective name for many kinds, with birds singing on the bushes, with various insects flitting about, these drifters—form the foundation of marine food webs. The plant-like phytoplankton turn and with worms crawling through the damp earth, and to reflect that these sunlight and carbon dioxide into sugars and release oxygen; zooplankton graze on them and, in turn, elaborately constructed forms, so different from each other, and dependent on feed fish, seabirds, and whales. These tiny organisms help regulate our climate by moving carbon from the surface to the deep ocean and by influencing the formation of clouds above. They produce each other in so complex a manner, have all been produced by laws acting chemical compounds that make us sick, but that can also be used to produce new medicines. They around us. […] There is grandeur in this view of life, with its several powers, are a barely tapped source of new materials, foods, and so much more. Small lives, large having been originally breathed into a few forms or into one; and that, whilst consequences is an understatement. As anthropogenic climate change accelerates in the coming this planet has gone cycling on according to the fixed law of gravity, from so century, understanding the ocean’s role in regulating heat, carbon, and life itself becomes essential for predicting and mitigating its far-reaching impacts. simple a beginning endless forms most beautiful and most wonderful have been, and are being, evolved. This thesis follows one thin thread through that immense tapestry: how chemical cues exuded by zooplankton grazers inform phytoplankton when to better defend themselves because their Charles Darwin predators are nearby, and how these indirect grazer-induced responses influence large-scale On the Origin of Species processes such as harmful algal bloom formation. A Kind of Magic – Queen 9 10 Background 1.1 A pale blue dot The astronomer and celebrated science communicator Carl Sagan described Earth—as seen from about 6 billion (109) kilometres away in the photograph taken by the Voyager 1 space probe on 14 February 1990—as a pale blue dot1. All that blue is ocean. About 71% of Earth’s surface is water, most of it gathered into a single, connected sea that wraps the continents and links every coast. The ocean is our planet’s largest living space and its great moderator: it stores heat, drives weather, shapes climate, and shuttles water and energy around the globe. This is not a still reservoir, but a moving world. Winds push surface currents; tides breathe in and out; great overturning flows carry surface waters downward and return deep waters to the light. The ocean has layers: a sunlit skin where life can harvest light; a dim, quieter middle; and the vast dark below. In all of these layers, from the coral shallows to the trenches deeper than any mountain is Chapter 1. high, life finds a way to persist. Background Why care? Because the ocean does not only feed its permanent inhabitants, it also feeds billions of people, it shapes our coastlines, it influences storms and droughts, it absorbs heat and carbon, and it sustains economies, cultures, and livelihoods across the planet. What happens offshore does not stay offshore: warming seas, shifting currents, and changing ecosystems ripple outward to affect climate, fisheries, and communities far beyond the shoreline. It is interesting to contemplate an entangled bank, clothed with many plants of Within this vast moving system, most life is small and drifting. Plankton—the collective name for many kinds, with birds singing on the bushes, with various insects flitting about, these drifters—form the foundation of marine food webs. The plant-like phytoplankton turn and with worms crawling through the damp earth, and to reflect that these sunlight and carbon dioxide into sugars and release oxygen; zooplankton graze on them and, in turn, elaborately constructed forms, so different from each other, and dependent on feed fish, seabirds, and whales. These tiny organisms help regulate our climate by moving carbon from the surface to the deep ocean and by influencing the formation of clouds above. They produce each other in so complex a manner, have all been produced by laws acting chemical compounds that make us sick, but that can also be used to produce new medicines. They around us. […] There is grandeur in this view of life, with its several powers, are a barely tapped source of new materials, foods, and so much more. Small lives, large having been originally breathed into a few forms or into one; and that, whilst consequences is an understatement. As anthropogenic climate change accelerates in the coming this planet has gone cycling on according to the fixed law of gravity, from so century, understanding the ocean’s role in regulating heat, carbon, and life itself becomes essential for predicting and mitigating its far-reaching impacts. simple a beginning endless forms most beautiful and most wonderful have been, and are being, evolved. This thesis follows one thin thread through that immense tapestry: how chemical cues exuded by zooplankton grazers inform phytoplankton when to better defend themselves because their Charles Darwin predators are nearby, and how these indirect grazer-induced responses influence large-scale On the Origin of Species processes such as harmful algal bloom formation. A Kind of Magic – Queen 9 10 Background Background 1.2 Plankton In January 1832, while traveling from Tenerife to the Cape Verde Islands aboard HMS Beagle, Charles Darwin wrote in his diary: “I proved to day the utility of a contrivance which will afford me many hours of amusement & work. — it is a bag four feet deep, made of bunting, & attached to semicircular bow this by lines is kept upright, & dragged behind the vessel. — this evening it brought up a mass of small animals, & tomorrow I look forward to a greater harvest. I am quite tired having worked all day at the produce of my net. — The number of animals that the net collects is very great & fully explains the manner so many animals of a large size live so far from land. — Many of these creatures so low in the scale of nature are most exquisite in their forms & rich colours. — It creates a feeling of wonder that so much beauty should be apparently created for such little purpose.” (Darwin, 1832: p. 55)2 The creatures that Darwin collected were plankton—from the Greek planáō (to drift, wander)—life forms that drift at the mercy of currents. Among them, photosynthetic phytoplankton form the base of marine food webs. Their importance extends far beyond the sea: they help produce the oxygen we breathe, shape global carbon cycles, and even influence weather patterns by releasing compounds that seed cloud formation. Despite making up less than 0.2% of global primary producer biomass (Bar-On et al., 2018), phytoplankton carry out roughly half of all photosynthesis on Earth (Field et al., 1998). Fig. 1: Simplified schematic of the marine food web. Credit: Jan Heuschele. Phytoplankton are grazed by many small animals and protists—mostly single-celled eukaryotic organisms (Fig. 1). Grazing in the pelagic, i.e. the free water column, is so intense that the average successful, I refer you to the excellent review by Kiørboe (2011). While some pelagic copepod turnover time of phytoplankton biomass in the ocean is only 2–6 days, which is roughly a thousand species have large, forward-facing eyes (e.g., Corycaeus), most rely on a single simple (naupliar) times faster than on land where plant material typically persists for 12–16 years (Falkowski and eye that senses light but cannot form images (Gregory et al., 1964). Because image-forming vision is limited in most plankton, they rely primarily on chemosensation—and, in many taxa, Raven, 1997). Similarly, the intensity of herbivory in pelagic systems is approximately three times mechanosensation—to locate mates, find food, and detect or respond to predation. higher than in terrestrial systems (Cyr and Pace, 1993). Microzooplankton (20–200 µm), such as ciliates and heterotrophic flagellates, consume an estimated 67% of marine primary production (Calbet and Landry, 2004). Mesozooplankton (0.2–20 mm), including copepods (from the Greek 1.3 Chemically mediated interactions and plant defences kōpē “oar/paddle”, and pod “foot”), account for a further 10–25% (Calbet, 2001). Copepods and other mesozooplankton also feed on the primary-consuming microzooplankton (Fig. 1) and are in A sea of chemistry turn consumed by larger crustaceans, small fish, and gelatinous macrozooplankton. In other words, Against this sensory backdrop, much of life in the sea is governed by chemistry. Organisms release copepods constitute a crucial trophic link in marine food webs. They transfer energy and biomass an enormous variety of molecules from diverse compound classed—e.g. alkaloids, terpenoids, from phytoplankton to larger consumers such as fish, seabirds and marine mammals, effectively lipids, and peptides—some of which can act as cues or signals. Chemically mediated interactions coupling microscopic primary production to the macroscopic animals that most people think of as can guide mate finding (van Duren and Videler, 1996; Bagøien and Kiørboe, 2005; Heuschele and “marine life”. Kiørboe, 2012; Seuront, 2013; Seuront and Stanley, 2014), mediate intraspecific communication (Nordeng, 2009; Boal et al., 2010; Herbert-Read et al., 2010), help locate or select resources and Pelagic copepods, the dominant mesozooplankton, are among the most abundant animals on Earth prey (Moore et al., 1999; Kozlowsky-Suzuki et al., 2006; Lombard et al., 2011; Sipler et al., 2014), (Humes, 1994; Turner, 2004). Estimating their global abundance at any given time is challenging, but first-order approximations—including my own—place it at roughly 5×1018 – 5×1019 individuals. and mediate predator avoidance and defence (Miralto et al., 1999; Bergkvist et al., 2008; Waggett Copepods are also diverse: more than 11 700 marine and 3 200 freshwater copepod species have et al., 2012; Tammilehto et al., 2015). been described, and many more are likely undescribed (Walter and Boxshall, 2025). These small Chemicals of information crustaceans, typically 0.2–5 mm long as adults, inhabit nearly all aquatic environments on Earth. For a thorough and detailed treatment of pelagic plankton biology and ecology, comprehensive In chemical ecology, infochemicals—chemicals that convey information—are classified by both textbooks are available (Castellani and Edwards, 2017; Suthers et al., 2019). For readers the taxonomic identity of the actors and by who benefits. Pheromones act within species, whereas specifically interested in the biology and ecology of pelagic copepods, and why these have been so allelochemicals act between species and include allomones (benefit the emitter), kairomones 11 12 Background Background 1.2 Plankton In January 1832, while traveling from Tenerife to the Cape Verde Islands aboard HMS Beagle, Charles Darwin wrote in his diary: “I proved to day the utility of a contrivance which will afford me many hours of amusement & work. — it is a bag four feet deep, made of bunting, & attached to semicircular bow this by lines is kept upright, & dragged behind the vessel. — this evening it brought up a mass of small animals, & tomorrow I look forward to a greater harvest. I am quite tired having worked all day at the produce of my net. — The number of animals that the net collects is very great & fully explains the manner so many animals of a large size live so far from land. — Many of these creatures so low in the scale of nature are most exquisite in their forms & rich colours. — It creates a feeling of wonder that so much beauty should be apparently created for such little purpose.” (Darwin, 1832: p. 55)2 The creatures that Darwin collected were plankton—from the Greek planáō (to drift, wander)—life forms that drift at the mercy of currents. Among them, photosynthetic phytoplankton form the base of marine food webs. Their importance extends far beyond the sea: they help produce the oxygen we breathe, shape global carbon cycles, and even influence weather patterns by releasing compounds that seed cloud formation. Despite making up less than 0.2% of global primary producer biomass (Bar-On et al., 2018), phytoplankton carry out roughly half of all photosynthesis on Earth (Field et al., 1998). Fig. 1: Simplified schematic of the marine food web. Credit: Jan Heuschele. Phytoplankton are grazed by many small animals and protists—mostly single-celled eukaryotic organisms (Fig. 1). Grazing in the pelagic, i.e. the free water column, is so intense that the average successful, I refer you to the excellent review by Kiørboe (2011). While some pelagic copepod turnover time of phytoplankton biomass in the ocean is only 2–6 days, which is roughly a thousand species have large, forward-facing eyes (e.g., Corycaeus), most rely on a single simple (naupliar) times faster than on land where plant material typically persists for 12–16 years (Falkowski and eye that senses light but cannot form images (Gregory et al., 1964). Because image-forming vision is limited in most plankton, they rely primarily on chemosensation—and, in many taxa, Raven, 1997). Similarly, the intensity of herbivory in pelagic systems is approximately three times mechanosensation—to locate mates, find food, and detect or respond to predation. higher than in terrestrial systems (Cyr and Pace, 1993). Microzooplankton (20–200 µm), such as ciliates and heterotrophic flagellates, consume an estimated 67% of marine primary production (Calbet and Landry, 2004). Mesozooplankton (0.2–20 mm), including copepods (from the Greek 1.3 Chemically mediated interactions and plant defences kōpē “oar/paddle”, and pod “foot”), account for a further 10–25% (Calbet, 2001). Copepods and other mesozooplankton also feed on the primary-consuming microzooplankton (Fig. 1) and are in A sea of chemistry turn consumed by larger crustaceans, small fish, and gelatinous macrozooplankton. In other words, Against this sensory backdrop, much of life in the sea is governed by chemistry. Organisms release copepods constitute a crucial trophic link in marine food webs. They transfer energy and biomass an enormous variety of molecules from diverse compound classed—e.g. alkaloids, terpenoids, from phytoplankton to larger consumers such as fish, seabirds and marine mammals, effectively lipids, and peptides—some of which can act as cues or signals. Chemically mediated interactions coupling microscopic primary production to the macroscopic animals that most people think of as can guide mate finding (van Duren and Videler, 1996; Bagøien and Kiørboe, 2005; Heuschele and “marine life”. Kiørboe, 2012; Seuront, 2013; Seuront and Stanley, 2014), mediate intraspecific communication (Nordeng, 2009; Boal et al., 2010; Herbert-Read et al., 2010), help locate or select resources and Pelagic copepods, the dominant mesozooplankton, are among the most abundant animals on Earth prey (Moore et al., 1999; Kozlowsky-Suzuki et al., 2006; Lombard et al., 2011; Sipler et al., 2014), (Humes, 1994; Turner, 2004). Estimating their global abundance at any given time is challenging, but first-order approximations—including my own—place it at roughly 5×1018 – 5×1019 individuals. and mediate predator avoidance and defence (Miralto et al., 1999; Bergkvist et al., 2008; Waggett Copepods are also diverse: more than 11 700 marine and 3 200 freshwater copepod species have et al., 2012; Tammilehto et al., 2015). been described, and many more are likely undescribed (Walter and Boxshall, 2025). These small Chemicals of information crustaceans, typically 0.2–5 mm long as adults, inhabit nearly all aquatic environments on Earth. For a thorough and detailed treatment of pelagic plankton biology and ecology, comprehensive In chemical ecology, infochemicals—chemicals that convey information—are classified by both textbooks are available (Castellani and Edwards, 2017; Suthers et al., 2019). For readers the taxonomic identity of the actors and by who benefits. Pheromones act within species, whereas specifically interested in the biology and ecology of pelagic copepods, and why these have been so allelochemicals act between species and include allomones (benefit the emitter), kairomones 11 12 Background Background (benefit the receiver), and synomones (benefit both). The same molecule can shift category with adjuncts. Resource-driven models argue that defence production is governed by resource supply context; a common feature in pelagic systems where encounters are brief and asymmetric. In marine and allocation. The carbon:nutrient balance model (Bryant et al., 1983; Tuomi et al., 1988, 1991) chemical ecology, we have increasingly adopted more evolutionarily explicit and transparent is perhaps the most influential of these. It predicts that when photosynthetic carbon accumulates— terminology. We use signal to denote infochemicals for which there is evidence of selection for relative to limiting nutrients—surplus carbon can be shunted into carbon-based secondary information transfer, and cue to denote infochemicals that receivers exploit to guide their behaviour metabolites (Bryant et al., 1983, 1989; Herms and Mattson, 1992). The key mechanistic intuition but for which there is no evidence of selection for communicative function. It should, however, be is simple: primary growth processes take priority; when these are limited by nutrients, excess carbon noted that evolutionary origins are often difficult to establish, and compounds may lie on a and nitrogen may be invested into defences with relatively low cost (Tuomi et al., 1988; Lerdau continuum. For example, exaptation—the evolutionary process by which a trait evolved for one and Coley, 2002). Meta-analyses show partial support for its predictions (see Stamp, 2003, for a function is co-opted or repurposed for a new one—can blur distinctions between cue and signal. detailed treatment) but many primary studies suffer from methodological issues that complicate Plant defences broad inference (Koricheva et al., 1998; Stamp, 2003) and numerous studies have failed to support Much of what we know about chemically mediated interactions was developed in terrestrial systems the predictions of the model entirely (reviewed in Herms and Mattson, 1992; Hamilton et al., 2001; (Sondheimer and Simeone, 1970; Rosenthal and Berenbaum, 1992; Karban and Baldwin, 1997; Koricheva, 2002). Hartmann, 2008), particularly plants and insects. This is especially true for the study of plant Demand-driven frameworks, such as the optimal defence model (ODM), take an evolutionary, chemical defences against herbivory, for which there is experimental evidence from as early as the cost–benefit approach instead. The ODM (McKey, 1974; McKey et al., 1979; Rhoades, 1979) treats late 1800s (Stahl, 1888). Although largely overlooked for more than half a century, secondary defence allocation as an evolved strategy that maximises fitness given herbivory risk, the value of metabolites—compounds not involved in primary metabolism— were recognised by entomologists plant parts, and the costs of defence (Rhoades, 1979; Roff, 1992; Stearns, 1992). A central in the 1950s as important mediators of plant–environment interactions (Hartmann, 2008), especially assumption is that defences are costly and trade off with growth and reproduction (Coley et al., for their roles as attractants and repellents of herbivorous insects (Butenandt et al., 1959; Fraenkel, 1985; Simms and Rausher, 1987). This yields a suite of predictions: defences should be inducible 1959). The very label “secondary” arguably encouraged scepticism about defensive functions, a when risk is variable, high-value tissues should be better protected, and genetic variation in defence potential example of the framing effect: a cognitive bias where the framing of information shapes traits should exist because of spatially or temporally heterogeneous selection (Karban et al., 1997). how people perceive and evaluate it (Tversky and Kahneman, 1981; Levin et al., 1998; Matthes The ODM is supported by several independent lines of empirical evidence. High intraspecific and Schemer, 2012). It should therefore not be surprising that many biologists viewed these genetic variation exists in the identity and amount of secondary-metabolites (Dirzo and Harper, compounds as little more than metabolic waste products well into the latter half of the 1900s 1982; Zangerl and Berenbaum, 1990), herbivores act as strong selective agents on those traits (Robinson, 1974; Haslam, 1985; Hartmann, 2008). Importantly, however, their work also (Simms and Rausher, 1989; Mauricio and Rausher, 1997), organisms deploy more defence where established the conceptual foundation for classifying and studying how plant defences. and when risk is higher (Baldwin and Karb, 1995; Zangerl and Rutledge, 1996), those defences Plant defences against herbivory can be broadly categorised as constitutive (continuously incur measurable costs in growth or reproduction when herbivory is absent (Vrieling and van Wijk, expressed), inducible (temporarily upregulated), or a combination of both (Karban and Baldwin, 1994; Strauss et al., 2002), and induced defences can increase producer fitness under attack 1997; Stamp, 2003). Constitutive defences include traits expressed regardless of immediate risk. (Baldwin et al., 1990; Agrawal, 1998). Taken together, these lines of evidence—variation, selection, They reduce damage from the first attack and are favoured when grazing risk is high and/or proportional defence allocation, costs, and fitness benefits—form the empirical backbone of the predictable, but require resources that are diverted away from growth and reproduction (Stamp, ODM. However, evidence against the ODM also exists, and designing experiments that accurately 2003). In contrast, inducible defences allow for phenotypic plasticity and may be advantageous test its predictions is often difficult (Fagerström et al., 1987; Stamp, 2003; McCall and Fordyce, when predator presence is unpredictable. This enables prey to scale the expression of potentially 2010). costly defences to the actual level of threat, and thereby the likelihood of predators evolving Beyond these two pillars, other frameworks have also proven useful: the growth-rate hypothesis counter-adaptations (Rhoades, 1979; Karban et al., 1997; Agrawal, 1999; Shelton, 2004; Preisser links defence strategy to intrinsic growth potential (Coley et al., 1985; Coley, 1987); the plant et al., 2007). Inducible defence expression depends on reliable yet short-lived cues that apparency hypothesis (Feeny, 1976) predicts that large, long-lived and conspicuous (i.e. apparent) reflect current predation risk (Karban and Baldwin, 1997; Stamp, 2003). In plankton systems, plants will rely more on constitutive defences; and the extended growth-differentiation balance physical parameters such as diffusion, turbulence, and rapid degradation of chemicals (Kiørboe, (Herms and Mattson, 1992) model blends growth and defence trade-offs in a continuous framework. 2008) constrain cue persistence and necessitate specific, detectable, and transient chemical signals. Collectively, these models provide a conceptual toolkit rather than a universal law. They help In other words: cues and signals must rise above chemical background noise, persist long enough predict how and when plants allocate energy and matter to secondary-metabolite production to to be detected and responded to, be specific enough to trigger the appropriate response, and dissipate defend against herbivores, and when constitutive versus inducible defence strategies should evolve quickly enough to prevent unnecessary or prolonged responses. (Stamp, 2003). Ecological frameworks of plant defences Observations across many predator–prey systems reveal a recurring asymmetry: predators often fail Ecologists have developed a rich set of theoretical frameworks to explain and predict when and to capture prey, whereas captured prey typically suffer severe fitness costs, often death. This how organisms invest in chemical defences. These frameworks fall roughly into two families: empirical pattern underpins the evolutionary arms-race metaphor described by Dawkins and Krebs resource-driven models and demand-driven (optimality) models, in addition to a few useful (1979). Predator–prey interactions are commonly framed as a continual cycle of adaptation and 13 14 Background Background (benefit the receiver), and synomones (benefit both). The same molecule can shift category with adjuncts. Resource-driven models argue that defence production is governed by resource supply context; a common feature in pelagic systems where encounters are brief and asymmetric. In marine and allocation. The carbon:nutrient balance model (Bryant et al., 1983; Tuomi et al., 1988, 1991) chemical ecology, we have increasingly adopted more evolutionarily explicit and transparent is perhaps the most influential of these. It predicts that when photosynthetic carbon accumulates— terminology. We use signal to denote infochemicals for which there is evidence of selection for relative to limiting nutrients—surplus carbon can be shunted into carbon-based secondary information transfer, and cue to denote infochemicals that receivers exploit to guide their behaviour metabolites (Bryant et al., 1983, 1989; Herms and Mattson, 1992). The key mechanistic intuition but for which there is no evidence of selection for communicative function. It should, however, be is simple: primary growth processes take priority; when these are limited by nutrients, excess carbon noted that evolutionary origins are often difficult to establish, and compounds may lie on a and nitrogen may be invested into defences with relatively low cost (Tuomi et al., 1988; Lerdau continuum. For example, exaptation—the evolutionary process by which a trait evolved for one and Coley, 2002). Meta-analyses show partial support for its predictions (see Stamp, 2003, for a function is co-opted or repurposed for a new one—can blur distinctions between cue and signal. detailed treatment) but many primary studies suffer from methodological issues that complicate Plant defences broad inference (Koricheva et al., 1998; Stamp, 2003) and numerous studies have failed to support Much of what we know about chemically mediated interactions was developed in terrestrial systems the predictions of the model entirely (reviewed in Herms and Mattson, 1992; Hamilton et al., 2001; (Sondheimer and Simeone, 1970; Rosenthal and Berenbaum, 1992; Karban and Baldwin, 1997; Koricheva, 2002). Hartmann, 2008), particularly plants and insects. This is especially true for the study of plant Demand-driven frameworks, such as the optimal defence model (ODM), take an evolutionary, chemical defences against herbivory, for which there is experimental evidence from as early as the cost–benefit approach instead. The ODM (McKey, 1974; McKey et al., 1979; Rhoades, 1979) treats late 1800s (Stahl, 1888). Although largely overlooked for more than half a century, secondary defence allocation as an evolved strategy that maximises fitness given herbivory risk, the value of metabolites—compounds not involved in primary metabolism— were recognised by entomologists plant parts, and the costs of defence (Rhoades, 1979; Roff, 1992; Stearns, 1992). A central in the 1950s as important mediators of plant–environment interactions (Hartmann, 2008), especially assumption is that defences are costly and trade off with growth and reproduction (Coley et al., for their roles as attractants and repellents of herbivorous insects (Butenandt et al., 1959; Fraenkel, 1985; Simms and Rausher, 1987). This yields a suite of predictions: defences should be inducible 1959). The very label “secondary” arguably encouraged scepticism about defensive functions, a when risk is variable, high-value tissues should be better protected, and genetic variation in defence potential example of the framing effect: a cognitive bias where the framing of information shapes traits should exist because of spatially or temporally heterogeneous selection (Karban et al., 1997). how people perceive and evaluate it (Tversky and Kahneman, 1981; Levin et al., 1998; Matthes The ODM is supported by several independent lines of empirical evidence. High intraspecific and Schemer, 2012). It should therefore not be surprising that many biologists viewed these genetic variation exists in the identity and amount of secondary-metabolites (Dirzo and Harper, compounds as little more than metabolic waste products well into the latter half of the 1900s 1982; Zangerl and Berenbaum, 1990), herbivores act as strong selective agents on those traits (Robinson, 1974; Haslam, 1985; Hartmann, 2008). Importantly, however, their work also (Simms and Rausher, 1989; Mauricio and Rausher, 1997), organisms deploy more defence where established the conceptual foundation for classifying and studying how plant defences. and when risk is higher (Baldwin and Karb, 1995; Zangerl and Rutledge, 1996), those defences Plant defences against herbivory can be broadly categorised as constitutive (continuously incur measurable costs in growth or reproduction when herbivory is absent (Vrieling and van Wijk, expressed), inducible (temporarily upregulated), or a combination of both (Karban and Baldwin, 1994; Strauss et al., 2002), and induced defences can increase producer fitness under attack 1997; Stamp, 2003). Constitutive defences include traits expressed regardless of immediate risk. (Baldwin et al., 1990; Agrawal, 1998). Taken together, these lines of evidence—variation, selection, They reduce damage from the first attack and are favoured when grazing risk is high and/or proportional defence allocation, costs, and fitness benefits—form the empirical backbone of the predictable, but require resources that are diverted away from growth and reproduction (Stamp, ODM. However, evidence against the ODM also exists, and designing experiments that accurately 2003). In contrast, inducible defences allow for phenotypic plasticity and may be advantageous test its predictions is often difficult (Fagerström et al., 1987; Stamp, 2003; McCall and Fordyce, when predator presence is unpredictable. This enables prey to scale the expression of potentially 2010). costly defences to the actual level of threat, and thereby the likelihood of predators evolving Beyond these two pillars, other frameworks have also proven useful: the growth-rate hypothesis counter-adaptations (Rhoades, 1979; Karban et al., 1997; Agrawal, 1999; Shelton, 2004; Preisser links defence strategy to intrinsic growth potential (Coley et al., 1985; Coley, 1987); the plant et al., 2007). Inducible defence expression depends on reliable yet short-lived cues that apparency hypothesis (Feeny, 1976) predicts that large, long-lived and conspicuous (i.e. apparent) reflect current predation risk (Karban and Baldwin, 1997; Stamp, 2003). In plankton systems, plants will rely more on constitutive defences; and the extended growth-differentiation balance physical parameters such as diffusion, turbulence, and rapid degradation of chemicals (Kiørboe, (Herms and Mattson, 1992) model blends growth and defence trade-offs in a continuous framework. 2008) constrain cue persistence and necessitate specific, detectable, and transient chemical signals. Collectively, these models provide a conceptual toolkit rather than a universal law. They help In other words: cues and signals must rise above chemical background noise, persist long enough predict how and when plants allocate energy and matter to secondary-metabolite production to to be detected and responded to, be specific enough to trigger the appropriate response, and dissipate defend against herbivores, and when constitutive versus inducible defence strategies should evolve quickly enough to prevent unnecessary or prolonged responses. (Stamp, 2003). Ecological frameworks of plant defences Observations across many predator–prey systems reveal a recurring asymmetry: predators often fail Ecologists have developed a rich set of theoretical frameworks to explain and predict when and to capture prey, whereas captured prey typically suffer severe fitness costs, often death. This how organisms invest in chemical defences. These frameworks fall roughly into two families: empirical pattern underpins the evolutionary arms-race metaphor described by Dawkins and Krebs resource-driven models and demand-driven (optimality) models, in addition to a few useful (1979). Predator–prey interactions are commonly framed as a continual cycle of adaptation and 13 14 Background Background counter-adaptation in which improvements in capture or avoidance select for reciprocal reproduction, or feeding preference, defined from the consumer’s point of view and is not improvements in the other party (Van Valen, 1973; Dawkins and Krebs, 1979). The “life-dinner” necessarily beneficial to the producer. Induced defences are responses that reduce the fitness costs principle (Dawkins and Krebs, 1979) provides a useful heuristic for this asymmetry. Because prey that consumer attacks impose on the producer. Mechanisms include limiting damage (resistance risk death while predators usually forfeit only a meal, selection can favour strong, often inducible sensu stricto), increasing tolerance to a given level of damage, or avoiding the consumer altogether. defences in prey, and predators may counter-adapt through improved foraging efficiency or by This definition is producer-centred and makes no assumption about the evolutionary origin of the evolving resistance to prey defences. That asymmetry, however, does not result in inevitable or trait. unbounded escalation. Although a useful and simple informal framework, the life-dinner principle is incomplete. The tempo and direction of reciprocal evolution are modulated by demographic and When to induce ecological factors—population size, encounter rates, gene flow, and spatial and temporal Inducible strategies are favoured when predation pressure varies, when defensive traits are costly, heterogeneity—which were not included in the original formulation of the concept. These factors and when alarm cues are reliable but short-lived. This enables defence expression to reflect current create coevolutionary hot- and cold-spots and can produce local adaptation, coevolutionary mosaics, risk rather than past conditions (Harvell, 1990; Karban and Baldwin, 1997; Karban et al., 1997; or stable equilibria rather than a simple, global arms race (Brodie and Brodie, 1999; Thompson, Tollrian and Harvell, 1999; Strauss et al., 2002; Stamp, 2003; Chen, 2008). Costs can be difficult 2005; McLean et al., 2024). Recognising these modifiers clarifies when inducible defences are to quantify. They include allocation costs—resource-based trade-offs between resistance to likely to evolve and persist, and when coevolutionary dynamics will be rapid, slow or spatially predators and metabolic functions that directly affect fitness—and ecological costs arising from heterogeneous. altered interactions in the environment (Strauss et al., 2002). In phytoplankton, allocation costs are often assumed to manifest as reduced growth rates (Pančić and Kiørboe, 2018; Ryderheim, 2021). Application in the plankton: similarities and differences Reliable cues are particularly important for phytoplankton because they generally cannot mount The ecological frameworks described previously were developed for terrestrial plants, but they effective defences once damage has occurred. translate surprisingly well—albeit with caveats—to phytoplankton. Resource effects (e.g. nutrients and light) clearly influence the production of phycotoxins (toxins produced by phytoplankton) and Examples of inducible defences remains a dominant focus of much of the harmful algal bloom (HAB) literature (Boyer et al., 1987; Inducible defences are common across taxonomically diverse groups; they include escape Anderson , 2002). However, growing empirical evidence shows that grazers and grazer- behaviours (Covich et al., 1994), the formation of spines (Gilbert and Stemberger, 1984) or et al. derived chemical cues can strongly modulate toxin levels and other defences. Consistent with winglike protrusions (Kuhlmann and Heckmann, 1985), and other morphological changes in form optimal-defence logic, when grazers are present or reliably sensed, inducible investment into or colouration that reduce predation risk (Krueger and Dodson, 1981; Dodson, 1989; Brönmark and defence traits can be favoured despite associated costs (Pančić and Kiørboe, 2018). The resource- Miner, 1992; McCollum and Van Buskirk, 1996; McCollum and Leimberger, 1997; Relyea and and demand-driven perspectives can be complementary: for example, resource supply may set the Werner, 2000). physiological capacity to produce defences, and demand-driven selection shapes when and how Phytoplankton are no exception. Unlike most terrestrial animals, larger plants, and macroalgae, much to invest given the predation risk. Recognising both axes is useful when interpreting single-celled organisms such as phytoplankton generally do not survive partial grazing: any phytoplankton defences—especially chemical ones—and the dynamics of harmful algal blooms. successful grazing event is lethal to the individual cell and cannot be compensated for by regrowth Finally, a note of caution: the plant-defence literature is large and nuanced, and many empirical (Karban and Baldwin, 1997; Tollrian and Harvell, 1999; van Donk et al., 2011). Because grazing studies suffer from methodological limitations that complicate meta-level inference (Stamp, 2003). events are lethal and because selective pressure to avoid consumption is so strong, these conditions Nevertheless, borrowing well-established ecological frameworks developed for terrestrial plant have led to the evolution of a range of inducible traits that putatively function as predator deterrents. defences provides clearer, testable models for understanding chemically mediated interactions These include life history traits, such as resting cyst formation (Rengefors et al., 1998; Toth et al., among plankton and for designing experiments that separate nutrient effects from grazer-induced 2004); morphological changes, such as colony plasticity (Hessen and van Donk, 1993; Lampert et responses. With that conceptual foundation, we now turn to inducible defences themselves: what al., 1994; Long et al., 2007; Bergkvist et al., 2008, 2012), shifts in cell size (Ryderheim et al., 2021), plastic trait changes look like, what costs they impose, and how grazers can shape prey phenotypes. shape and cell-wall modifications (Grønning and Kiørboe, 2020; Ryderheim, Grønning, et al., 2022), and spines (Hessen and van Donk, 1993); behavioural responses, such as altered swimming (Selander et al., 2011); and biochemical responses, including toxin production (Guisande et al., 2002; Jang et al., 2003; Selander et al., 2008, 2019; Yang et al., 2011; Tammilehto et al., 2015; 1.4 Inducible defences Lundholm et al., 2018) and increased bioluminescence (Lindström et al., 2017; Prevett et al., 2019). Definitions The first published demonstration of grazer-induced morphological change in phytoplankton was Induced trait changes are widespread across plants and animals. Following Karban and Baldwin when Hessen and van Donk (1993). They found that the green alga Desmodesmus subspicatus (1997, p. 3), I use three terms with distinct meanings. Induced responses are any plastic changes (formerly Scenedesmus subspicatus) shifted from single cells to multicellular colonies when expressed after damage, stress, or detection of reliable risk cues. This definition does not assume exposed to the diplostracan Daphnia magna (formerly, and still commonly, called “cladoceran(s)”; fitness effects for either consumers or producers. While the phenotype is plastic, the capacity to water fleas) or to filtered water previously containing D. magna; an effect replicated the following respond may be under genetic control (Schlichting, 1986; Sultan, 1987; Bradshaw and Hardwick, year by Lampert and colleagues (1994). The compounds believed responsible were later isolated 1989). Induced resistance is the subset of induced responses that reduce consumer survival, from frozen, commercially available Daphnia material and identified as aliphatic sulphates; these 15 16 Background Background counter-adaptation in which improvements in capture or avoidance select for reciprocal reproduction, or feeding preference, defined from the consumer’s point of view and is not improvements in the other party (Van Valen, 1973; Dawkins and Krebs, 1979). The “life-dinner” necessarily beneficial to the producer. Induced defences are responses that reduce the fitness costs principle (Dawkins and Krebs, 1979) provides a useful heuristic for this asymmetry. Because prey that consumer attacks impose on the producer. Mechanisms include limiting damage (resistance risk death while predators usually forfeit only a meal, selection can favour strong, often inducible sensu stricto), increasing tolerance to a given level of damage, or avoiding the consumer altogether. defences in prey, and predators may counter-adapt through improved foraging efficiency or by This definition is producer-centred and makes no assumption about the evolutionary origin of the evolving resistance to prey defences. That asymmetry, however, does not result in inevitable or trait. unbounded escalation. Although a useful and simple informal framework, the life-dinner principle is incomplete. The tempo and direction of reciprocal evolution are modulated by demographic and When to induce ecological factors—population size, encounter rates, gene flow, and spatial and temporal Inducible strategies are favoured when predation pressure varies, when defensive traits are costly, heterogeneity—which were not included in the original formulation of the concept. These factors and when alarm cues are reliable but short-lived. This enables defence expression to reflect current create coevolutionary hot- and cold-spots and can produce local adaptation, coevolutionary mosaics, risk rather than past conditions (Harvell, 1990; Karban and Baldwin, 1997; Karban et al., 1997; or stable equilibria rather than a simple, global arms race (Brodie and Brodie, 1999; Thompson, Tollrian and Harvell, 1999; Strauss et al., 2002; Stamp, 2003; Chen, 2008). Costs can be difficult 2005; McLean et al., 2024). Recognising these modifiers clarifies when inducible defences are to quantify. They include allocation costs—resource-based trade-offs between resistance to likely to evolve and persist, and when coevolutionary dynamics will be rapid, slow or spatially predators and metabolic functions that directly affect fitness—and ecological costs arising from heterogeneous. altered interactions in the environment (Strauss et al., 2002). In phytoplankton, allocation costs are often assumed to manifest as reduced growth rates (Pančić and Kiørboe, 2018; Ryderheim, 2021). Application in the plankton: similarities and differences Reliable cues are particularly important for phytoplankton because they generally cannot mount The ecological frameworks described previously were developed for terrestrial plants, but they effective defences once damage has occurred. translate surprisingly well—albeit with caveats—to phytoplankton. Resource effects (e.g. nutrients and light) clearly influence the production of phycotoxins (toxins produced by phytoplankton) and Examples of inducible defences remains a dominant focus of much of the harmful algal bloom (HAB) literature (Boyer et al., 1987; Inducible defences are common across taxonomically diverse groups; they include escape Anderson , 2002). However, growing empirical evidence shows that grazers and grazer- behaviours (Covich et al., 1994), the formation of spines (Gilbert and Stemberger, 1984) or et al. derived chemical cues can strongly modulate toxin levels and other defences. Consistent with winglike protrusions (Kuhlmann and Heckmann, 1985), and other morphological changes in form optimal-defence logic, when grazers are present or reliably sensed, inducible investment into or colouration that reduce predation risk (Krueger and Dodson, 1981; Dodson, 1989; Brönmark and defence traits can be favoured despite associated costs (Pančić and Kiørboe, 2018). The resource- Miner, 1992; McCollum and Van Buskirk, 1996; McCollum and Leimberger, 1997; Relyea and and demand-driven perspectives can be complementary: for example, resource supply may set the Werner, 2000). physiological capacity to produce defences, and demand-driven selection shapes when and how Phytoplankton are no exception. Unlike most terrestrial animals, larger plants, and macroalgae, much to invest given the predation risk. Recognising both axes is useful when interpreting single-celled organisms such as phytoplankton generally do not survive partial grazing: any phytoplankton defences—especially chemical ones—and the dynamics of harmful algal blooms. successful grazing event is lethal to the individual cell and cannot be compensated for by regrowth Finally, a note of caution: the plant-defence literature is large and nuanced, and many empirical (Karban and Baldwin, 1997; Tollrian and Harvell, 1999; van Donk et al., 2011). Because grazing studies suffer from methodological limitations that complicate meta-level inference (Stamp, 2003). events are lethal and because selective pressure to avoid consumption is so strong, these conditions Nevertheless, borrowing well-established ecological frameworks developed for terrestrial plant have led to the evolution of a range of inducible traits that putatively function as predator deterrents. defences provides clearer, testable models for understanding chemically mediated interactions These include life history traits, such as resting cyst formation (Rengefors et al., 1998; Toth et al., among plankton and for designing experiments that separate nutrient effects from grazer-induced 2004); morphological changes, such as colony plasticity (Hessen and van Donk, 1993; Lampert et responses. With that conceptual foundation, we now turn to inducible defences themselves: what al., 1994; Long et al., 2007; Bergkvist et al., 2008, 2012), shifts in cell size (Ryderheim et al., 2021), plastic trait changes look like, what costs they impose, and how grazers can shape prey phenotypes. shape and cell-wall modifications (Grønning and Kiørboe, 2020; Ryderheim, Grønning, et al., 2022), and spines (Hessen and van Donk, 1993); behavioural responses, such as altered swimming (Selander et al., 2011); and biochemical responses, including toxin production (Guisande et al., 2002; Jang et al., 2003; Selander et al., 2008, 2019; Yang et al., 2011; Tammilehto et al., 2015; 1.4 Inducible defences Lundholm et al., 2018) and increased bioluminescence (Lindström et al., 2017; Prevett et al., 2019). Definitions The first published demonstration of grazer-induced morphological change in phytoplankton was Induced trait changes are widespread across plants and animals. Following Karban and Baldwin when Hessen and van Donk (1993). They found that the green alga Desmodesmus subspicatus (1997, p. 3), I use three terms with distinct meanings. Induced responses are any plastic changes (formerly Scenedesmus subspicatus) shifted from single cells to multicellular colonies when expressed after damage, stress, or detection of reliable risk cues. This definition does not assume exposed to the diplostracan Daphnia magna (formerly, and still commonly, called “cladoceran(s)”; fitness effects for either consumers or producers. While the phenotype is plastic, the capacity to water fleas) or to filtered water previously containing D. magna; an effect replicated the following respond may be under genetic control (Schlichting, 1986; Sultan, 1987; Bradshaw and Hardwick, year by Lampert and colleagues (1994). The compounds believed responsible were later isolated 1989). Induced resistance is the subset of induced responses that reduce consumer survival, from frozen, commercially available Daphnia material and identified as aliphatic sulphates; these 15 16 Background Background compounds reproduced the colony response seen with live Daphnia and culture water (Yasumoto consumption (Abrams, 1990, 2000)—and can explain why similar defensive shifts appear even et al., 2005, 2008). However, it remains unproven that live Daphnia release these compounds in when grazers and prey are physically separated. For example, A. minutum exposed to waterborne detectable amounts into their surrounding environment (van Donk et al., 2011). cues from the grazing copepod Acartia tonsa across a mesh barrier contained 140% more PSTs than Different grazers can induce opposite expressions of the same trait. A beautiful example of this controls (Selander et al., 2006). Similarly, the freshwater cyanobacterium Microcystis aeruginosa comes from the haptophyte likewise increased its production of hepatotoxic microcystins in response to Daphnia cues (Jang et Phaeocystis globosa, which forms larger colonies in response to cues from grazing ciliates (which preferentially feed on single cells), but suppresses colony formation al., 2003). The chain-forming diatom Skeletonema marinoi was shown to suppress chain formation by 60–90% in response to chemical cues from larger copepod grazers that feed up to four times when exposed to copepod chemical cues across a mesh barrier (Bergkvist et al., 2012). Copepods more on colonies than on single cells (Jakobsen and Tang, 2002; Long , 2007). Among and the chemical cues they exude have since been found to induce varying defensive traits in several et al. planktonic (or planktic; see discussions in Emiliani, 1952, 1991) chemically mediated predator– phytoplankton and protists, mainly autotrophic diatoms and mixotrophic dinoflagellates. These prey interactions, particularly in marine systems, phytoplankton responses to copepods are the best findings demonstrate that phytoplankton sense and respond to chemical cues from zooplankton described to date and include many of the induced defences mentioned earlier in this section. For grazers, and that those cues alone are sufficient to induce defensive traits. an overview, see the excellent article series Chemical ecology of the marine plankton periodically Dawn of a new era? updated by the Kubanek group (Poulson et al., 2009; Sieg et al., 2011; Roy et al., 2013; Schwartz Although we have good evidence for chemically mediated, grazer-induced responses in several et al., 2016; Brown et al., 2019). plankton, few of the responsible signalling molecules are known. The cueing compounds released by copepods represent one notable exception. They were first described by Selander and colleagues (2015) as a group of taurine-conjugated polar lipids, named copepodamides. These compounds 1.5 Defences against smelly copepods: Copepodamide-mediated responses were purified from commercially available, freeze-dried Calanus copepods used as fish feed in Norwegian salmon farms. To my knowledge, Selander and colleagues (2015) provided the first Fear as an adaptation description of chemical compounds that mediate interactions between marine zooplankton and Predation—and sometimes the mere risk of predation—is one of the strongest selective pressures phytoplankton. in nature and has driven the evolution of diverse morphological, chemical, and behavioural defences that reduce the likelihood of being detected and consumed (Karban and Baldwin, 1997; Kats and Copepodamides have one of two molecular scaffolds that differ at position C3 by either a methyl Dill, 1998). Crucially, predation risk alone can reshape prey behaviour and physiology (see ‘the group (dihydro-copepodamide, dhCA) or a methylene group (copepodamide, CA). Henceforth I ecology of fear’: Brown et al., 1999; Preisser et al., 2005; Zanette et al., 2011; Suraci et al., 2016; use copepodamides for the compound class, and dhCA(s) or CA(s) for scaffold-specific groups. Zanette and Clinchy, 2019). Such trait-mediated effects can cascade through food webs and alter Regardless of scaffold, all copepodamides contain a fatty acyl group at position C5 (Fig. 2). The both community structure and ecosystem function (Adrian and Schneider-Olt, 1999; Suraci et al., fatty acyl chain at C5 varies with diet, whereas the scaffold appears diet-independent and is likely 2016; Tiselius and Møller, 2017). genetically determined (Selander et al., 2015; Grebner et al., 2019). A large Calanus sp. (prosome length 2-3 mm) can exude up to 120 pmol copepodamides per day (Selander et al., 2015). Et tu, kōpēpod? To my knowledge, the first experimental evidence of copepod-induced defensive traits in Measurements in the field show that, for much of the year, copepodamide concentrations reach phytoplankton was reported in 2002, when the dinoflagellate Alexandrium minutum doubled its levels used in some of the experiments mentioned above and are demonstrably bioactive (Selander cellular toxin content after direct exposure to the grazing copepod Acartia clausi (Guisande et al., et al., 2019). Copepodamide degradation is temperature dependent and rapid (half-life on the order 2002). Similar induction effects, for other traits and species, have been repeatedly demonstrated of hours). The effective average concentration in experiments over 24 h is approximately 0.5–2% since (Pondaven et al., 2007; Bergkvist et al., 2008, 2012; Selander et al., 2012; Bjærke et al., of the nominal concentration (Selander et al., 2019). Rapid degradation is one of the features 2015). However, experiments that involve direct grazing can be confounded by selective predation: grazers may, for example, preferentially consume larger or less-toxic individuals, producing shifts in population composition (and therefore the population mean of a trait) without individual-level physiological induction. Thus, observed changes in mean trait values may reflect either true grazer- induced phenotypic plasticity or compositional shifts caused by selective grazing. Incarcerated copepods Building on this, researchers next investigated whether such responses are chemically mediated or dependent on physical predator-prey contact. For example, they incubated phytoplankton and Fig. 2: General structure of copepodamides. Two main subgroups exist, determined by the copepods together and placed the copepods in mesh “cages” that physically separated them from presence of methylene (copepodamide/CA) or methyl (dihydro-copepodamide/dhCA) at R1. The the algae—allowing water and chemical cues to pass through. Alternatively, researchers used blend is species specific, but the fatty acid side chain (at position R2) changes with diet. culture medium previously inhabited by copepods, which therefore contained copepod chemical Copepodamides are named by the acyl group (Grebner et al., 2019) followed by the scaffold name cues. Non-lethal, information-driven effects matter—sometimes as much as or more than direct e.g. 22:6 dihydro-copepodamide for a dhCA scaffold with a docosahexaenoic acyl group in position R2. Figure caption source: Paper I. 17 18 Background Background compounds reproduced the colony response seen with live Daphnia and culture water (Yasumoto consumption (Abrams, 1990, 2000)—and can explain why similar defensive shifts appear even et al., 2005, 2008). However, it remains unproven that live Daphnia release these compounds in when grazers and prey are physically separated. For example, A. minutum exposed to waterborne detectable amounts into their surrounding environment (van Donk et al., 2011). cues from the grazing copepod Acartia tonsa across a mesh barrier contained 140% more PSTs than Different grazers can induce opposite expressions of the same trait. A beautiful example of this controls (Selander et al., 2006). Similarly, the freshwater cyanobacterium Microcystis aeruginosa comes from the haptophyte likewise increased its production of hepatotoxic microcystins in response to Daphnia cues (Jang et Phaeocystis globosa, which forms larger colonies in response to cues from grazing ciliates (which preferentially feed on single cells), but suppresses colony formation al., 2003). The chain-forming diatom Skeletonema marinoi was shown to suppress chain formation by 60–90% in response to chemical cues from larger copepod grazers that feed up to four times when exposed to copepod chemical cues across a mesh barrier (Bergkvist et al., 2012). Copepods more on colonies than on single cells (Jakobsen and Tang, 2002; Long , 2007). Among and the chemical cues they exude have since been found to induce varying defensive traits in several et al. planktonic (or planktic; see discussions in Emiliani, 1952, 1991) chemically mediated predator– phytoplankton and protists, mainly autotrophic diatoms and mixotrophic dinoflagellates. These prey interactions, particularly in marine systems, phytoplankton responses to copepods are the best findings demonstrate that phytoplankton sense and respond to chemical cues from zooplankton described to date and include many of the induced defences mentioned earlier in this section. For grazers, and that those cues alone are sufficient to induce defensive traits. an overview, see the excellent article series Chemical ecology of the marine plankton periodically Dawn of a new era? updated by the Kubanek group (Poulson et al., 2009; Sieg et al., 2011; Roy et al., 2013; Schwartz Although we have good evidence for chemically mediated, grazer-induced responses in several et al., 2016; Brown et al., 2019). plankton, few of the responsible signalling molecules are known. The cueing compounds released by copepods represent one notable exception. They were first described by Selander and colleagues (2015) as a group of taurine-conjugated polar lipids, named copepodamides. These compounds 1.5 Defences against smelly copepods: Copepodamide-mediated responses were purified from commercially available, freeze-dried Calanus copepods used as fish feed in Norwegian salmon farms. To my knowledge, Selander and colleagues (2015) provided the first Fear as an adaptation description of chemical compounds that mediate interactions between marine zooplankton and Predation—and sometimes the mere risk of predation—is one of the strongest selective pressures phytoplankton. in nature and has driven the evolution of diverse morphological, chemical, and behavioural defences that reduce the likelihood of being detected and consumed (Karban and Baldwin, 1997; Kats and Copepodamides have one of two molecular scaffolds that differ at position C3 by either a methyl Dill, 1998). Crucially, predation risk alone can reshape prey behaviour and physiology (see ‘the group (dihydro-copepodamide, dhCA) or a methylene group (copepodamide, CA). Henceforth I ecology of fear’: Brown et al., 1999; Preisser et al., 2005; Zanette et al., 2011; Suraci et al., 2016; use copepodamides for the compound class, and dhCA(s) or CA(s) for scaffold-specific groups. Zanette and Clinchy, 2019). Such trait-mediated effects can cascade through food webs and alter Regardless of scaffold, all copepodamides contain a fatty acyl group at position C5 (Fig. 2). The both community structure and ecosystem function (Adrian and Schneider-Olt, 1999; Suraci et al., fatty acyl chain at C5 varies with diet, whereas the scaffold appears diet-independent and is likely 2016; Tiselius and Møller, 2017). genetically determined (Selander et al., 2015; Grebner et al., 2019). A large Calanus sp. (prosome length 2-3 mm) can exude up to 120 pmol copepodamides per day (Selander et al., 2015). Et tu, kōpēpod? To my knowledge, the first experimental evidence of copepod-induced defensive traits in Measurements in the field show that, for much of the year, copepodamide concentrations reach phytoplankton was reported in 2002, when the dinoflagellate Alexandrium minutum doubled its levels used in some of the experiments mentioned above and are demonstrably bioactive (Selander cellular toxin content after direct exposure to the grazing copepod Acartia clausi (Guisande et al., et al., 2019). Copepodamide degradation is temperature dependent and rapid (half-life on the order 2002). Similar induction effects, for other traits and species, have been repeatedly demonstrated of hours). The effective average concentration in experiments over 24 h is approximately 0.5–2% since (Pondaven et al., 2007; Bergkvist et al., 2008, 2012; Selander et al., 2012; Bjærke et al., of the nominal concentration (Selander et al., 2019). Rapid degradation is one of the features 2015). However, experiments that involve direct grazing can be confounded by selective predation: grazers may, for example, preferentially consume larger or less-toxic individuals, producing shifts in population composition (and therefore the population mean of a trait) without individual-level physiological induction. Thus, observed changes in mean trait values may reflect either true grazer- induced phenotypic plasticity or compositional shifts caused by selective grazing. Incarcerated copepods Building on this, researchers next investigated whether such responses are chemically mediated or dependent on physical predator-prey contact. For example, they incubated phytoplankton and Fig. 2: General structure of copepodamides. Two main subgroups exist, determined by the copepods together and placed the copepods in mesh “cages” that physically separated them from presence of methylene (copepodamide/CA) or methyl (dihydro-copepodamide/dhCA) at R1. The the algae—allowing water and chemical cues to pass through. Alternatively, researchers used blend is species specific, but the fatty acid side chain (at position R2) changes with diet. culture medium previously inhabited by copepods, which therefore contained copepod chemical Copepodamides are named by the acyl group (Grebner et al., 2019) followed by the scaffold name cues. Non-lethal, information-driven effects matter—sometimes as much as or more than direct e.g. 22:6 dihydro-copepodamide for a dhCA scaffold with a docosahexaenoic acyl group in position R2. Figure caption source: Paper I. 17 18 Background Background expected of “good” cues, because long-lasting cues would risk inducing defensive traits even after the danger has passed and result in unnecessary costs (Rhoades, 1979; Karban and Baldwin, 1997; Stamp, 2003). Good cues thus enable prey organisms to regulate the expression of defence traits to match predation risk. The function of copepodamides in copepods is still unknown, as is their site of production. It has been hypothesised that copepodamides could play a role in digestion or lipid processing. Supporting evidence is that the fatty acyl group attached at C5 reflects the composition of phytoplankton consumed and changes with diet, and that exudation rates decline within hours of starvation (Grebner et al., 2019). Importantly, similar taurine-conjugated compounds are implicated in digestion and lipid uptake in other marine organisms (Kunimitsu and Keiko, 1986; Huxtable, 1992). Since their description in 2015, copepodamides have been shown experimentally to reproduce phytoplankton responses observed under direct grazing or with copepod-conditioned water containing their chemical cues. Thus, copepodamides provide a plausible mechanistic link between grazer presence and chemically inducible phytoplankton traits. Copepodamides as defence inducers Defence traits demonstrably induced by isolated and purified copepodamides include increased toxin production (Selander et al., 2015, 2019; Grebner et al., 2019; Ryderheim et al., 2021; Brown et al., 2022; Olesen et al., 2022, 2024; Zhang et al., 2024; Jia et al., 2025), suppressed chain formation (Grebner et al., 2019; Selander et al., 2019; Rigby and Selander, 2021; Rigby et al., Fig. 3: Schematic illustrating some of the responses that copepodamides can induce in marine 2022), increased bioluminescence (Lindström et al., 2017; Prevett et al., 2019), altered diel feeding phytoplankton. Credit: Jan Heuschele. rhythm (Arias et al., 2021), increased frustule silicification in diatoms (Grønning and Kiørboe, 2020), increased diatom aggregation due to enhanced “stickiness” (Grønning and Kiørboe, 2022), lengths by up to 70% when exposed to copepodamides (Grebner et al., 2019; Selander et al., 2019; and shifts in prokaryotic and eukaryotic plankton community structure (Rigby et al., 2024). In short, Rigby and Selander, 2021; Rigby et al., 2022), and other chain-forming diatoms similarly shorten phytoplankton can change form, physical structure, chemistry and timing in response to chains by tens of percent (Rigby and Selander, 2021). These morphological shifts reduce copepodamides. Fig. 3 illustrates some responses induced by copepodamides, and Table 1 lists all hydromechanical and chemical signatures, which lower the encounter rate with copepods species shown to respond in published experiments to date. (Bergkvist et al., 2012; Bjærke et al., 2015; Rigby and Selander, 2021) and reduces their clearance These responses have direct ecological consequences. Paralytic shellfish toxin (PST) production by rate (Ryderheim, Hansen, et al., 2022). Such size shifts may move prey outside the particle-size dinoflagellates such as Alexandrium increases by up to 1 600% when exposed to copepodamides range that copepods can efficiently handle, although they may instead move within the prey size (Selander et al., 2015; Grebner et al., 2019; Ryderheim et al., 2021). These elevated PST levels spectrum of smaller zooplankton grazers. Similarly, the chain-forming dinoflagellate Alexandrium demonstrably correlate to copepods rejecting toxic cells and selectively feeding on less-toxic tamarense shortens chains and reduces swimming velocity when challenged with water-borne cues alternatives when available (Teegarden, 1999; Selander et al., 2006; Wohlrab et al., 2017; Xu and from caged copepods (Selander et al., 2011, 2012), effectively reducing their encounter rate with Kiørboe, 2018; Ryderheim et al., 2021). Similarly, amnesic shellfish toxin (AST, domoic acid) predators. Whether this response is induced by purified copepodamides alone remains unexplored. production by diatoms such as Pseudo-nitzschia increases by up to 10 000% when exposed to copepodamides (Grebner et al., 2019; Selander et al., 2019; Olesen et al., 2022, unpublished Bioluminescence is the production of visible light by living organisms. In marine dinoflagellates, manuscript (2024) in PhD thesis: Olesen, 2024). Increased ASTs, similarly to PSTs, result in this takes the form of brief flashes produced by a chemical reaction between luciferin and luciferase rejection rates of 70–80% compared with 5–25% in less-toxic controls (Olesen et al., 2022; Olesen, inside small cytoplasmic structures called scintillons (DeSa and Hastings, 1968; Hamman and 2024). The combination of induced toxin production and selective rejection of more toxic cells Seliger, 1972; Valiadi and Iglesias-Rodriguez, 2013). The flash is triggered by mechanical favours the persistence and dominance of toxin-producing taxa, and thus may contribute to HAB- disturbances, e.g. contact with a grazer’s feeding appendages. Dinoflagellate bioluminescence acts formation. However, rejection of defended cells is not total, and some copepods appear relatively at the point of capture and appears to be an efficient grazer deterrent (Esaias and Curl, 1972; Prevett unaffected by consuming PST- and AST-rich prey; these toxins can accumulate in copepods and act et al., 2019), but the proximate mechanism of this remains unresolved. Three main hypotheses have as vectors of toxins to higher trophic levels (Teegarden and Cembella, 1996; Hamasaki et al., 2003; been proposed to date. Burkenroad (1943) suggested bioluminescence serves as a “burglar alarm”, Teegarden et al., 2003; Leandro et al., 2010; Tammilehto et al., 2012; Harðardóttir et al., 2015). attracting visual predators of the grazers. Esaias and Curl (1972) suggested that the bioluminescent flash startles the copepod, enabling prey escape. Finally, bioluminescence is proposed to act as Other trait responses act either by lowering the encounter rate with predators—which depends in aposematism—a signal of toxicity or unpalatability—to predators (Hanley and Widder, 2017). part on prey size and on the swimming velocities of both predator and prey—or by directly reducing predator handling success. Chain-forming diatoms, such as Skeletonema marinoi, reduce their chain 19 20 Background Background expected of “good” cues, because long-lasting cues would risk inducing defensive traits even after the danger has passed and result in unnecessary costs (Rhoades, 1979; Karban and Baldwin, 1997; Stamp, 2003). Good cues thus enable prey organisms to regulate the expression of defence traits to match predation risk. The function of copepodamides in copepods is still unknown, as is their site of production. It has been hypothesised that copepodamides could play a role in digestion or lipid processing. Supporting evidence is that the fatty acyl group attached at C5 reflects the composition of phytoplankton consumed and changes with diet, and that exudation rates decline within hours of starvation (Grebner et al., 2019). Importantly, similar taurine-conjugated compounds are implicated in digestion and lipid uptake in other marine organisms (Kunimitsu and Keiko, 1986; Huxtable, 1992). Since their description in 2015, copepodamides have been shown experimentally to reproduce phytoplankton responses observed under direct grazing or with copepod-conditioned water containing their chemical cues. Thus, copepodamides provide a plausible mechanistic link between grazer presence and chemically inducible phytoplankton traits. Copepodamides as defence inducers Defence traits demonstrably induced by isolated and purified copepodamides include increased toxin production (Selander et al., 2015, 2019; Grebner et al., 2019; Ryderheim et al., 2021; Brown et al., 2022; Olesen et al., 2022, 2024; Zhang et al., 2024; Jia et al., 2025), suppressed chain formation (Grebner et al., 2019; Selander et al., 2019; Rigby and Selander, 2021; Rigby et al., Fig. 3: Schematic illustrating some of the responses that copepodamides can induce in marine 2022), increased bioluminescence (Lindström et al., 2017; Prevett et al., 2019), altered diel feeding phytoplankton. Credit: Jan Heuschele. rhythm (Arias et al., 2021), increased frustule silicification in diatoms (Grønning and Kiørboe, 2020), increased diatom aggregation due to enhanced “stickiness” (Grønning and Kiørboe, 2022), lengths by up to 70% when exposed to copepodamides (Grebner et al., 2019; Selander et al., 2019; and shifts in prokaryotic and eukaryotic plankton community structure (Rigby et al., 2024). In short, Rigby and Selander, 2021; Rigby et al., 2022), and other chain-forming diatoms similarly shorten phytoplankton can change form, physical structure, chemistry and timing in response to chains by tens of percent (Rigby and Selander, 2021). These morphological shifts reduce copepodamides. Fig. 3 illustrates some responses induced by copepodamides, and Table 1 lists all hydromechanical and chemical signatures, which lower the encounter rate with copepods species shown to respond in published experiments to date. (Bergkvist et al., 2012; Bjærke et al., 2015; Rigby and Selander, 2021) and reduces their clearance These responses have direct ecological consequences. Paralytic shellfish toxin (PST) production by rate (Ryderheim, Hansen, et al., 2022). Such size shifts may move prey outside the particle-size dinoflagellates such as Alexandrium increases by up to 1 600% when exposed to copepodamides range that copepods can efficiently handle, although they may instead move within the prey size (Selander et al., 2015; Grebner et al., 2019; Ryderheim et al., 2021). These elevated PST levels spectrum of smaller zooplankton grazers. Similarly, the chain-forming dinoflagellate Alexandrium demonstrably correlate to copepods rejecting toxic cells and selectively feeding on less-toxic tamarense shortens chains and reduces swimming velocity when challenged with water-borne cues alternatives when available (Teegarden, 1999; Selander et al., 2006; Wohlrab et al., 2017; Xu and from caged copepods (Selander et al., 2011, 2012), effectively reducing their encounter rate with Kiørboe, 2018; Ryderheim et al., 2021). Similarly, amnesic shellfish toxin (AST, domoic acid) predators. Whether this response is induced by purified copepodamides alone remains unexplored. production by diatoms such as Pseudo-nitzschia increases by up to 10 000% when exposed to copepodamides (Grebner et al., 2019; Selander et al., 2019; Olesen et al., 2022, unpublished Bioluminescence is the production of visible light by living organisms. In marine dinoflagellates, manuscript (2024) in PhD thesis: Olesen, 2024). Increased ASTs, similarly to PSTs, result in this takes the form of brief flashes produced by a chemical reaction between luciferin and luciferase rejection rates of 70–80% compared with 5–25% in less-toxic controls (Olesen et al., 2022; Olesen, inside small cytoplasmic structures called scintillons (DeSa and Hastings, 1968; Hamman and 2024). The combination of induced toxin production and selective rejection of more toxic cells Seliger, 1972; Valiadi and Iglesias-Rodriguez, 2013). The flash is triggered by mechanical favours the persistence and dominance of toxin-producing taxa, and thus may contribute to HAB- disturbances, e.g. contact with a grazer’s feeding appendages. Dinoflagellate bioluminescence acts formation. However, rejection of defended cells is not total, and some copepods appear relatively at the point of capture and appears to be an efficient grazer deterrent (Esaias and Curl, 1972; Prevett unaffected by consuming PST- and AST-rich prey; these toxins can accumulate in copepods and act et al., 2019), but the proximate mechanism of this remains unresolved. Three main hypotheses have as vectors of toxins to higher trophic levels (Teegarden and Cembella, 1996; Hamasaki et al., 2003; been proposed to date. Burkenroad (1943) suggested bioluminescence serves as a “burglar alarm”, Teegarden et al., 2003; Leandro et al., 2010; Tammilehto et al., 2012; Harðardóttir et al., 2015). attracting visual predators of the grazers. Esaias and Curl (1972) suggested that the bioluminescent flash startles the copepod, enabling prey escape. Finally, bioluminescence is proposed to act as Other trait responses act either by lowering the encounter rate with predators—which depends in aposematism—a signal of toxicity or unpalatability—to predators (Hanley and Widder, 2017). part on prey size and on the swimming velocities of both predator and prey—or by directly reducing predator handling success. Chain-forming diatoms, such as Skeletonema marinoi, reduce their chain 19 20 Background Background The dinoflagellate Lingulaulax polyedra (previously Lingulodinium polyedra) responds to Table 1: List of organisms that respond to copepodamides by inducing putative defence traits. copepodamides by increasing bioluminescence up to 200% (Lindström et al., 2017; Prevett et al., AST/PST denote amnesic/paralytic shellfish toxins, DPT denotes Dinophysis-produced toxins, 2019). This enhanced bioluminescence effectively deters grazers: flashing cells are significantly +/– denote increase/decrease, roman numerals denote thesis papers. Adapted from Rigby (2022). more rejected than non-flashing ones. High-speed, low-light videos of tethered individuals of the copepod Temora longicornis show that flashes—which occur on contact with the copepod’s Species Group Response References mouth—trigger immediate prey rejection accompanied by vigorous beating of the copepods’ swimming appendages (Prevett et al., 2019). Although the tethered copepod cannot escape, the Pseudo-nitzschia seriata Diatom AST + Grebner et al., 2019; Selander et al., 2019; same response would propel free-swimming copepods away from the flash site. This behaviour Olesen et al., 2022; Olesen et al., 2024; Jia et al., 2025 provides mechanistic support for the burglar alarm hypothesis, as the flash recruits predators by eliciting the grazer’s escape response and movement. The burglar alarm hypothesis has recently Grebner et al., 2019; Selander et al., 2019; been further supported by Huang and colleagues (2024), who demonstrated that increased Skeletonema marinoi Diatom Chain length – Rigby et al., 2022, Grønning & Kiørboe, bioluminescence reduces clearance and ingestion rates of Temora longicornis nauplii (young 2022; II crustacean larvae) while increasing their escape jump frequency, velocity, and duration. These more Aggregation Grønning & Kiørboe, 2022 vigorous jumps make the grazer more detectable to flow-sensing predators such as the (stickiness) + copepod Centropages typicus, resulting in higher grazer mortality due to their own predators. Chaetoceros curvicetus Diatom Chain length – Rigby & Selander 2021, Rigby et al., 2022 Unlike the classical burglar alarm hypothesis, this “behavioural cascade” induced by single-cell flashes recruits predators indirectly through the grazer’s own escape behaviour. The authors argue Thalassiosira rotula Diatom Chain length – Rigby & Selander 2021, Rigby et al., 2022 that individual cell defence via startling is the primary adaptive trait of bioluminescence in dinoflagellates, with the burglar alarm as a secondary but ecologically significant adaptation. Thalassiosira weissflogii Diatom Frustule silica + Grønning & Kiørboe, 2020 Together, these studies provide some mechanistic support for the burglar alarm and startling Amphiprora paludosa Diatom Frustule silica + Grønning & Kiørboe, 2020 hypotheses for bioluminescence as grazer deterrents, while evidence for aposematism remains scarce. Ditylum brightwellii Diatom Frustule silica + Grønning & Kiørboe, 2020 Finally, some diatoms respond to copepodamides by increasing frustule silicification, i.e. the Nitzschia laevis Diatom Frustule silica + Grønning & Kiørboe, 2020 amount of silica in their shells (Grønning and Kiørboe, 2020; Ryderheim, Grønning, et al., 2022). Navicula incerta Diatom Frustule silica + Grønning & Kiørboe, 2020 Diatoms with more silicified frustules withstand copepod feeding better by increasing their Cyclotella meneghiniana Diatom Frustule silica + Grønning & Kiørboe, 2020 handling time (Ryderheim, Grønning, et al., 2022) and causing wear on copepod silica-lined teeth (Ryderheim et al., 2024), which impairs copepod feeding efficiency and may reduce their long- Cyclotella cryptica Diatom Frustule silica + Grønning & Kiørboe, 2020 term fitness. Consequently, copepods tend to reject more-silicified cells (Liu et al., 2016; Pančić et Aggregation Grønning & Kiørboe, 2022 al., 2019; Ryderheim, Grønning, et al., 2022; Ryderheim et al., 2024). Additionally, the anti-grazer (stickiness) + benefit of this defence appear to incur growth costs (Pančić et al., 2019; Grønning and Kiørboe, 2020). Selander et al., 2015; Grebner et al., 2019; Alexandrium minutum Dinoflagellate PST + Ryderheim et al., 2021; Brown et al., Overall, the body of work comprising ten years of research since the description of copepodamides 2022; Rigby et al., 2022; Zhang et al., illustrates a general point: chemically mediated information (not just consumption) is a major driver 2024 of phytoplankton defence trait expression, with consequences that may cascade through food webs Alexandrium catenella Dinoflagellate PST + III to affect community structure and biogeochemical fluxes. Isolated copepodamides provide a Bioluminescence + III repeatable way to ‘switch on’ defensive phenotypes in the lab, avoiding confounding effects of live grazers and removing the need to maintain copepod cultures. This may help resolve long-standing Alexandrium tamarense Dinoflagellate Bioluminescence + Lindström et al., 2017 questions about the evolution of anti-grazer defences, a proposed explanation to Hutchinson’s Lingulaulax polyedra Dinoflagellate Bioluminescence + Lindström et al., 2017 “paradox of the plankton” (Hutchinson, 1961). The paradox refers to the observation that Dinophysis sacculus Dinoflagellate DPT + IV phytoplankton communities maintain far more species than simple resource-competition theory predicts. If two species compete for a single limiting nutrient, the species that can persist at the Protoceratium reticulatum Dinoflagellate Bioluminescence + III lowest resource concentration should exclude the other (Hardin, 1960; Hutchinson, 1961; Tilman, Gymnodinium catenatum Dinoflagellate PST + III 1982). Yet, we observe high plankton diversity supported on a small number of limiting resources, implying that additional ecological processes must prevent competitive exclusion. Predation and Strombidium arenicola Ciliate Enhanced diel Arias et al 2020 the evolution of anti-predator defences provide one explanation to the paradox. If two species differ feeding rhythm in energy allocation, one being a competition specialist that allocates more to growth and the other 21 22 Background Background The dinoflagellate Lingulaulax polyedra (previously Lingulodinium polyedra) responds to Table 1: List of organisms that respond to copepodamides by inducing putative defence traits. copepodamides by increasing bioluminescence up to 200% (Lindström et al., 2017; Prevett et al., AST/PST denote amnesic/paralytic shellfish toxins, DPT denotes Dinophysis-produced toxins, 2019). This enhanced bioluminescence effectively deters grazers: flashing cells are significantly +/– denote increase/decrease, roman numerals denote thesis papers. Adapted from Rigby (2022). more rejected than non-flashing ones. High-speed, low-light videos of tethered individuals of the copepod Temora longicornis show that flashes—which occur on contact with the copepod’s Species Group Response References mouth—trigger immediate prey rejection accompanied by vigorous beating of the copepods’ swimming appendages (Prevett et al., 2019). Although the tethered copepod cannot escape, the Pseudo-nitzschia seriata Diatom AST + Grebner et al., 2019; Selander et al., 2019; same response would propel free-swimming copepods away from the flash site. This behaviour Olesen et al., 2022; Olesen et al., 2024; Jia et al., 2025 provides mechanistic support for the burglar alarm hypothesis, as the flash recruits predators by eliciting the grazer’s escape response and movement. The burglar alarm hypothesis has recently Grebner et al., 2019; Selander et al., 2019; been further supported by Huang and colleagues (2024), who demonstrated that increased Skeletonema marinoi Diatom Chain length – Rigby et al., 2022, Grønning & Kiørboe, bioluminescence reduces clearance and ingestion rates of Temora longicornis nauplii (young 2022; II crustacean larvae) while increasing their escape jump frequency, velocity, and duration. These more Aggregation Grønning & Kiørboe, 2022 vigorous jumps make the grazer more detectable to flow-sensing predators such as the (stickiness) + copepod Centropages typicus, resulting in higher grazer mortality due to their own predators. Chaetoceros curvicetus Diatom Chain length – Rigby & Selander 2021, Rigby et al., 2022 Unlike the classical burglar alarm hypothesis, this “behavioural cascade” induced by single-cell flashes recruits predators indirectly through the grazer’s own escape behaviour. The authors argue Thalassiosira rotula Diatom Chain length – Rigby & Selander 2021, Rigby et al., 2022 that individual cell defence via startling is the primary adaptive trait of bioluminescence in dinoflagellates, with the burglar alarm as a secondary but ecologically significant adaptation. Thalassiosira weissflogii Diatom Frustule silica + Grønning & Kiørboe, 2020 Together, these studies provide some mechanistic support for the burglar alarm and startling Amphiprora paludosa Diatom Frustule silica + Grønning & Kiørboe, 2020 hypotheses for bioluminescence as grazer deterrents, while evidence for aposematism remains scarce. Ditylum brightwellii Diatom Frustule silica + Grønning & Kiørboe, 2020 Finally, some diatoms respond to copepodamides by increasing frustule silicification, i.e. the Nitzschia laevis Diatom Frustule silica + Grønning & Kiørboe, 2020 amount of silica in their shells (Grønning and Kiørboe, 2020; Ryderheim, Grønning, et al., 2022). Navicula incerta Diatom Frustule silica + Grønning & Kiørboe, 2020 Diatoms with more silicified frustules withstand copepod feeding better by increasing their Cyclotella meneghiniana Diatom Frustule silica + Grønning & Kiørboe, 2020 handling time (Ryderheim, Grønning, et al., 2022) and causing wear on copepod silica-lined teeth (Ryderheim et al., 2024), which impairs copepod feeding efficiency and may reduce their long- Cyclotella cryptica Diatom Frustule silica + Grønning & Kiørboe, 2020 term fitness. Consequently, copepods tend to reject more-silicified cells (Liu et al., 2016; Pančić et Aggregation Grønning & Kiørboe, 2022 al., 2019; Ryderheim, Grønning, et al., 2022; Ryderheim et al., 2024). Additionally, the anti-grazer (stickiness) + benefit of this defence appear to incur growth costs (Pančić et al., 2019; Grønning and Kiørboe, 2020). Selander et al., 2015; Grebner et al., 2019; Alexandrium minutum Dinoflagellate PST + Ryderheim et al., 2021; Brown et al., Overall, the body of work comprising ten years of research since the description of copepodamides 2022; Rigby et al., 2022; Zhang et al., illustrates a general point: chemically mediated information (not just consumption) is a major driver 2024 of phytoplankton defence trait expression, with consequences that may cascade through food webs Alexandrium catenella Dinoflagellate PST + III to affect community structure and biogeochemical fluxes. Isolated copepodamides provide a Bioluminescence + III repeatable way to ‘switch on’ defensive phenotypes in the lab, avoiding confounding effects of live grazers and removing the need to maintain copepod cultures. This may help resolve long-standing Alexandrium tamarense Dinoflagellate Bioluminescence + Lindström et al., 2017 questions about the evolution of anti-grazer defences, a proposed explanation to Hutchinson’s Lingulaulax polyedra Dinoflagellate Bioluminescence + Lindström et al., 2017 “paradox of the plankton” (Hutchinson, 1961). The paradox refers to the observation that Dinophysis sacculus Dinoflagellate DPT + IV phytoplankton communities maintain far more species than simple resource-competition theory predicts. If two species compete for a single limiting nutrient, the species that can persist at the Protoceratium reticulatum Dinoflagellate Bioluminescence + III lowest resource concentration should exclude the other (Hardin, 1960; Hutchinson, 1961; Tilman, Gymnodinium catenatum Dinoflagellate PST + III 1982). Yet, we observe high plankton diversity supported on a small number of limiting resources, implying that additional ecological processes must prevent competitive exclusion. Predation and Strombidium arenicola Ciliate Enhanced diel Arias et al 2020 the evolution of anti-predator defences provide one explanation to the paradox. If two species differ feeding rhythm in energy allocation, one being a competition specialist that allocates more to growth and the other 21 22 Background a defence specialist that allocates more to anti-predator traits, predation can suppress the fast grower. This releases resources for the well-defended, slower-growing species. This “killing the winner” dynamic (Thingstad and Lignell, 1997; Winter et al., 2010), also known as keystone-predation (Paine, 1966), promotes coexistence by making competitive dominance context dependent. Inducible defences—which are expressed only when predators are present—add further temporal and spatial context, because defence expression depends on local risk and resource conditions. Such plasticity broadens niche differences and slows competitive exclusion, helping to explain species richness seemingly resource-limited plankton assemblages (Leibold et al., 2017). Beyond improving our basic understanding of plankton interactions, this young subfield of plankton ecology is already yielding useful practical applications. Efforts to monitor copepodamides in the field—including adaptations of SPATT-style (Solid Phase Adsorption Toxin Tracking) passive samplers and analyses of routinely collected bivalve-monitoring samples—show promise for improving HAB surveillance and modelling (Trapp et al., 2021, 2023). Trapp and colleagues (2021) Chapter 2. found that adding copepod metrics (copepod biomass and copepodamides measured in mussel tissue) improved forecasts of Dinophysis-produced okadaic acid in mussels along the Swedish west Aims of thesis coast, relative to models using only Dinophysis abundance. These additions also increase forecast lead time. Finally, copepodamides hold potential uses in nascent biotechnological and biomedical applications. For example, researchers exploring algal toxins and toxin derivatives as medical agents (e.g. neosaxitoxin as a long-acting local anaesthetic; Greenhough et al., 2025) could benefit from methods that increase yields in toxin-producing bioreactors. Plants are not just food for animals […] The world is not green. It is colored All told, the copepodamide-mediated copepod–phytoplankton system is both an elegant example lectin, tannin, cyanide, caffeine, aflatoxin, and canavanine. of information-driven ecology and the basis of an emerging practical toolbox for studying predator– prey interactions. Daniel H. Janzen, 1977 Kyss! – Albin Lee Meldau 23 24 Background a defence specialist that allocates more to anti-predator traits, predation can suppress the fast grower. This releases resources for the well-defended, slower-growing species. This “killing the winner” dynamic (Thingstad and Lignell, 1997; Winter et al., 2010), also known as keystone-predation (Paine, 1966), promotes coexistence by making competitive dominance context dependent. Inducible defences—which are expressed only when predators are present—add further temporal and spatial context, because defence expression depends on local risk and resource conditions. Such plasticity broadens niche differences and slows competitive exclusion, helping to explain species richness seemingly resource-limited plankton assemblages (Leibold et al., 2017). Beyond improving our basic understanding of plankton interactions, this young subfield of plankton ecology is already yielding useful practical applications. Efforts to monitor copepodamides in the field—including adaptations of SPATT-style (Solid Phase Adsorption Toxin Tracking) passive samplers and analyses of routinely collected bivalve-monitoring samples—show promise for improving HAB surveillance and modelling (Trapp et al., 2021, 2023). Trapp and colleagues (2021) Chapter 2. found that adding copepod metrics (copepod biomass and copepodamides measured in mussel tissue) improved forecasts of Dinophysis-produced okadaic acid in mussels along the Swedish west Aims of thesis coast, relative to models using only Dinophysis abundance. These additions also increase forecast lead time. Finally, copepodamides hold potential uses in nascent biotechnological and biomedical applications. For example, researchers exploring algal toxins and toxin derivatives as medical agents (e.g. neosaxitoxin as a long-acting local anaesthetic; Greenhough et al., 2025) could benefit from methods that increase yields in toxin-producing bioreactors. Plants are not just food for animals […] The world is not green. It is colored All told, the copepodamide-mediated copepod–phytoplankton system is both an elegant example lectin, tannin, cyanide, caffeine, aflatoxin, and canavanine. of information-driven ecology and the basis of an emerging practical toolbox for studying predator– prey interactions. Daniel H. Janzen, 1977 Kyss! – Albin Lee Meldau 23 24 Aims The study of copepodamide‑mediated interactions is a young and rapidly evolving field in plankton ecology, with much still to be done. My aim in this thesis is to expand our current understanding of copepodamides by investigating who produces them, which organisms detect and respond to them, and the ecological consequences of these interactions. By bridging experimental studies with integrative synthesis, I also aim to evaluate the importance of grazer‑induced defences for harmful algal bloom formation compared to the better‑studied resource‑driven effects. To achieve these goals, the individual papers employ a combination of observational, experimental and synthesis methods and address the following objectives: I. Determine whether freshwater copepods also produce copepodamides, and test whether copepodamide composition differs between freshwater and marine copepods, including patterns attributable to taxonomy and sampling sites. Based on anecdotal evidence from a small pond in the Botanical Garden of Gothenburg (Sweden), we hypothesise that Chapter 3. freshwater copepods also produce copepodamides. Organisms and methods II. Test whether copepodamides suppress chain formation in three previously untested chain- forming diatom species, and quantify species-specific responses. We hypothesise that all three species will reduce chain length in response to copepodamides, consistent with responses reported for Skeletonema marinoi and other diatoms. III. Determine whether bloom-forming dinoflagellates (Alexandrium catenella, And do you think that unto such as you Protoceratium reticulatum, and Gymnodinium catenatum) induce bioluminescence and A maggot-minded, starved, fanatic crew toxin production in response to copepodamides, and assess whether responses are God gave a secret, and denied it me? associated with allocation costs. We hypothesise that copepodamides will induce toxin Well, well—what matters it? Believe that, too! production in G. catenatum, bioluminescence in P. reticulatum, and both traits in A. catenella, and that co‑expression will incur the greatest growth cost. Rubaiyât of Omar Khayyâm IV. Test whether two Dinophysis species (D. sacculus and D. acuminata) increase production of diarrhetic shellfish toxins when exposed to copepodamides or live copepods, determine the partitioning of toxins between cells and the surrounding medium, and evaluate whether toxin induction is accompanied by metabolome-wide changes. We hypothesise that both species will increase toxin production in response to copepodamides and to live copepods. V. Quantify and compare the effects of grazing risk (demand-driven) versus relative nitrogen enrichment (increased N:P ratio; resource-driven) on phycotoxin induction in Alexandrium dinoflagellates and Pseudo‑nitzschia diatoms, and situate the harmful algal bloom literature within the ecological plant-defence frameworks developed for terrestrial plants. We hypothesise that demand-driven induction is as strong as, or stronger than, resource-driven induction in both genera, particularly in Pseudo-nitzschia. The Book of Love – Peter Gabriel 25 26 Aims The study of copepodamide‑mediated interactions is a young and rapidly evolving field in plankton ecology, with much still to be done. My aim in this thesis is to expand our current understanding of copepodamides by investigating who produces them, which organisms detect and respond to them, and the ecological consequences of these interactions. By bridging experimental studies with integrative synthesis, I also aim to evaluate the importance of grazer‑induced defences for harmful algal bloom formation compared to the better‑studied resource‑driven effects. To achieve these goals, the individual papers employ a combination of observational, experimental and synthesis methods and address the following objectives: I. Determine whether freshwater copepods also produce copepodamides, and test whether copepodamide composition differs between freshwater and marine copepods, including patterns attributable to taxonomy and sampling sites. Based on anecdotal evidence from a small pond in the Botanical Garden of Gothenburg (Sweden), we hypothesise that Chapter 3. freshwater copepods also produce copepodamides. Organisms and methods II. Test whether copepodamides suppress chain formation in three previously untested chain- forming diatom species, and quantify species-specific responses. We hypothesise that all three species will reduce chain length in response to copepodamides, consistent with responses reported for Skeletonema marinoi and other diatoms. III. Determine whether bloom-forming dinoflagellates (Alexandrium catenella, And do you think that unto such as you Protoceratium reticulatum, and Gymnodinium catenatum) induce bioluminescence and A maggot-minded, starved, fanatic crew toxin production in response to copepodamides, and assess whether responses are God gave a secret, and denied it me? associated with allocation costs. We hypothesise that copepodamides will induce toxin Well, well—what matters it? Believe that, too! production in G. catenatum, bioluminescence in P. reticulatum, and both traits in A. catenella, and that co‑expression will incur the greatest growth cost. Rubaiyât of Omar Khayyâm IV. Test whether two Dinophysis species (D. sacculus and D. acuminata) increase production of diarrhetic shellfish toxins when exposed to copepodamides or live copepods, determine the partitioning of toxins between cells and the surrounding medium, and evaluate whether toxin induction is accompanied by metabolome-wide changes. We hypothesise that both species will increase toxin production in response to copepodamides and to live copepods. V. Quantify and compare the effects of grazing risk (demand-driven) versus relative nitrogen enrichment (increased N:P ratio; resource-driven) on phycotoxin induction in Alexandrium dinoflagellates and Pseudo‑nitzschia diatoms, and situate the harmful algal bloom literature within the ecological plant-defence frameworks developed for terrestrial plants. We hypothesise that demand-driven induction is as strong as, or stronger than, resource-driven induction in both genera, particularly in Pseudo-nitzschia. The Book of Love – Peter Gabriel 25 26 Organisms and methods Organisms and methods In this chapter, I provide a general overview of the main methods used and organisms studied as might be underrepresented in vertical tows. Horizontal tows are also useful for mapping horizontal part of this thesis. I broadly outline sampling approaches, experimental methods, and analytical heterogeneity and bloom dynamics over large spatial scales. The main drawback of horizontal tows techniques—without delving into step-by-step protocols, which are detailed in the individual papers. is that they sample only a narrow depth slice. Also, use of a calibrated rotor-based flow-metre is essential to obtain quantitative estimates of filtered volume in horizontal tow; otherwise catch data 3.1 Organisms are qualitative only. Why diatoms and dinoflagellates? Extraction and purification of copepodamides Diatoms and dinoflagellates underpin all marine food webs: they are numerically abundant, The process (described in Selander 2015) begins with freeze‑dried Calanus finmarchicus taxonomically diverse, and together drive a large fraction of oceanic primary production and commercially fished off the coast of Norway (Calanus AS, Tromsø, Norway). Copepod material is particle fluxes. Their ubiquity means that trait changes in these groups ripple up through food webs extracted twice in methanol, each extraction lasting 24–48 h. The methanolic phase is collected and and biogeochemical cycles. Understanding how—and why—they alter form, chemistry, or filtered to remove particulates. The clarified crude extract goes through a liquid–liquid separation behaviour under threat therefore matters for interpreting large-scale processes and food web process to remove non-polar lipids, following a protocol adapted from Löfgren and colleagues dynamics, affecting ecosystems and humans alike. (2012). Ammonia is added to Milli-Q water and mixed with the methanol to yield methanol:water Both groups are powerful model systems for exploring inducible defences: they combine with 1% ammonia (95:5), which is then mixed with heptane:methanol (98:2) in a separation funnel. experimental tractability with ecological and evolutionary significance because they are abundant The mixture is shaken vigorously for a few minutes and then allowed to separate into fractions. The in marine ecosystems and subject to intense grazing pressure. Both groups can be cultured reliably methanol fraction—containing the copepodamides—is collected and undergoes the same procedure and express a range of plastic traits—from morphological shifts like chain formation and with heptane:methanol (98:2) one or two more times to further remove non-polar lipids. The final silicification in diatoms to behavioural changes, cyst formation and toxin production in methanol fraction is adjusted to 20% methanol with water and loaded onto a solid‑phase extraction dinoflagellates—in response to grazers and grazer cues. Some species in each group also produce (SPE) column. Compounds more polar than copepodamides (including lysolipids) are removed harmful algal blooms, creating a direct link between defence induction and real-world concerns with 50% methanol, and the fraction containing copepodamides is eluted using 100% methanol such as toxin outbreaks and ecosystem disruption. (Selander 2015). A 70% methanol intermediary step can also be employed before the final elution. The 100% methanol eluate is dried down in a rotary evaporator until just dry, redissolved in a small Their contrasting life histories and defence modes allow experiments tease apart how organisms volume of methanol, and transferred to HPLC vials before gradient purification (Selander et al., allocate energy between growth and protection. Tey also permit studies of how trait-mediated 2015). responses influence community structure, nutrient cycling, interactions with competitors, and the initiation and persistence of bloom events. Their versatility and fast growth relative to terrestrial Samples are further purified with a gradient elution on a high-performance liquid chromatography plants make diatoms and dinoflagellates powerful model systems for testing general hypotheses of system (HP-LC) fitted with a reverse-phase C18 column using a 25‑min gradient from 100% eluent inducible defence and plant-herbivore interactions, examining trade-offs under predation stress, and A (methanol:acetonitrile:water, 45:40:15) to 100% eluent B (2‑propanol), with 0.2% formic acid developing predictive models for bloom dynamics. added to both eluents and a flow of 1 mL min−1; with a 5 minute hold at 100% B and re‑equilibrates in 100% A for 7 min before subsequent injections. Fractions are collected every 15 s, and copepodamide‑containing fractions are confirmed by direct‑infusion mass spectrometry on an LC- 3.2 Methods system coupled to a triple quadrupole mass-spectrometer with electrospray ionization, after which verified fractions are pooled (Selander et al., 2015). Sampling plankton Plankton sampling commonly relies on towing nets through the water column3, with two principal Analysis of copepodamides from water samples and in individual copepods approaches: vertical and horizontal tows. In a vertical tow, a plankton net—typically with a fine Experimental and field water samples are analysed by concentrating copepodamides on mesh of 20–500 µm—is lowered from the surface to a fixed depth (for example, 50–200 m) and reversed-phase SPE columns (Evolute ABN, 100 mg of sorbent), desalted with Milli-Q water, and then hauled up at a controlled speed. This method integrates organisms over the sampled depth eluted with 100% methanol divided into two equal additions separated by a 30 s soak step to range, providing a composite sample that is useful for estimating total abundance and community maximise yield. The eluate is concentrated under a stream of nitrogen gas—while in a 40°C heat composition across strata. Vertical tows are straightforward to perform from small vessels, require block—and redissolved in a small amount of methanol (~50-80 µL) for mass spectrometry analysis. minimal manoeuvring, and yield quantitative estimates of plankton metrics per unit volume filtered. Individual copepods are extracted by transferring them into 1.5 mL HPLC glass vials, removing the However, vertical tows can obscure fine-scale vertical distribution patterns, leading to potential water, adding 1 mL methanol, and extracting overnight at −20°C. Extracts are then concentrated by under- or overestimation of certain taxa. evaporation until dry under nitrogen at 40°C and redissolved in a small amount of methanol before Horizontal tows, by contrast, involve towing the net at a constant depth—often near the surface (0– storage at −20°C until analysis. 5 m) or at specific midwater layers—while the vessel moves at a steady speed (e.g. 1–2 knots) for Copepodamides are analysed on an LC-system coupled to a triple quadrupole detector with a fixed distance or time. This approach captures a “snapshot” of plankton communities at a chosen electrospray ionization. Separation uses a C18 column, heated to 50°C, with a gradient from eluent depth and is particularly effective for sampling fast-swimming or patchily distributed species that A (methanol:acetonitrile:water, 35:35:30) to eluent B (2-propanol) at 0.2 mL min−1, with 0.2% 27 28 Organisms and methods Organisms and methods In this chapter, I provide a general overview of the main methods used and organisms studied as might be underrepresented in vertical tows. Horizontal tows are also useful for mapping horizontal part of this thesis. I broadly outline sampling approaches, experimental methods, and analytical heterogeneity and bloom dynamics over large spatial scales. The main drawback of horizontal tows techniques—without delving into step-by-step protocols, which are detailed in the individual papers. is that they sample only a narrow depth slice. Also, use of a calibrated rotor-based flow-metre is essential to obtain quantitative estimates of filtered volume in horizontal tow; otherwise catch data 3.1 Organisms are qualitative only. Why diatoms and dinoflagellates? Extraction and purification of copepodamides Diatoms and dinoflagellates underpin all marine food webs: they are numerically abundant, The process (described in Selander 2015) begins with freeze‑dried Calanus finmarchicus taxonomically diverse, and together drive a large fraction of oceanic primary production and commercially fished off the coast of Norway (Calanus AS, Tromsø, Norway). Copepod material is particle fluxes. Their ubiquity means that trait changes in these groups ripple up through food webs extracted twice in methanol, each extraction lasting 24–48 h. The methanolic phase is collected and and biogeochemical cycles. Understanding how—and why—they alter form, chemistry, or filtered to remove particulates. The clarified crude extract goes through a liquid–liquid separation behaviour under threat therefore matters for interpreting large-scale processes and food web process to remove non-polar lipids, following a protocol adapted from Löfgren and colleagues dynamics, affecting ecosystems and humans alike. (2012). Ammonia is added to Milli-Q water and mixed with the methanol to yield methanol:water Both groups are powerful model systems for exploring inducible defences: they combine with 1% ammonia (95:5), which is then mixed with heptane:methanol (98:2) in a separation funnel. experimental tractability with ecological and evolutionary significance because they are abundant The mixture is shaken vigorously for a few minutes and then allowed to separate into fractions. The in marine ecosystems and subject to intense grazing pressure. Both groups can be cultured reliably methanol fraction—containing the copepodamides—is collected and undergoes the same procedure and express a range of plastic traits—from morphological shifts like chain formation and with heptane:methanol (98:2) one or two more times to further remove non-polar lipids. The final silicification in diatoms to behavioural changes, cyst formation and toxin production in methanol fraction is adjusted to 20% methanol with water and loaded onto a solid‑phase extraction dinoflagellates—in response to grazers and grazer cues. Some species in each group also produce (SPE) column. Compounds more polar than copepodamides (including lysolipids) are removed harmful algal blooms, creating a direct link between defence induction and real-world concerns with 50% methanol, and the fraction containing copepodamides is eluted using 100% methanol such as toxin outbreaks and ecosystem disruption. (Selander 2015). A 70% methanol intermediary step can also be employed before the final elution. The 100% methanol eluate is dried down in a rotary evaporator until just dry, redissolved in a small Their contrasting life histories and defence modes allow experiments tease apart how organisms volume of methanol, and transferred to HPLC vials before gradient purification (Selander et al., allocate energy between growth and protection. Tey also permit studies of how trait-mediated 2015). responses influence community structure, nutrient cycling, interactions with competitors, and the initiation and persistence of bloom events. Their versatility and fast growth relative to terrestrial Samples are further purified with a gradient elution on a high-performance liquid chromatography plants make diatoms and dinoflagellates powerful model systems for testing general hypotheses of system (HP-LC) fitted with a reverse-phase C18 column using a 25‑min gradient from 100% eluent inducible defence and plant-herbivore interactions, examining trade-offs under predation stress, and A (methanol:acetonitrile:water, 45:40:15) to 100% eluent B (2‑propanol), with 0.2% formic acid developing predictive models for bloom dynamics. added to both eluents and a flow of 1 mL min−1; with a 5 minute hold at 100% B and re‑equilibrates in 100% A for 7 min before subsequent injections. Fractions are collected every 15 s, and copepodamide‑containing fractions are confirmed by direct‑infusion mass spectrometry on an LC- 3.2 Methods system coupled to a triple quadrupole mass-spectrometer with electrospray ionization, after which verified fractions are pooled (Selander et al., 2015). Sampling plankton Plankton sampling commonly relies on towing nets through the water column3, with two principal Analysis of copepodamides from water samples and in individual copepods approaches: vertical and horizontal tows. In a vertical tow, a plankton net—typically with a fine Experimental and field water samples are analysed by concentrating copepodamides on mesh of 20–500 µm—is lowered from the surface to a fixed depth (for example, 50–200 m) and reversed-phase SPE columns (Evolute ABN, 100 mg of sorbent), desalted with Milli-Q water, and then hauled up at a controlled speed. This method integrates organisms over the sampled depth eluted with 100% methanol divided into two equal additions separated by a 30 s soak step to range, providing a composite sample that is useful for estimating total abundance and community maximise yield. The eluate is concentrated under a stream of nitrogen gas—while in a 40°C heat composition across strata. Vertical tows are straightforward to perform from small vessels, require block—and redissolved in a small amount of methanol (~50-80 µL) for mass spectrometry analysis. minimal manoeuvring, and yield quantitative estimates of plankton metrics per unit volume filtered. Individual copepods are extracted by transferring them into 1.5 mL HPLC glass vials, removing the However, vertical tows can obscure fine-scale vertical distribution patterns, leading to potential water, adding 1 mL methanol, and extracting overnight at −20°C. Extracts are then concentrated by under- or overestimation of certain taxa. evaporation until dry under nitrogen at 40°C and redissolved in a small amount of methanol before Horizontal tows, by contrast, involve towing the net at a constant depth—often near the surface (0– storage at −20°C until analysis. 5 m) or at specific midwater layers—while the vessel moves at a steady speed (e.g. 1–2 knots) for Copepodamides are analysed on an LC-system coupled to a triple quadrupole detector with a fixed distance or time. This approach captures a “snapshot” of plankton communities at a chosen electrospray ionization. Separation uses a C18 column, heated to 50°C, with a gradient from eluent depth and is particularly effective for sampling fast-swimming or patchily distributed species that A (methanol:acetonitrile:water, 35:35:30) to eluent B (2-propanol) at 0.2 mL min−1, with 0.2% 27 28 Organisms and methods Organisms and methods formic acid added to each eluent. The gradient starts with 5 min isocratic elution at 95% eluent A, Cell densities and chain lengths can be enumerated under light microscopy—often using counting followed by a linear increase of eluent B to 82% over 18 min, held for 1 min, then re-equilibrated chambers such as the Sedgewick-Rafter and Utermöhl chamber (Fig. 4C)—and/or by automated for 8 min with 5% eluent B. The ion source operates in negative mode at 300°C and 4.5 kV with image analysis in software such as ImageJ. Machine-based methods such as Coulter counters and nitrogen gas flow of 7 L min−1 at 35 psi. Known copepodamides are targeted through multiple flow cytometry can also be used, and provide rapid, high-throughput counts. Here, we enumerated reaction monitoring, confirmed by retention time and diagnostic fragments using fragmentation cells manually in Sedgewick-Rafter chambers (Paper II) and multiwell plates (Papers III & IV) voltage of 250 V and collision energy of 44 eV. under inverted light microscopy, or by analysing microscopy images using Fiji ImageJ (Paper III; Experimental methods Schindelin et al., 2012). We expose phytoplankton to copepodamides—which are dissolved in methanol—by coating the Bioluminescence capacity is measured using a luminometer, with light emission triggered either by inside of the culture vessel, then allowing solvent to evaporate before adding the cells. This can be gentle aeration through a capillary to mimic hydromechanical disturbance or by injection of acetic done under a gentle nitrogen stream for quicker evaporation while minimising oxidation. Controls acid to elicit a maximal bioluminescent response. are coated with methanol only and otherwise treated identically. For re-exposure, we carefully transfer each replicate into a freshly coated glass vessel to maintain consistent cue levels. Because Toxins are quantified by high-performance liquid chromatography coupled to mass spectrometry copepodamides desorb slowly from glass surfaces and degrade within hours in water, we use and calibrated against certified reference materials (e.g. from National Research Council Canada) nominal concentrations higher than the target concentration to ensure sustained exposure over the to ensure accuracy and comparability across studies. course of experiments. Software Plankton experiments typically run on motorised plankton wheels that rotate at 0.5–1 rpm, creating I have used R (R Core Team, 2024) through RStudio (Posit team, 2024) for all statistical analyses a gentle, continuous motion after a brief spin-up, ensuring non-motile cells remain in suspension and most visualisations across the papers in this thesis. All code and data used (for published and (Fig. 4A & B). Experiments are performed in controlled temperature and light conditions suited to preprinted papers) are available in openly accessible repositories (e.g., Zenodo). the taxa in question. A B C Fig. 4: Small (A) and large (B) motorised plankton wheels rotating at 0.5–2.5 rpm, with incubation vessels attached. (C) Sedgewick-Rafter counting chamber that holds 1 mL of sample and features 1000 squares, each square thus corresponds to 1 µL of the sample. 29 30 Organisms and methods Organisms and methods formic acid added to each eluent. The gradient starts with 5 min isocratic elution at 95% eluent A, Cell densities and chain lengths can be enumerated under light microscopy—often using counting followed by a linear increase of eluent B to 82% over 18 min, held for 1 min, then re-equilibrated chambers such as the Sedgewick-Rafter and Utermöhl chamber (Fig. 4C)—and/or by automated for 8 min with 5% eluent B. The ion source operates in negative mode at 300°C and 4.5 kV with image analysis in software such as ImageJ. Machine-based methods such as Coulter counters and nitrogen gas flow of 7 L min−1 at 35 psi. Known copepodamides are targeted through multiple flow cytometry can also be used, and provide rapid, high-throughput counts. Here, we enumerated reaction monitoring, confirmed by retention time and diagnostic fragments using fragmentation cells manually in Sedgewick-Rafter chambers (Paper II) and multiwell plates (Papers III & IV) voltage of 250 V and collision energy of 44 eV. under inverted light microscopy, or by analysing microscopy images using Fiji ImageJ (Paper III; Experimental methods Schindelin et al., 2012). We expose phytoplankton to copepodamides—which are dissolved in methanol—by coating the Bioluminescence capacity is measured using a luminometer, with light emission triggered either by inside of the culture vessel, then allowing solvent to evaporate before adding the cells. This can be gentle aeration through a capillary to mimic hydromechanical disturbance or by injection of acetic done under a gentle nitrogen stream for quicker evaporation while minimising oxidation. Controls acid to elicit a maximal bioluminescent response. are coated with methanol only and otherwise treated identically. For re-exposure, we carefully transfer each replicate into a freshly coated glass vessel to maintain consistent cue levels. Because Toxins are quantified by high-performance liquid chromatography coupled to mass spectrometry copepodamides desorb slowly from glass surfaces and degrade within hours in water, we use and calibrated against certified reference materials (e.g. from National Research Council Canada) nominal concentrations higher than the target concentration to ensure sustained exposure over the to ensure accuracy and comparability across studies. course of experiments. Software Plankton experiments typically run on motorised plankton wheels that rotate at 0.5–1 rpm, creating I have used R (R Core Team, 2024) through RStudio (Posit team, 2024) for all statistical analyses a gentle, continuous motion after a brief spin-up, ensuring non-motile cells remain in suspension and most visualisations across the papers in this thesis. All code and data used (for published and (Fig. 4A & B). Experiments are performed in controlled temperature and light conditions suited to preprinted papers) are available in openly accessible repositories (e.g., Zenodo). the taxa in question. A B C Fig. 4: Small (A) and large (B) motorised plankton wheels rotating at 0.5–2.5 rpm, with incubation vessels attached. (C) Sedgewick-Rafter counting chamber that holds 1 mL of sample and features 1000 squares, each square thus corresponds to 1 µL of the sample. 29 30 Summary of papers Paper I: First description of copepodamides in freshwater copepods Copepodamides have been found in all marine cyclopoid and calanoid copepods tested to date (Selander et al., 2015; Grebner et al., 2019; Paper I), except perhaps in the strictly carnivorous Paraeuchaeta norvegica (Lundholm et al., 2018), and they seem to act as general alarm cues of copepod presence to taxonomically diverse prey organisms. However, the presence of copepodamides in freshwater copepods has previously consisted of anecdotal evidence from a small pond in the Gothenburg Botanical Garden. For this study, we sampled freshwater copepods from six Swedish lakes and marine copepods from the west coast of Sweden (Fig. 5). We measured copepodamides in individual copepods and in bulk zooplankton samples (dominated by copepods) using targeted and non-targeted high-resolution mass spectrometry, respectively. We show that copepodamides are consistently present across all Chapter 4. freshwater copepod samples at concentrations around 0.1‰ of dry mass in millimetre-sized copepods (~100 µg dry mass), comparable to similarly sized marine copepods (Fig. 6e). This Summary of papers supports our hypothesis. Freshwater copepods were dominated by dihydro-copepodamides (dhCAs; see Chapter 1.5: Dawn of a new era? for details on copepodamide structure), whereas marine copepods contained mixed dhCAs and CAs (Fig 7). Lower variability in copepodamide composition—likely reflecting population-level bottlenecks observed in population genetic variation—is perhaps unsurprising, given that freshwater copepods derive from marine and brackish ancestors that invaded freshwater systems multiple times (Lee and Bell, 1999; Lee, 2016). The universe we observe has precisely the properties we should expect if there is, at bottom, no design, no purpose, no evil, no good, nothing but blind, pitiless indifference. Richard Dawkins River Out of Eden Fig. 5: Map of sampling stations (left) with names and positions (right table). Freshwater sites are shown in green and marine in blue. Stations F1–F3 markers overlap, F5 is located on the island Brännö. M3 and M4 markers overlap and are in the Koster Fjord. Maps produced in MATLAB 2023a. Figure caption source: Paper I. Let There Be Rock – AC/DC 31 32 Summary of papers Paper I: First description of copepodamides in freshwater copepods Copepodamides have been found in all marine cyclopoid and calanoid copepods tested to date (Selander et al., 2015; Grebner et al., 2019; Paper I), except perhaps in the strictly carnivorous Paraeuchaeta norvegica (Lundholm et al., 2018), and they seem to act as general alarm cues of copepod presence to taxonomically diverse prey organisms. However, the presence of copepodamides in freshwater copepods has previously consisted of anecdotal evidence from a small pond in the Gothenburg Botanical Garden. For this study, we sampled freshwater copepods from six Swedish lakes and marine copepods from the west coast of Sweden (Fig. 5). We measured copepodamides in individual copepods and in bulk zooplankton samples (dominated by copepods) using targeted and non-targeted high-resolution mass spectrometry, respectively. We show that copepodamides are consistently present across all Chapter 4. freshwater copepod samples at concentrations around 0.1‰ of dry mass in millimetre-sized copepods (~100 µg dry mass), comparable to similarly sized marine copepods (Fig. 6e). This Summary of papers supports our hypothesis. Freshwater copepods were dominated by dihydro-copepodamides (dhCAs; see Chapter 1.5: Dawn of a new era? for details on copepodamide structure), whereas marine copepods contained mixed dhCAs and CAs (Fig 7). Lower variability in copepodamide composition—likely reflecting population-level bottlenecks observed in population genetic variation—is perhaps unsurprising, given that freshwater copepods derive from marine and brackish ancestors that invaded freshwater systems multiple times (Lee and Bell, 1999; Lee, 2016). The universe we observe has precisely the properties we should expect if there is, at bottom, no design, no purpose, no evil, no good, nothing but blind, pitiless indifference. Richard Dawkins River Out of Eden Fig. 5: Map of sampling stations (left) with names and positions (right table). Freshwater sites are shown in green and marine in blue. Stations F1–F3 markers overlap, F5 is located on the island Brännö. M3 and M4 markers overlap and are in the Koster Fjord. Maps produced in MATLAB 2023a. Figure caption source: Paper I. Let There Be Rock – AC/DC 31 32 Summary of papers Summary of papers Fig. 6: (a) Representative mass spectra from precursor ion scans of copepodamides in bulk Fig. 7: Composition of zooplankton samples from limnic (F5) and (b) marine (M4). Note the absence of CA (blue) in the copepodamides in individual limnic samples. c: Non-metric dimensional scaling (nMDS) ordination of copepodamide copepods from targeted LC–MS composition in bulk zooplankton samples for each site (smaller points) and (d) in individual analysis of the most abundant copepods (smaller points). Larger points denote the centroid for each group (i.e. habitat type copepodamides. Organised by average in c and sampling site average in d), coloured ellipses are 95% confidence intervals of habitat (marine & freshwater), centroids. Stress values for the nMDS models were 0.04 and 0.11 respectively. (e) Total taxon and sampling site. Blue copepodamide content in individual copepods (pmol ind−1, filled circles) plotted against their and green bars denote CAs and estimated dry mass (µg). Thin lines denote regression lines for the individual sampling sites, dhCAs respectively. Figure thicker coloured lines denote regression lines for the two habitat types and the dotted black line caption source: Paper I. denotes the global regression (Ln(CA pmol) = 0.296 * Ln(dry mass µg) + 0.25, R2 = 0.087). Shaded error bands denote 95% confidence intervals for the habitat regressions. Figure caption source: Paper I. 33 34 Summary of papers Summary of papers Fig. 6: (a) Representative mass spectra from precursor ion scans of copepodamides in bulk Fig. 7: Composition of zooplankton samples from limnic (F5) and (b) marine (M4). Note the absence of CA (blue) in the copepodamides in individual limnic samples. c: Non-metric dimensional scaling (nMDS) ordination of copepodamide copepods from targeted LC–MS composition in bulk zooplankton samples for each site (smaller points) and (d) in individual analysis of the most abundant copepods (smaller points). Larger points denote the centroid for each group (i.e. habitat type copepodamides. Organised by average in c and sampling site average in d), coloured ellipses are 95% confidence intervals of habitat (marine & freshwater), centroids. Stress values for the nMDS models were 0.04 and 0.11 respectively. (e) Total taxon and sampling site. Blue copepodamide content in individual copepods (pmol ind−1, filled circles) plotted against their and green bars denote CAs and estimated dry mass (µg). Thin lines denote regression lines for the individual sampling sites, dhCAs respectively. Figure thicker coloured lines denote regression lines for the two habitat types and the dotted black line caption source: Paper I. denotes the global regression (Ln(CA pmol) = 0.296 * Ln(dry mass µg) + 0.25, R2 = 0.087). Shaded error bands denote 95% confidence intervals for the habitat regressions. Figure caption source: Paper I. 33 34 Summary of papers Summary of papers Species-specific copepodamide profiles were temporally and spatially stable, and compositional variety among marine copepods was driven more by differences between taxa than within taxa (Figs. 6c-d). Fatty acyl moieties in the copepodamides of freshwater copepods varied more in saturation compared to their marine counterparts, and included odd-numbered carbon chains (e.g. C15 and C17) more commonly found in bacteria (Kaneda, 1991). Finally, we identified ten putative novel copepodamides, including four unique to freshwater species. These results establish that copepodamides are ubiquitous among grazing copepods and conserved across aquatic ecosystems, and they raise questions about their ecophysiological significance of copepodamides in freshwater environments. Because dhCAs have been suggested to be more potent inducers of toxin production than CAs in at least one dinoflagellate species (Selander et al., 2015), our findings could have direct implications for limnic systems dominated by copepods, if limnic phytoplankton respond similarly to their marine counterparts. Paper II: Chain suppression of new diatom species Exposure to copepodamides has been shown to shorten chains in at least three chain-forming species of diatoms (Rigby and Selander, 2021; Rigby et al., 2022 and references therein) and one dinoflagellate (Selander et al., 2011, 2012), which reduces encounter rates with—and subsequent grazing by—copepods. However, the prevalence of this defence across chain-forming diatoms remains unknown. Here we exposed three previously untested chain-forming diatoms—Skeletonema subsalsum, a newly isolated strain of Chaetoceros sp., and Eupyxidicula turris—alongside S. marinoi (positive control) to copepodamides (1 nM nominal) for approximately 80 h in controlled conditions, then quantified chain lengths microscopically. We found that S. marinoi chains shortened by 37% (from 4 to 2.5 cells per chain) relative to controls when exposed to copepodamides, confirming the expected response (Figs. 8A-B). In contrast, the three novel taxa showed no to minor changes. S. subsalsum chain lengths decreased by 3%, and although Chaetoceros sp. and E. turris both trended toward shorter chains (~14% reductions), these changes were not statistically significant. Chain- length distributions shifted substantially only in S. marinoi: under cue exposure more than half the cells were in short chains (≤2 cells per chain), compared with fewer than one third in controls (Fig. 9). Only the response of S. marinoi conforms to our hypotheses. We conclude that copepodamide-induced chain suppression is not a universal defence trait across chain-forming diatoms, pointing to species-specific variation in anti-copepod responses. The lack of response in most of the taxa suggests that these species may prioritise other defence mechanisms, such as enhanced frustule silicification or spine modification, when sensing copepods nearby. Fig. 8: Effects of copepodamide exposure on chain length and growth. (A) Percent change in mean Alternatively, they do not employ any anti-grazer defences. Even subtle trends toward shorter chains in sp. and may alter grazer encounter rates and feeding efficiency. chain length relative to control. Filled circles are mean percent change from control, back-Chaetoceros E. turris transformed from log response ratios (Hedges et al., 1999) and error bars denote 95% confidence However, the primary defence burden in these taxa—if they express any—is likely borne by traits intervals (CI) calculated on the log scale and back-transformed. (B) Chain length of individual other than chain suppression. The species-specific patterns we observed raise questions about the replicates (semi-transparent diamonds) and group means (filled circles) for control (grey) and evolutionary history and ecological context that shape which defence traits evolve. copepodamide (black) treatments; error bars denote 95% CI of means. (C) Growth rate of individual replicates and group means (symbols as in B); error bars denote 95% CI of means. Sample sizes: n = 5 replicates per treatment for Skeletonema marinoi and S. subsalsum; n = 3 per treatment for Chaetoceros sp. and Eupyxidicula turris. Asterisk (*) denotes a statistically significant difference between treatments (permutation test, two-sided, α = 0.05). Figure caption source: Paper II. 35 36 Summary of papers Summary of papers Species-specific copepodamide profiles were temporally and spatially stable, and compositional variety among marine copepods was driven more by differences between taxa than within taxa (Figs. 6c-d). Fatty acyl moieties in the copepodamides of freshwater copepods varied more in saturation compared to their marine counterparts, and included odd-numbered carbon chains (e.g. C15 and C17) more commonly found in bacteria (Kaneda, 1991). Finally, we identified ten putative novel copepodamides, including four unique to freshwater species. These results establish that copepodamides are ubiquitous among grazing copepods and conserved across aquatic ecosystems, and they raise questions about their ecophysiological significance of copepodamides in freshwater environments. Because dhCAs have been suggested to be more potent inducers of toxin production than CAs in at least one dinoflagellate species (Selander et al., 2015), our findings could have direct implications for limnic systems dominated by copepods, if limnic phytoplankton respond similarly to their marine counterparts. Paper II: Chain suppression of new diatom species Exposure to copepodamides has been shown to shorten chains in at least three chain-forming species of diatoms (Rigby and Selander, 2021; Rigby et al., 2022 and references therein) and one dinoflagellate (Selander et al., 2011, 2012), which reduces encounter rates with—and subsequent grazing by—copepods. However, the prevalence of this defence across chain-forming diatoms remains unknown. Here we exposed three previously untested chain-forming diatoms—Skeletonema subsalsum, a newly isolated strain of Chaetoceros sp., and Eupyxidicula turris—alongside S. marinoi (positive control) to copepodamides (1 nM nominal) for approximately 80 h in controlled conditions, then quantified chain lengths microscopically. We found that S. marinoi chains shortened by 37% (from 4 to 2.5 cells per chain) relative to controls when exposed to copepodamides, confirming the expected response (Figs. 8A-B). In contrast, the three novel taxa showed no to minor changes. S. subsalsum chain lengths decreased by 3%, and although Chaetoceros sp. and E. turris both trended toward shorter chains (~14% reductions), these changes were not statistically significant. Chain- length distributions shifted substantially only in S. marinoi: under cue exposure more than half the cells were in short chains (≤2 cells per chain), compared with fewer than one third in controls (Fig. 9). Only the response of S. marinoi conforms to our hypotheses. We conclude that copepodamide-induced chain suppression is not a universal defence trait across chain-forming diatoms, pointing to species-specific variation in anti-copepod responses. The lack of response in most of the taxa suggests that these species may prioritise other defence mechanisms, such as enhanced frustule silicification or spine modification, when sensing copepods nearby. Fig. 8: Effects of copepodamide exposure on chain length and growth. (A) Percent change in mean Alternatively, they do not employ any anti-grazer defences. Even subtle trends toward shorter chains in sp. and may alter grazer encounter rates and feeding efficiency. chain length relative to control. Filled circles are mean percent change from control, back-Chaetoceros E. turris transformed from log response ratios (Hedges et al., 1999) and error bars denote 95% confidence However, the primary defence burden in these taxa—if they express any—is likely borne by traits intervals (CI) calculated on the log scale and back-transformed. (B) Chain length of individual other than chain suppression. The species-specific patterns we observed raise questions about the replicates (semi-transparent diamonds) and group means (filled circles) for control (grey) and evolutionary history and ecological context that shape which defence traits evolve. copepodamide (black) treatments; error bars denote 95% CI of means. (C) Growth rate of individual replicates and group means (symbols as in B); error bars denote 95% CI of means. Sample sizes: n = 5 replicates per treatment for Skeletonema marinoi and S. subsalsum; n = 3 per treatment for Chaetoceros sp. and Eupyxidicula turris. Asterisk (*) denotes a statistically significant difference between treatments (permutation test, two-sided, α = 0.05). Figure caption source: Paper II. 35 36 Summary of papers Summary of papers Paper III: Simultaneous induction of multiple defence traits Bioluminescence and toxin production are known to function as defence traits against grazing by copepods in several dinoflagellate species (Prevett et al., 2019; Ryderheim et al., 2021), and copepodamides can demonstrably induce these individually. However, whether copepodamides can induce simultaneous multi-trait expression. and whether this carries fitness (allocation) costs to growth, has remained untested. Here we exposed three bloom-forming dinoflagellates—Alexandrium catenella (toxic and bioluminescent), Protoceratium reticulatum (bioluminescent), and Gymnodinium catenatum (toxic)—to increasing concentrations of purified copepodamides (0.1–5 nM nominal), re-exposing every 48 h to compensate for degradation, and measured bioluminescence, PSTs, and growth over 1–8 days. All three species upregulated defence traits, relative to controls: P. reticulatum and A. catenella increased in bioluminescence by ~31–83% over five days (Figs. 10a-b); G. catenatum significantly increased in PST content (peaking at ~165% on day 4), and A. catenella roughly doubled its PSTs (Fig. 11a-b). Although the increase in A. catenella toxins was technically not statistically significant at the 5% level (p = 0.053), the 95% confidence interval of the effect size (23–236 %) suggests a true increase; reminding me of a quote by Rosnow and Rosenthal— critiquing the almost religious obsession many researchers had become with an arbitrary cut-off— which goes “We want to underscore that, surely, God loves the .06 nearly as much as the .05” (Rosnow and Rosenthal, 1989, 1277). Notably, this is the first experimental demonstration that copepodamides induce GC-type toxin production in G. catenatum, and that A. catenella simultaneously increase bioluminescence and toxins—one of few experimental examples of multi-trait defence expression in phytoplankton. Contrary to predictions rooted in theoretical models of inducible defences, induced bioluminescence and toxin production showed no detectable growth costs under our nutrient-replete laboratory conditions (Figs. 10e-f & 11e-f). This absence of measurable costs aligns with some recent studies but contrasts with theoretical expectations that defences should be costly, raising questions about whether costs manifest only under resource limitation or involve unmeasured traits (Ryderheim et al., 2021). We thus expand the set of copepodamide-responsive species and show that single-trait studies may miss important interactions in multi-trait expressing taxa. They also suggest that copepods may chemically and indirectly influence harmful-algal-bloom dynamics by changing grazer–prey interactions when inducing defended morphotypes (e.g., by selectively grazing on the non- defended), thereby potentially increasing the persistence and competitive success of defended dinoflagellates. These results give support for our hypotheses regarding defence trait induction, but Fig. 9: Relative chain-length frequency distributions for four taxa under control (grey) and not regarding the costs of those. copepodamide (Cue; black) treatments: Skeletonema marinoi (A), S. subsalsum (B), Chaetoceros sp. (C), and Eupyxidicula turris (D). Bars show mean relative frequencies of chains in each length class (number of chains of a given length divided by the total number of chains for that taxon and treatment), averaged across replicates. Means are based on n = 5 replicates per treatment for S. marinoi and S. subsalsum, and n = 3 per treatment for Chaetoceros sp. and E. turris. Error bars are 95% confidence intervals of the mean frequencies. White circles in panel B indicate the proportion of cells in that group that were non-viable (non-vegetative, potentially dead) and are calculated as (bar height) × (mean within-class proportion non-viable). For example, a bar height of 20% combined with 50% within-class non-viability yields a white circle at 20 × 0.50 = 10%. Figure caption source: Paper II. 37 38 Summary of papers Summary of papers Paper III: Simultaneous induction of multiple defence traits Bioluminescence and toxin production are known to function as defence traits against grazing by copepods in several dinoflagellate species (Prevett et al., 2019; Ryderheim et al., 2021), and copepodamides can demonstrably induce these individually. However, whether copepodamides can induce simultaneous multi-trait expression. and whether this carries fitness (allocation) costs to growth, has remained untested. Here we exposed three bloom-forming dinoflagellates—Alexandrium catenella (toxic and bioluminescent), Protoceratium reticulatum (bioluminescent), and Gymnodinium catenatum (toxic)—to increasing concentrations of purified copepodamides (0.1–5 nM nominal), re-exposing every 48 h to compensate for degradation, and measured bioluminescence, PSTs, and growth over 1–8 days. All three species upregulated defence traits, relative to controls: P. reticulatum and A. catenella increased in bioluminescence by ~31–83% over five days (Figs. 10a-b); G. catenatum significantly increased in PST content (peaking at ~165% on day 4), and A. catenella roughly doubled its PSTs (Fig. 11a-b). Although the increase in A. catenella toxins was technically not statistically significant at the 5% level (p = 0.053), the 95% confidence interval of the effect size (23–236 %) suggests a true increase; reminding me of a quote by Rosnow and Rosenthal— critiquing the almost religious obsession many researchers had become with an arbitrary cut-off— which goes “We want to underscore that, surely, God loves the .06 nearly as much as the .05” (Rosnow and Rosenthal, 1989, 1277). Notably, this is the first experimental demonstration that copepodamides induce GC-type toxin production in G. catenatum, and that A. catenella simultaneously increase bioluminescence and toxins—one of few experimental examples of multi-trait defence expression in phytoplankton. Contrary to predictions rooted in theoretical models of inducible defences, induced bioluminescence and toxin production showed no detectable growth costs under our nutrient-replete laboratory conditions (Figs. 10e-f & 11e-f). This absence of measurable costs aligns with some recent studies but contrasts with theoretical expectations that defences should be costly, raising questions about whether costs manifest only under resource limitation or involve unmeasured traits (Ryderheim et al., 2021). We thus expand the set of copepodamide-responsive species and show that single-trait studies may miss important interactions in multi-trait expressing taxa. They also suggest that copepods may chemically and indirectly influence harmful-algal-bloom dynamics by changing grazer–prey interactions when inducing defended morphotypes (e.g., by selectively grazing on the non- defended), thereby potentially increasing the persistence and competitive success of defended dinoflagellates. These results give support for our hypotheses regarding defence trait induction, but Fig. 9: Relative chain-length frequency distributions for four taxa under control (grey) and not regarding the costs of those. copepodamide (Cue; black) treatments: Skeletonema marinoi (A), S. subsalsum (B), Chaetoceros sp. (C), and Eupyxidicula turris (D). Bars show mean relative frequencies of chains in each length class (number of chains of a given length divided by the total number of chains for that taxon and treatment), averaged across replicates. Means are based on n = 5 replicates per treatment for S. marinoi and S. subsalsum, and n = 3 per treatment for Chaetoceros sp. and E. turris. Error bars are 95% confidence intervals of the mean frequencies. White circles in panel B indicate the proportion of cells in that group that were non-viable (non-vegetative, potentially dead) and are calculated as (bar height) × (mean within-class proportion non-viable). For example, a bar height of 20% combined with 50% within-class non-viability yields a white circle at 20 × 0.50 = 10%. Figure caption source: Paper II. 37 38 Summary of papers Summary of papers Fig. 10: (a-b) Paralytic shellfish toxin (PST) amounts and composition (GC6: hydroxyl-benzoyl Fig. 11: (a-b) Paralytic shellfish toxin (PST) amounts and composition (GC6: hydroxyl-benzoyl analogue 6, C2: N-Sulfocarbamoyl-gonyautoxin-2, GTX3: Gonyautoxin-3, GTX4: Gonyautoxin- analogue 6, C2: N-Sulfocarbamoyl-gonyautoxin-2, GTX3: Gonyautoxin-3, GTX4: Gonyautoxin- 4, NEO: Neosaxitoxin, STX: Saxitoxin) for (a) Gymnodinium catenatum and (b) Alexandrium 4, NEO: Neosaxitoxin, STX: Saxitoxin) for (a) Gymnodinium catenatum and (b) Alexandrium catenella across copepodamide treatments and days (4-8). Colored bars are means of each toxin catenella across copepodamide treatments and days (4-8). Colored bars are means of each toxin congener based on n = 4 replicates for A. catenella and n = 5 replicates for G. catenatum, error congener based on n = 4 replicates for A. catenella and n = 5 replicates for G. catenatum, error bars denote 95% confidence intervals of pooled toxins, and asterisks (*) denote statistically bars denote 95% confidence intervals of pooled toxins, and asterisks (*) denote statistically significant differences in total toxins compared to control (Table 3). An outlier replicate in CA significant differences in total toxins compared to control (Table 3). An outlier replicate in CA treatment 0.5 nM on day 8 is not visible in b. (c-d) Cell concentrations for each copepodamide treatment 0.5 nM on day 8 is not visible in b. (c-d) Cell concentrations for each copepodamide treatment after 4 and 8 days for (c) G. catenatum and (d) A. catenella. Bars are mean values of n treatment after 4 and 8 days for (c) G. catenatum and (d) A. catenella. Bars are mean values of n = 4 and 5 replicates, respectively, and error bars denote 95% confidence intervals. (e-f) Scatter = 4 and 5 replicates, respectively, and error bars denote 95% confidence intervals. (e-f) Scatter plots of growth rates and net toxin production rates for (e) G. catenatum and (f) A. catenella. plots of growth rates and net toxin production rates for (e) G. catenatum and (f) A. catenella. Figure caption source: Paper III. Figure caption source: Paper III. 39 40 Summary of papers Summary of papers Fig. 10: (a-b) Paralytic shellfish toxin (PST) amounts and composition (GC6: hydroxyl-benzoyl Fig. 11: (a-b) Paralytic shellfish toxin (PST) amounts and composition (GC6: hydroxyl-benzoyl analogue 6, C2: N-Sulfocarbamoyl-gonyautoxin-2, GTX3: Gonyautoxin-3, GTX4: Gonyautoxin- analogue 6, C2: N-Sulfocarbamoyl-gonyautoxin-2, GTX3: Gonyautoxin-3, GTX4: Gonyautoxin- 4, NEO: Neosaxitoxin, STX: Saxitoxin) for (a) Gymnodinium catenatum and (b) Alexandrium 4, NEO: Neosaxitoxin, STX: Saxitoxin) for (a) Gymnodinium catenatum and (b) Alexandrium catenella across copepodamide treatments and days (4-8). Colored bars are means of each toxin catenella across copepodamide treatments and days (4-8). Colored bars are means of each toxin congener based on n = 4 replicates for A. catenella and n = 5 replicates for G. catenatum, error congener based on n = 4 replicates for A. catenella and n = 5 replicates for G. catenatum, error bars denote 95% confidence intervals of pooled toxins, and asterisks (*) denote statistically bars denote 95% confidence intervals of pooled toxins, and asterisks (*) denote statistically significant differences in total toxins compared to control (Table 3). An outlier replicate in CA significant differences in total toxins compared to control (Table 3). An outlier replicate in CA treatment 0.5 nM on day 8 is not visible in b. (c-d) Cell concentrations for each copepodamide treatment 0.5 nM on day 8 is not visible in b. (c-d) Cell concentrations for each copepodamide treatment after 4 and 8 days for (c) G. catenatum and (d) A. catenella. Bars are mean values of n treatment after 4 and 8 days for (c) G. catenatum and (d) A. catenella. Bars are mean values of n = 4 and 5 replicates, respectively, and error bars denote 95% confidence intervals. (e-f) Scatter = 4 and 5 replicates, respectively, and error bars denote 95% confidence intervals. (e-f) Scatter plots of growth rates and net toxin production rates for (e) G. catenatum and (f) A. catenella. plots of growth rates and net toxin production rates for (e) G. catenatum and (f) A. catenella. Figure caption source: Paper III. Figure caption source: Paper III. 39 40 Summary of papers Summary of papers Paper IV: Inducing Dinophysis Copepods may contribute to harmful algal bloom formation by selectively rejecting harmful cells (Teegarden, 1999; Xu and Kiørboe, 2018). Copepods, and copepodamides alone, have been shown to induce several-fold increases in toxin production in PST- and AST-producers such as Alexandrium (Ryderheim et al., 2021) and Pseudo-nitzschia (Olesen et al., 2022). However, it remains unknown if diarrhetic shellfish toxin (DST) producing dinoflagellates—such as Dinophysis—respond to copepods or copepodamides in a similar fashion. Studying this genus has historically been a challenge because of their difficulty to maintain in culture (Park et al., 2006; Reguera et al., 2012). Notably, Trapp and colleagues (2021) showed that including copepod biomass—and more so, copepodamide concentrations measured in blue mussel samples— improved forecasts of okadaic acid (a DST) in mussels on the Swedish west coast, suggesting a tangible connection between copepod presence and harmful Dinophysis dynamics. Here we exposed laboratory cultures of Dinophysis sacculus and D. acuminata to direct grazing by Acartia sp. copepods, or to one of three copepodamide treatments (1, 5, 10 nM nominal) for three Fig. 13: Proportion of intracellular (grey) and extracellular (white) toxins for Dinophysis days, with fresh cue exposure after 48 h to maintain stimuli throughout the experimental period. sacculus (left) and Dinophysis acuminata (right) after 68.5 h of exposure to 1–10 nM We monitored copepods daily and replaced non-motile ones. We then measured intracellular and concentrations of copepodamides, a living Acartia sp. copepod, or control conditions without extracellular Dinophysis-produced toxins (DPTs; okadaic-acid group toxins and pectenotoxin–2) copepods or copepod cues. Bars are the mean value of three replicates (n = 3) and error bars via LC-MS/MS, estimated growth from microscopical enumeration, and performed untargeted denote the 95 % confidence intervals of the means. Letters denote statistically homogeneous metabolomic analyses to examine broader physiological responses. treatments. Note that only the lower confidence limits are shown for extracellular bars. Figure caption source: Paper IV. Total DPTs increased by 8–45% (relative to methanol control) in D. sacculus across treatments, but only the highest copepodamide treatment (10 nM) differed significantly from controls (Fig. 12). D. acuminata was not affected by any of the treatments. Growth rates were low across all groups and explained up to 91% of the variation in toxin content. This supports the notion that growth rate has affects cell and volume normalised toxin content measures, which we statistically corrected for. Fig. 12: Total Dinophysis produced toxins, normalised by sample volume, for Dinophysis sacculus (black) and Dinophysis acuminata (grey) after 68.5 h of exposure to 1–10 nM concentrations of copepodamides, a living Acartia sp. copepod, or control conditions without copepods or copepod cues. Circles represent the covariate (growth rate) adjusted mean values (n = 3), predicted at growth rate = 0, and error bars denote the 95 % confidence intervals of the predicted means. Fig. 14: PCA score plot from LC–HRMS derived metabolomic profiles for D. sacculus (left, +MS Transparent diamonds show the measured data) and D. acuminata (right, -MS data) after 68.5 h of exposure to three concentrations of toxin content in each replicate (i.e., not chemical cues purified from copepods (copepodamides, triangles), a living Acartia sp. copepod covariate adjusted). Letters denote (circles), or control conditions (squares). Transparent symbols without borders are individual statistically homogeneous subgroups for D replicate dimensionality reductions, opaque symbols with black border denote the centroids sacculus. There were no significantly (averages) for each group. Figure caption source: Paper IV. different subgroups in D acuminata. Figure caption source: Paper IV. 41 42 Summary of papers Summary of papers Paper IV: Inducing Dinophysis Copepods may contribute to harmful algal bloom formation by selectively rejecting harmful cells (Teegarden, 1999; Xu and Kiørboe, 2018). Copepods, and copepodamides alone, have been shown to induce several-fold increases in toxin production in PST- and AST-producers such as Alexandrium (Ryderheim et al., 2021) and Pseudo-nitzschia (Olesen et al., 2022). However, it remains unknown if diarrhetic shellfish toxin (DST) producing dinoflagellates—such as Dinophysis—respond to copepods or copepodamides in a similar fashion. Studying this genus has historically been a challenge because of their difficulty to maintain in culture (Park et al., 2006; Reguera et al., 2012). Notably, Trapp and colleagues (2021) showed that including copepod biomass—and more so, copepodamide concentrations measured in blue mussel samples— improved forecasts of okadaic acid (a DST) in mussels on the Swedish west coast, suggesting a tangible connection between copepod presence and harmful Dinophysis dynamics. Here we exposed laboratory cultures of Dinophysis sacculus and D. acuminata to direct grazing by Acartia sp. copepods, or to one of three copepodamide treatments (1, 5, 10 nM nominal) for three Fig. 13: Proportion of intracellular (grey) and extracellular (white) toxins for Dinophysis days, with fresh cue exposure after 48 h to maintain stimuli throughout the experimental period. sacculus (left) and Dinophysis acuminata (right) after 68.5 h of exposure to 1–10 nM We monitored copepods daily and replaced non-motile ones. We then measured intracellular and concentrations of copepodamides, a living Acartia sp. copepod, or control conditions without extracellular Dinophysis-produced toxins (DPTs; okadaic-acid group toxins and pectenotoxin–2) copepods or copepod cues. Bars are the mean value of three replicates (n = 3) and error bars via LC-MS/MS, estimated growth from microscopical enumeration, and performed untargeted denote the 95 % confidence intervals of the means. Letters denote statistically homogeneous metabolomic analyses to examine broader physiological responses. treatments. Note that only the lower confidence limits are shown for extracellular bars. Figure caption source: Paper IV. Total DPTs increased by 8–45% (relative to methanol control) in D. sacculus across treatments, but only the highest copepodamide treatment (10 nM) differed significantly from controls (Fig. 12). D. acuminata was not affected by any of the treatments. Growth rates were low across all groups and explained up to 91% of the variation in toxin content. This supports the notion that growth rate has affects cell and volume normalised toxin content measures, which we statistically corrected for. Fig. 12: Total Dinophysis produced toxins, normalised by sample volume, for Dinophysis sacculus (black) and Dinophysis acuminata (grey) after 68.5 h of exposure to 1–10 nM concentrations of copepodamides, a living Acartia sp. copepod, or control conditions without copepods or copepod cues. Circles represent the covariate (growth rate) adjusted mean values (n = 3), predicted at growth rate = 0, and error bars denote the 95 % confidence intervals of the predicted means. Fig. 14: PCA score plot from LC–HRMS derived metabolomic profiles for D. sacculus (left, +MS Transparent diamonds show the measured data) and D. acuminata (right, -MS data) after 68.5 h of exposure to three concentrations of toxin content in each replicate (i.e., not chemical cues purified from copepods (copepodamides, triangles), a living Acartia sp. copepod covariate adjusted). Letters denote (circles), or control conditions (squares). Transparent symbols without borders are individual statistically homogeneous subgroups for D replicate dimensionality reductions, opaque symbols with black border denote the centroids sacculus. There were no significantly (averages) for each group. Figure caption source: Paper IV. different subgroups in D acuminata. Figure caption source: Paper IV. 41 42 Summary of papers Summary of papers DPTs were redistributed from internal compartments to the extracellular medium in the highest copepodamide treatments (5 and 10 nM), with extracellular toxin levels two to three times higher than controls, indicating either active release or passive leakage of toxins (Fig. 13). Untargeted analysis of cellular metabolomes revealed significant changes in metabolite profiles for both species in response to the highest copepodamide treatments (Fig. 14), independent of known toxins, though it remains unclear whether these represent stress responses or more complex mechanisms. The relatively small grazer-induced effect in one of the two Dinophysis species tested here (45%)— especially compared to effects in harmful species of e.g. Alexandrium reported previously— suggests that DPT production in Dinophysis is likely not induced by copepods en masse, except perhaps in patches with high copepod densities. Our results raise questions about the primary purpose of Dinophysis toxins. Paper V: Bottom-up versus top-down induction of phycotoxins: A meta-analysis Phycotoxin production is controlled by both bottom-up nutrient availability and top-down grazer presence, but few studies have directly compared the induction capacity of these two drivers. Whilst HAB research has long emphasised nutrient availability as a key factor controlling growth and toxin production, recent evidence shows that harmful algae can sense the presence of zooplankton grazers and respond by dramatically increasing toxin production. However, despite decades of research the relative importance of these two drivers remains unclear. We conducted a meta-analysis of 113 control-treatment contrasts from 37 peer-reviewed experimental studies (Fig. 15), comparing phycotoxin induction due to relative nitrogen enrichment (increased N:P ratio relative to control) and elevated grazing risk (exposure to zooplankton grazers or their chemical cues). We focused on the two most studied marine HAB-forming genera: Alexandrium dinoflagellates producing PSTs, and Pseudo-nitzschia diatoms producing the AST domoic acid. We used multi-level mixed-effect meta-analytical models, accounting for hierarchical structure and between-study variance, and estimated the maximum induction capacity for each driver. We show that phycotoxins increase in response to both relative nitrogen enrichment and elevated grazing risk, by 267% (95% CI: 125–498%) and 388% (230–622%) respectively, when pooled across genera (Fig. 16). Although elevated grazing risk produced a 121 percentage-point (absolute percentage difference) larger mean increase than relative nitrogen enrichment, this difference was not statistically significant. However, marked differences emerged when phytoplankton genus was included as a moderator (factor), with Pseudo-nitzschia ASTs increasing ten times more in response to grazers than Alexandrium PSTs did, whilst both genera responded similarly to relative nitrogen enrichment (Fig. 17). In Pseudo-nitzschia, toxin increase in response to grazing risk was four times Fig. 15: Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) flow greater than that induced by relative nitrogen enrichment, but this difference was marginally non- chart showing the number of articles found, screened, and included/excluded. r = resource (relative significant. nitrogen enrichment/increased N:P ratio), d = demand (increased grazing risk). Figure caption These findings challenge traditional nutrient-centric views on the drivers of phycotoxin production. source: Paper V. The strong genus-specific differences in grazer responses show that Pseudo-nitzschia is more plastic in their production of phycotoxins compared to Alexandrium. This supports the idea that small, inconspicuous taxa tend to be more inducible in chemical defence expression than larger, conspicuous taxa. 43 44 Summary of papers Summary of papers DPTs were redistributed from internal compartments to the extracellular medium in the highest copepodamide treatments (5 and 10 nM), with extracellular toxin levels two to three times higher than controls, indicating either active release or passive leakage of toxins (Fig. 13). Untargeted analysis of cellular metabolomes revealed significant changes in metabolite profiles for both species in response to the highest copepodamide treatments (Fig. 14), independent of known toxins, though it remains unclear whether these represent stress responses or more complex mechanisms. The relatively small grazer-induced effect in one of the two Dinophysis species tested here (45%)— especially compared to effects in harmful species of e.g. Alexandrium reported previously— suggests that DPT production in Dinophysis is likely not induced by copepods en masse, except perhaps in patches with high copepod densities. Our results raise questions about the primary purpose of Dinophysis toxins. Paper V: Bottom-up versus top-down induction of phycotoxins: A meta-analysis Phycotoxin production is controlled by both bottom-up nutrient availability and top-down grazer presence, but few studies have directly compared the induction capacity of these two drivers. Whilst HAB research has long emphasised nutrient availability as a key factor controlling growth and toxin production, recent evidence shows that harmful algae can sense the presence of zooplankton grazers and respond by dramatically increasing toxin production. However, despite decades of research the relative importance of these two drivers remains unclear. We conducted a meta-analysis of 113 control-treatment contrasts from 37 peer-reviewed experimental studies (Fig. 15), comparing phycotoxin induction due to relative nitrogen enrichment (increased N:P ratio relative to control) and elevated grazing risk (exposure to zooplankton grazers or their chemical cues). We focused on the two most studied marine HAB-forming genera: Alexandrium dinoflagellates producing PSTs, and Pseudo-nitzschia diatoms producing the AST domoic acid. We used multi-level mixed-effect meta-analytical models, accounting for hierarchical structure and between-study variance, and estimated the maximum induction capacity for each driver. We show that phycotoxins increase in response to both relative nitrogen enrichment and elevated grazing risk, by 267% (95% CI: 125–498%) and 388% (230–622%) respectively, when pooled across genera (Fig. 16). Although elevated grazing risk produced a 121 percentage-point (absolute percentage difference) larger mean increase than relative nitrogen enrichment, this difference was not statistically significant. However, marked differences emerged when phytoplankton genus was included as a moderator (factor), with Pseudo-nitzschia ASTs increasing ten times more in response to grazers than Alexandrium PSTs did, whilst both genera responded similarly to relative nitrogen enrichment (Fig. 17). In Pseudo-nitzschia, toxin increase in response to grazing risk was four times Fig. 15: Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) flow greater than that induced by relative nitrogen enrichment, but this difference was marginally non- chart showing the number of articles found, screened, and included/excluded. r = resource (relative significant. nitrogen enrichment/increased N:P ratio), d = demand (increased grazing risk). Figure caption These findings challenge traditional nutrient-centric views on the drivers of phycotoxin production. source: Paper V. The strong genus-specific differences in grazer responses show that Pseudo-nitzschia is more plastic in their production of phycotoxins compared to Alexandrium. This supports the idea that small, inconspicuous taxa tend to be more inducible in chemical defence expression than larger, conspicuous taxa. 43 44 Summary of papers Summary of papers Fig. 16: Effects of relative nitrogen enrichment (resource) and elevated grazing risk (demand) on phycotoxin induction in two harmful algal bloom-forming marine phytoplankton genera. Effect size (x-axis) is the small sample bias-corrected log response ratio (LRRΔ) proposed by Lajeunesse (2015). White diamonds represent the mean effect size, thick black boxes denote the 95% confidence intervals (95% CI) of the mean effect, and thin black lines denote the 95% prediction intervals (95% PI; where 95% of new effect sizes are expected to fall with repeated sampling of Fig. 17: Effects of relative nitrogen enrichment (resource) and elevated grazing risk (demand) on the literature). Circles denote individual effect sizes (k) from a given number of studies (n), sized phycotoxin induction, partitioned by phytoplankton genus (Alexandrium: orange; Pseudo- inversely proportional to their sampling error (1/SE). Back-transformed mean values, 95% CI nitzschia: green). Effect size (x-axis) is the small sample bias-corrected log response ratio (LRRΔ) limits, and 95% PI limits are presented as corresponding percentages in the annotated text. Figure proposed by Lajeunesse (2015). Orange squares represent mean effect sizes for Alexandrium, and caption source: Paper V. green diamonds represent mean effect sizes for Pseudo-nitzschia. Thick black lines denote 95% confidence intervals (95% CI) for the mean effects, and thin black lines denote 95% prediction intervals (95% PI; where 95% of new effect sizes are expected to fall with repeated sampling of We conclude that grazing risk appears to rival, and in some cases supersede, the well-established the literature). Circles denote individual effect sizes (k) from a given number of studies (n), sized phycotoxin-inducing effects of relative nitrogen enrichment in marine harmful algae. This has direct inversely proportional to their sampling error (1/SE). Back-transformed mean values, 95% CI implications for harmful algal bloom forecasting and management, suggesting that grazer limits, and 95% PI limits are presented as corresponding percentages in the annotated text for each community composition and density should be considered alongside traditional nutrient monitoring. group. Figure caption source: Paper V. Future attempts to understand phycotoxin evolution and production dynamics should aim to integrate both bottom-up nutrient availability and top-down selective pressures, drawing insights from well-established plant defence frameworks to better predict and manage these ecologically and economically impactful events. 45 46 Summary of papers Summary of papers Fig. 16: Effects of relative nitrogen enrichment (resource) and elevated grazing risk (demand) on phycotoxin induction in two harmful algal bloom-forming marine phytoplankton genera. Effect size (x-axis) is the small sample bias-corrected log response ratio (LRRΔ) proposed by Lajeunesse (2015). White diamonds represent the mean effect size, thick black boxes denote the 95% confidence intervals (95% CI) of the mean effect, and thin black lines denote the 95% prediction intervals (95% PI; where 95% of new effect sizes are expected to fall with repeated sampling of Fig. 17: Effects of relative nitrogen enrichment (resource) and elevated grazing risk (demand) on the literature). Circles denote individual effect sizes (k) from a given number of studies (n), sized phycotoxin induction, partitioned by phytoplankton genus (Alexandrium: orange; Pseudo- inversely proportional to their sampling error (1/SE). Back-transformed mean values, 95% CI nitzschia: green). Effect size (x-axis) is the small sample bias-corrected log response ratio (LRRΔ) limits, and 95% PI limits are presented as corresponding percentages in the annotated text. Figure proposed by Lajeunesse (2015). Orange squares represent mean effect sizes for Alexandrium, and caption source: Paper V. green diamonds represent mean effect sizes for Pseudo-nitzschia. Thick black lines denote 95% confidence intervals (95% CI) for the mean effects, and thin black lines denote 95% prediction intervals (95% PI; where 95% of new effect sizes are expected to fall with repeated sampling of We conclude that grazing risk appears to rival, and in some cases supersede, the well-established the literature). Circles denote individual effect sizes (k) from a given number of studies (n), sized phycotoxin-inducing effects of relative nitrogen enrichment in marine harmful algae. This has direct inversely proportional to their sampling error (1/SE). Back-transformed mean values, 95% CI implications for harmful algal bloom forecasting and management, suggesting that grazer limits, and 95% PI limits are presented as corresponding percentages in the annotated text for each community composition and density should be considered alongside traditional nutrient monitoring. group. Figure caption source: Paper V. Future attempts to understand phycotoxin evolution and production dynamics should aim to integrate both bottom-up nutrient availability and top-down selective pressures, drawing insights from well-established plant defence frameworks to better predict and manage these ecologically and economically impactful events. 45 46 Conclusions and implications In this thesis, I show that copepodamides are broadly conserved across freshwater and marine copepods (Paper I), that taxonomically diverse marine phytoplankton have—albeit not universally—co-opted these compounds as a general alarm cue to induce defensive traits (Papers II-IV), and that grazer-driven phycotoxin induction can match or exceed the well-established effects of relative nitrogen enrichment in the two best-studied HAB-forming genera, Alexandrium and Pseudo-nitzschia (Paper V). Paper I is the first systematic description of copepodamides in freshwater copepods. We found that freshwater copepods contain copepodamides in amounts comparable to similarly sized marine species, and that their composition differs markedly from marine copepods by being dominated by dhCAs. This could have large implications in freshwater systems, as dhCAs have been reported to be more potent inducers of paralytic shellfish toxins in at least one Alexandrium species (Selander Chapter 5. et al., 2015). If that potency generalises, and if freshwater phytoplankton respond to copepodamides similarly to marine taxa, copepodamide-driven induction could influence HAB dynamics and Conclusions and implications community structure in lakes. Regardless, the ubiquity of copepodamides among freshwater copepods opens a new and exciting research frontier. I suggest that future work test whether freshwater phytoplankton detect and respond to copepodamides. If they do, identification of the expressed traits and comparisons between lakes with differing nutrient regimes and zooplankton assemblages should follow. I believe that comparative experiments that expose freshwater phytoplankton assemblages from copepod-dominated and Diplostraca-dominated lakes to It has often and confidently been asserted, that man's origin can never be copepodamide blends matching local copepod profiles would be particularly interesting. Such paired, comparative experiments could reveal whether differences in signal composition and known: but ignorance more frequently begets confidence than does consumer community identity produce predictable, arms-race-style divergence in prey defences and knowledge: it is those who know little, and not those who know much, who so grazer tolerance. positively assert that this or that problem will never be solved by science. In Paper II, we exposed four species of chain-forming diatoms to purified copepodamides and Charles Darwin demonstrated that copepodamide-mediated chain shortening is species-specific. Skeletonema The Descent of Man marinoi showed the expected strong, ecologically meaningful chain shortening, whereas the three taxa tested for the first time—Skeletonema subsalsum, Chaetoceros sp., and Eupyxidicula turris— showed minimal to no change in chain length. It is possible that these same taxa would respond to other chemical cues exuded by live copepods, particularly from copepod species with which they co-occur and that represent their main predators. I would, however, still caution against describing chain suppression as a broadly utilised defence trait, based on the limited set of species shown to employ this strategy to date. I suggest that future studies, when relevant, measure multiple putative defence traits concurrently—e.g., chain length, frustule silicification, setae morphology, and toxin production—across strains and resource regimes to characterise trait covariation and context dependence. Exposure to relevant live predators is also warranted. I conclude that chain suppression should not be assumed a universal anti-copepod defence mediated by copepodamides in chain- forming diatoms Paper III describes the first experimental evidence that copepodamide exposure induces bioluminescence in Protoceratium reticulatum, PSTs in Gymnodinium catenatum, and that Alexandrium catenella simultaneously upregulate both bioluminescence and PSTs in response to copepodamides. This expands the set of copepodamide-responsive taxa and shows that single dinoflagellate species can co-express multiple defensive modalities when exposed to copepod cues. Satisfy My Soul – Bob Marley The relative shift in toxin composition of G. catenatum, driven primarily by increased GC congeners, has toxicological relevance because congener changes may alter consumer risk even when total PST load is unchanged. We found no measurable allocation cost to growth under 47 48 Conclusions and implications In this thesis, I show that copepodamides are broadly conserved across freshwater and marine copepods (Paper I), that taxonomically diverse marine phytoplankton have—albeit not universally—co-opted these compounds as a general alarm cue to induce defensive traits (Papers II-IV), and that grazer-driven phycotoxin induction can match or exceed the well-established effects of relative nitrogen enrichment in the two best-studied HAB-forming genera, Alexandrium and Pseudo-nitzschia (Paper V). Paper I is the first systematic description of copepodamides in freshwater copepods. We found that freshwater copepods contain copepodamides in amounts comparable to similarly sized marine species, and that their composition differs markedly from marine copepods by being dominated by dhCAs. This could have large implications in freshwater systems, as dhCAs have been reported to be more potent inducers of paralytic shellfish toxins in at least one Alexandrium species (Selander Chapter 5. et al., 2015). If that potency generalises, and if freshwater phytoplankton respond to copepodamides similarly to marine taxa, copepodamide-driven induction could influence HAB dynamics and Conclusions and implications community structure in lakes. Regardless, the ubiquity of copepodamides among freshwater copepods opens a new and exciting research frontier. I suggest that future work test whether freshwater phytoplankton detect and respond to copepodamides. If they do, identification of the expressed traits and comparisons between lakes with differing nutrient regimes and zooplankton assemblages should follow. I believe that comparative experiments that expose freshwater phytoplankton assemblages from copepod-dominated and Diplostraca-dominated lakes to It has often and confidently been asserted, that man's origin can never be copepodamide blends matching local copepod profiles would be particularly interesting. Such paired, comparative experiments could reveal whether differences in signal composition and known: but ignorance more frequently begets confidence than does consumer community identity produce predictable, arms-race-style divergence in prey defences and knowledge: it is those who know little, and not those who know much, who so grazer tolerance. positively assert that this or that problem will never be solved by science. In Paper II, we exposed four species of chain-forming diatoms to purified copepodamides and Charles Darwin demonstrated that copepodamide-mediated chain shortening is species-specific. Skeletonema The Descent of Man marinoi showed the expected strong, ecologically meaningful chain shortening, whereas the three taxa tested for the first time—Skeletonema subsalsum, Chaetoceros sp., and Eupyxidicula turris— showed minimal to no change in chain length. It is possible that these same taxa would respond to other chemical cues exuded by live copepods, particularly from copepod species with which they co-occur and that represent their main predators. I would, however, still caution against describing chain suppression as a broadly utilised defence trait, based on the limited set of species shown to employ this strategy to date. I suggest that future studies, when relevant, measure multiple putative defence traits concurrently—e.g., chain length, frustule silicification, setae morphology, and toxin production—across strains and resource regimes to characterise trait covariation and context dependence. Exposure to relevant live predators is also warranted. I conclude that chain suppression should not be assumed a universal anti-copepod defence mediated by copepodamides in chain- forming diatoms Paper III describes the first experimental evidence that copepodamide exposure induces bioluminescence in Protoceratium reticulatum, PSTs in Gymnodinium catenatum, and that Alexandrium catenella simultaneously upregulate both bioluminescence and PSTs in response to copepodamides. This expands the set of copepodamide-responsive taxa and shows that single dinoflagellate species can co-express multiple defensive modalities when exposed to copepod cues. Satisfy My Soul – Bob Marley The relative shift in toxin composition of G. catenatum, driven primarily by increased GC congeners, has toxicological relevance because congener changes may alter consumer risk even when total PST load is unchanged. We found no measurable allocation cost to growth under 47 48 Conclusions and implications Conclusions and implications nutrient-replete conditions, but I note that an absence of costs is likely context dependent and may Paper V is the first meta-analysis comparing bottom-up nutrient-driven phycotoxin induction become apparent under resource limitation or in complex communities. effects with top-down grazer-driven effects. In it, we demonstrate that relative nitrogen enrichment Future studies should aim to measure multiple putative defence traits and growth concurrently, and and elevated grazing risk induce phycotoxins to a similar extent when pooled across the two best explicitly account for growth-rate effects when analysing toxin induction. This can be done studied marine HAB genera (Alexandrium and Pseudo-nitzschia), but that this pattern is genus- and statistically, by including growth rate as a covariate in linear models, or preferably by using driver-dependent. The synthesised estimates are robust despite considerable methodological production-rate response variables such as toxin or bioluminescence produced per cell per unit time heterogeneity in the underlying literature, indicating that top-down drivers deserve equal attention (Anderson et al., 1990). Note that per-cell metrics are most appropriate for mechanistic studies of alongside nutrient dynamics when seeking mechanistic explanations for toxin variability or prey defence. If the focus is on ecosystem or societal outcomes—which is arguably of more interest discussing ecological and evolutionary drivers of toxin production. if one is e.g., a mussel or oyster farmer—toxin content per volume of seawater or amount of toxins The marine HAB research field has long sat firmly within a bottom-up, resource-centred paradigm accumulated in biota would be more informative. I suggest that congener-level analyses become of phycotoxin dynamics, which effectively and accurately describes how and when phycotoxins routine to enable study of toxin-composition effects, and that grazing experiments with the novel may be produced but does not explain why such compounds evolved. To be fair, however, this is species reported here test whether induced traits confer measurable anti-grazing benefits. not a flaw of the underlying framework but a feature; resource-based models of plant-defence were never intended to explain evolutionary drivers. I argue that a top-down, grazer-dependent Paper IV represents the first experimental study of how the DST-producing dinoflagellate perspective better captures the selective context that has favoured the evolution of phycotoxins (the Dinophysis responds to a copepod grazer and to copepodamides. We observed a modest increase in why), and that it can also better explain the when with regard to phenotypically plastic traits. Dinophysis-produced toxins (DPTs) in D. sacculus at the highest cue concentration and no clear Combining both perspectives is likely the most productive way forward. The expanded growth– response in D. acuminata, and a cue-dependent redistribution of toxins to the extracellular medium differentiation balance (GDB) model described by Herms and Mattson (1992) provides a useful, at high cue exposures in both species. Growth was low for both species in all treatments and integrative framework to aid in this because it subsumes both resource- and demand-based explained much of the variation in toxins, which we statistically accounted for in the analyses. I hypotheses and perspectives (Stamp, 2003). Under the GDB, resource availability sets conclude that copepod-induced DPT production in Dinophysis is modest and species specific under physiological bounds for synthesis of chemical defences while grazing risk modulates allocation the conditions we tested, and that it is unlikely to be a pervasive, population-scale driver of DST within those bounds; life history and apparency effects (size, motility, growth rate) can further bias dynamics. The relatively small induction prompts alternative hypotheses about the function of these species towards more constitutive or more inducible strategies. toxins. For example, Dinophysis may rely largely on constitutive defence expression if these toxins I propose three priorities for future research based on this synthesis. First, multifactorial, fully serve as grazer deterrents at all. DPTs may primarily target different grazer groups not included crossed experiments manipulating stoichiometry and grazing risk are particularly valuable for here (such as microzooplankton); they could aid in immobilising and capturing ciliate prey making intraspecific comparisons. They should be used to test predictions from plant-defence (Giménez Papiol et al., 2016; Mafra Jr. et al., 2016); or they may have a different function altogether. frameworks (the GDB being a leading candidate) and use phycotoxin production rate as the The observed extracellular increase could reflect active export or passive leakage from damaged response variable when the primary interest is toxin synthesis per se. Second, future syntheses cells, but our data do not allow us to distinguish among these. Experiments combining high- should broaden taxonomic scope beyond Alexandrium and Pseudo-nitzschia once sufficient resolution toxin localisation (for example imaging mass-spectrometry or labelled toxin tracers), primary literature exists to support them. Similarly, an analogous synthesis for freshwater HAB membrane-integrity assays, and simultaneous intra- and extracellular metabolomics may assist in taxa is justified; one obvious candidate is the microcystin-producing cyanobacterium Microcystis, separating active export from leakage. although enough primary studies on grazer-induced microcystin production are still lacking to Notably, Trapp and colleagues (2021) found that okadaic acid levels in mussels on the Swedish permit such a comparative synthesis. Finally, periodic updates of this synthesis as new studies west coast correlated with copepod biomass and—even more strongly—with copepodamide accumulate would refine the resource–demand comparison and better define its limits for these concentrations measured in the mussel tissues. There are several non-mutually exclusive genera. explanations for why the in situ patterns they reported were not corroborated by grazer- or cue- mediated toxin induction in our experiment. First, selective grazing in the field may have driven Closing remarks relative increases in Dinophysis abundance by removing less-defended competitors, increasing The use of purified copepodamides allow for controlled, repeatable induction assays and accurate toxins measured in the mussels without requiring strong grazer-induced toxin production in dose–response characterisation of inducible traits. This enables estimation of evolutionary costs and Dinophysis. Second, strain composition and cryptic genetic variation in natural Dinophysis benefits (trade-offs) of defence traits, and provides a powerful tool to identify the molecular populations could cause larger between-population variability than captured by our strains. Indeed, machinery leading to trait expression. At the same time, laboratory simplifications such as mono- variation in toxin content between strains of Dinophysis species and clades can be up to ten-fold specific cultures, fixed cue concentrations, and nutrient-replete media limit immediate extrapolation (Séchet et al., 2021). Finally, accumulation processes in mussels integrate over time and space, and to field dynamics. Thus, benchmarking experiments that link laboratory results with field may amplify small or intermittent toxin releases that appear modest in short laboratory exposures. observations are necessary to establish the ecological relevance of experimental findings. I suggest that future work combine high-resolution field sampling with targeted grazing Regardless, future copepodamide research should target the phytoplankton side by identifying experiments that manipulate community composition to distinguish among these mechanisms and receptors and signalling pathways for copepodamides using transcriptomics and genetic tools, and link mechanistic responses to monitoring observations. the grazer side by determining copepodamide biosynthesis, physiological function, and release 49 50 Conclusions and implications Conclusions and implications nutrient-replete conditions, but I note that an absence of costs is likely context dependent and may Paper V is the first meta-analysis comparing bottom-up nutrient-driven phycotoxin induction become apparent under resource limitation or in complex communities. effects with top-down grazer-driven effects. In it, we demonstrate that relative nitrogen enrichment Future studies should aim to measure multiple putative defence traits and growth concurrently, and and elevated grazing risk induce phycotoxins to a similar extent when pooled across the two best explicitly account for growth-rate effects when analysing toxin induction. This can be done studied marine HAB genera (Alexandrium and Pseudo-nitzschia), but that this pattern is genus- and statistically, by including growth rate as a covariate in linear models, or preferably by using driver-dependent. The synthesised estimates are robust despite considerable methodological production-rate response variables such as toxin or bioluminescence produced per cell per unit time heterogeneity in the underlying literature, indicating that top-down drivers deserve equal attention (Anderson et al., 1990). Note that per-cell metrics are most appropriate for mechanistic studies of alongside nutrient dynamics when seeking mechanistic explanations for toxin variability or prey defence. If the focus is on ecosystem or societal outcomes—which is arguably of more interest discussing ecological and evolutionary drivers of toxin production. if one is e.g., a mussel or oyster farmer—toxin content per volume of seawater or amount of toxins The marine HAB research field has long sat firmly within a bottom-up, resource-centred paradigm accumulated in biota would be more informative. I suggest that congener-level analyses become of phycotoxin dynamics, which effectively and accurately describes how and when phycotoxins routine to enable study of toxin-composition effects, and that grazing experiments with the novel may be produced but does not explain why such compounds evolved. To be fair, however, this is species reported here test whether induced traits confer measurable anti-grazing benefits. not a flaw of the underlying framework but a feature; resource-based models of plant-defence were never intended to explain evolutionary drivers. I argue that a top-down, grazer-dependent Paper IV represents the first experimental study of how the DST-producing dinoflagellate perspective better captures the selective context that has favoured the evolution of phycotoxins (the Dinophysis responds to a copepod grazer and to copepodamides. We observed a modest increase in why), and that it can also better explain the when with regard to phenotypically plastic traits. Dinophysis-produced toxins (DPTs) in D. sacculus at the highest cue concentration and no clear Combining both perspectives is likely the most productive way forward. The expanded growth– response in D. acuminata, and a cue-dependent redistribution of toxins to the extracellular medium differentiation balance (GDB) model described by Herms and Mattson (1992) provides a useful, at high cue exposures in both species. Growth was low for both species in all treatments and integrative framework to aid in this because it subsumes both resource- and demand-based explained much of the variation in toxins, which we statistically accounted for in the analyses. I hypotheses and perspectives (Stamp, 2003). Under the GDB, resource availability sets conclude that copepod-induced DPT production in Dinophysis is modest and species specific under physiological bounds for synthesis of chemical defences while grazing risk modulates allocation the conditions we tested, and that it is unlikely to be a pervasive, population-scale driver of DST within those bounds; life history and apparency effects (size, motility, growth rate) can further bias dynamics. The relatively small induction prompts alternative hypotheses about the function of these species towards more constitutive or more inducible strategies. toxins. For example, Dinophysis may rely largely on constitutive defence expression if these toxins I propose three priorities for future research based on this synthesis. First, multifactorial, fully serve as grazer deterrents at all. DPTs may primarily target different grazer groups not included crossed experiments manipulating stoichiometry and grazing risk are particularly valuable for here (such as microzooplankton); they could aid in immobilising and capturing ciliate prey making intraspecific comparisons. They should be used to test predictions from plant-defence (Giménez Papiol et al., 2016; Mafra Jr. et al., 2016); or they may have a different function altogether. frameworks (the GDB being a leading candidate) and use phycotoxin production rate as the The observed extracellular increase could reflect active export or passive leakage from damaged response variable when the primary interest is toxin synthesis per se. Second, future syntheses cells, but our data do not allow us to distinguish among these. Experiments combining high- should broaden taxonomic scope beyond Alexandrium and Pseudo-nitzschia once sufficient resolution toxin localisation (for example imaging mass-spectrometry or labelled toxin tracers), primary literature exists to support them. Similarly, an analogous synthesis for freshwater HAB membrane-integrity assays, and simultaneous intra- and extracellular metabolomics may assist in taxa is justified; one obvious candidate is the microcystin-producing cyanobacterium Microcystis, separating active export from leakage. although enough primary studies on grazer-induced microcystin production are still lacking to Notably, Trapp and colleagues (2021) found that okadaic acid levels in mussels on the Swedish permit such a comparative synthesis. Finally, periodic updates of this synthesis as new studies west coast correlated with copepod biomass and—even more strongly—with copepodamide accumulate would refine the resource–demand comparison and better define its limits for these concentrations measured in the mussel tissues. There are several non-mutually exclusive genera. explanations for why the in situ patterns they reported were not corroborated by grazer- or cue- mediated toxin induction in our experiment. First, selective grazing in the field may have driven Closing remarks relative increases in Dinophysis abundance by removing less-defended competitors, increasing The use of purified copepodamides allow for controlled, repeatable induction assays and accurate toxins measured in the mussels without requiring strong grazer-induced toxin production in dose–response characterisation of inducible traits. This enables estimation of evolutionary costs and Dinophysis. Second, strain composition and cryptic genetic variation in natural Dinophysis benefits (trade-offs) of defence traits, and provides a powerful tool to identify the molecular populations could cause larger between-population variability than captured by our strains. Indeed, machinery leading to trait expression. At the same time, laboratory simplifications such as mono- variation in toxin content between strains of Dinophysis species and clades can be up to ten-fold specific cultures, fixed cue concentrations, and nutrient-replete media limit immediate extrapolation (Séchet et al., 2021). Finally, accumulation processes in mussels integrate over time and space, and to field dynamics. Thus, benchmarking experiments that link laboratory results with field may amplify small or intermittent toxin releases that appear modest in short laboratory exposures. observations are necessary to establish the ecological relevance of experimental findings. I suggest that future work combine high-resolution field sampling with targeted grazing Regardless, future copepodamide research should target the phytoplankton side by identifying experiments that manipulate community composition to distinguish among these mechanisms and receptors and signalling pathways for copepodamides using transcriptomics and genetic tools, and link mechanistic responses to monitoring observations. the grazer side by determining copepodamide biosynthesis, physiological function, and release 49 50 Conclusions and implications dynamics in relation to diet. The detection of copepodamides in freshwater copepods opens an References exciting new research frontier that should be investigated further. Abrams, P. A. (1990) The evolution of anti-predator traits in prey in response to evolutionary In summary, chemical cues from grazers are likely a ubiquitous and powerful influence on change in predators. Oikos, 59, 147. https://doi.org/10.2307/3545529. phytoplankton form and function. My thesis moves the young field of copepodamide ecology Abrams, P. A. (2000) The evolution of predator-prey interactions: Theory and evidence. Annual beyond isolated demonstrations of cue responses, and towards an integrated view in which grazer Review of Ecology, Evolution, and Systematics, 31, 79–105. cues, nutrients, and species identity jointly determine defensive phenotypes and their putative https://doi.org/10.1146/annurev.ecolsys.31.1.79. ecological consequences. Appreciating and quantifying zooplankton-phytoplankton interactions are Adrian, R. and Schneider-Olt, B. (1999) Top-down effects of crustacean zooplankton on pelagic essential steps towards improved forecasting of harmful algal blooms, better prediction of shifts in microorganisms in a mesotrophic lake. 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