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Phd Birgitte Nilsson.Pdf Roskilde University Molecular Stress Physiology in the Calanoid Copepod Acartia tonsa Nilsson, Birgitte Publication date: 2018 Document Version Publisher's PDF, also known as Version of record Citation for published version (APA): Nilsson, B. (2018). Molecular Stress Physiology in the Calanoid Copepod Acartia tonsa. Roskilde Universitet. General rights Copyright and moral rights for the publications made accessible in the public portal are retained by the authors and/or other copyright owners and it is a condition of accessing publications that users recognise and abide by the legal requirements associated with these rights. • Users may download and print one copy of any publication from the public portal for the purpose of private study or research. • You may not further distribute the material or use it for any profit-making activity or commercial gain. • You may freely distribute the URL identifying the publication in the public portal. Take down policy If you believe that this document breaches copyright please contact [email protected] providing details, and we will remove access to the work immediately and investigate your claim. Download date: 23. Sep. 2021 Molecular Stress Physiology in the Calanoid copepod Acartia tonsa Ph.D. thesis by Birgitte Nilsson Department of Science and Environment Universitetsvej 1, DK-4000 Roskilde, Denmark Supervisor: Professor Benni Winding Hansen This thesis has been submitted to the Doctoral School of Science and Environment, Roskilde University on the 22nd of December 2017. Molecular Stress Physiology in the Calanoid Copepod Acartia tonsa Ph.D. dissertation by Birgitte Nilsson Department of Science and Environment Universitetsvej 1, DK-4000 Roskilde, Denmark Supervisor: Professor Benni Winding Hansen This dissertation has been submitted to the Doctoral School of Science and Environment, Roskilde University on the 30th of December 2017. ISBN: 978-87-7349-997-9 1 2 Preface This Ph.D. dissertation is a part of the project: Acartia tonsa Molecular PHysiology Imple- mentation of novel and fast tools to assess COPepod physiological states (AMPHICOP). The project was supported by a research grant (No. 8689) from VILLUM FONDEN. Roskilde University Department of Science and Environment and Graduate School of En- vironmental Stress Studies (GESS) was responsible for hosting me during the Ph.D. jour- ney. University of Connecticut Department of Marine Sciences and Professor Ann Bucklin hosted me during my research stay in the U.S. The research group included following members: Ph.D. student Birgitte Nilsson, Roskilde University Professor Benni Winding Hansen, Roskilde University Post-doctoral fellow Tue Sparholdt Jørgensen, Roskilde University Professor Lars Hestbjerg Hansen, Aarhus University Professor Ann Bucklin provided supervision at the University of Connecticut. Research field: Molecular stress physiology 3 4 Abstract The human population is growing which causes major changes in marine ecosystems in terms of thermal stress, acidification, and pollution. This is and will inevitably have a nega- tive impact on marine plants and animals. Copepods are intermediaries of the marine food web, providing a link of energy transfer from phytoplankton to higher trophic levels. If copepods suffer due to environmental changes, it will affect the whole food web negatively. It is, thus, important to understand how stressors are affecting copepods. The aim of the present Ph.D. dissertation is to provide new information for understanding how environmental stress affects the calanoid copepod, Acartia tonsa, at the transcriptional level. Targeted - (i.e. real-time quantitative PCR) and transcriptome-wide methods for ex- amining gene expression are able to give an early indication of when organisms experience a condition as stressful. The three produced manuscripts of the present Ph.d. dissertation covers different aspects of molecular stress physiology in Acartia tonsa: Producing Acartia tonsa in high densities, as live feed for fish-larvae, is a major obstacle in the aquaculture industries. In manuscript I, Acartia tonsa was exposed to very high densities (up to 10,000 ind. L-1) for 12 h. The short-term exposure to elevated densities did not cause any significant changes in swimming behavior, respiration, or gene expression of heat shock protein 70, and 90 kDa, as well as ferritin. In gene expression studies, handling has the potential of affecting the transcription of stress-related biomarkers. In manuscript II, the whole-transcriptome response towards handling stress and salinity shock (i.e., positive control) were examined in comparison to a negative control for Acartia tonsa. The effect of the treatments were evaluated 15 min and 24 h post-exposure, since it was not clear when to expect a transcriptional response. Han- dling did affect the transcriptional pattern in Acartia tonsa, especially after 24 h. Among the differential expressed genes, a number of commonly-used copepod biomarkers were identi- fied. Thus, the biomarkers for stress-related studies should be chosen carefully. Acartia tonsa is capable of producing embryonic quiescence in response to unfavorable envi- ronmental conditions. Manuscript III, provides a detailed description of subitaneous devel- opment and quiescence in Acartia tonsa eggs. Furthermore, it was demonstrated that timing of quiescence determines egg viability. 5 Resumé (Danish abstract) Væksten af den menneskelige population er årsag til store ændringer i det marine økosy- stem, såsom termisk stress, forsuring og forurening. Dette vil unægtelig resultere i en nega- tiv påvirkning af marine planter og dyr. Vandlopper er et mellemled i den marine fødekæde, hvor de kobler energi fra fytoplankton til højere trofiske niveauer. Hvis vandlopperne bliver belastet af miljømæssige ændringer vil det påvirke den marine fødekæde negativt. Formålet med Ph.D. afhandlingen var at bidrage med ny viden til at kunne forstå hvordan miljømæssig stress påvirker vandloppen, Acartia tonsa, på gen-niveau. Målrettede – (dvs. re- al-time quantitative PCR), såvel som ”whole-transcriptome” metoder blev benyttet for at undersøge gen-ekspression. Gen-ekspression kan give en tidlig indikation på hvornår en or- ganisme oplever stress. De tre producerede manuskripter af denne Ph.D. afhandling dækker forskellige aspekter indenfor molekylær stress-fysiologi af Acartia tonsa. Produktionen af Acartia tonsa i høje densiteter, til brug som levende foder for fiskelarver, er en stor hindring i akvakultur-industrien. I manuskript I blev Acartia tonsa udsat for høje densiteter (op til 10,000 ind. L-1) i 12 timer. Den kortvarige eksponering til øgede densiteter gav ikke nogen signifikante ændringer i svømme-adfærd, respiration, eller gen-ekspression af heat-shock protein 70-, og 90 kDa, så vel som ferritin. Håndtering af dyr har potentialet til at påvirke gen-ekspression af stress-relaterede biomar- kører. I manuskript II blev stress-responsen mod håndterings - og salt stress undersøgt med RNA-sekvensering. Effekten af behandlingerne blev evalueret 15 min og 24 timer efter ek- sponering da det var uklart hvornår der kunne forventes en respons på det molekylære ni- veau. Håndtering havde en klar påvirkning på gen-ekspression for Acartia tonsa, specielt 24 timer efter eksponering. Blandt de differentierede udtrykte gener blev der identificeret et antal biomarkører som typisk bliver brugt i vandloppe-studier. Valget af biomarkører i gen- ekspressions studier bør derfor vælges nøje for at undgå en håndterings-effekt. Acartia tonsa er i stand til at inducere et embryonisk hvilestadie, kaldt quiescens, i respons til ugunstige forhold. Manuskript III giver en detaljeret beskrivelse af den subitane udvikling, såvel som quiescens i Acartia tonsa æg. Det var desuden demonstreret at timing for indukti- on af quiescens bestemmer æggenes levedygtighed. 6 Acknowledgements My dissertation was about stressing copepods in different ways to uncover what happens at the molecular level – and thereby provide cues to how this tiny creature “works” and copes with its surroundings. But to be honest, sometimes the copepods were stressing me more than I was stressing them. This work is a part of the AMPHICOP project and was carried out at Roskilde University (Roskilde, Denmark) for three years, with a six months research stay at the University of Connecticut (UConn, Groton, USA). Many people have supported my journey, and I would like to thank them with gratitude. Especially I would like to thank: • Professor Benni W. Hansen for supervision and guidance during this amazing journey. • Professor Ann Bucklin for taking me in and introducing me to her network at UConn. Thanks for helping me with all the practical and personal issues there is when moving to another country, and for all your useful feedback and support. • My big brother in science, Per M. Jepsen. Thank you for always being helpful in professional relations, but besides that also being a very dear friend. I like how we cooperate and create magic in the lab. • Jennifer M. Questel. Thank you for all your help in the U.S. and for being an amaz- ing person. Also thanks to Odin for cuddly support. • My amazing co-authors! Especially thanks to Peter Stief for the visit at University of Southern Denmark (Department of Biology, Odense, Denmark), and Hans H. Jakobsen for letting me borrow your epi-fluorescence microscope at Institute for Bioscience (Aarhus University, Roskilde, Denmark), for bringing me coffee and be- ing just as excited about pictures of copepod eggs as I am. • Hans Ramløv.
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