Impact Objectives • Relevance of autotrophic versus heterotrophic components in the fitness of mixotrophic sacoglossan sea slugs

• Fate of -derived carbon and nitrogen inside these animal cells

• Impact of photoprotection mechanisms on the functionality of acquired

Functional chloroplasts inside animal cells

Dr Sónia Cruz and her collaborators, Drs Paulo Cartaxana, Bruno Jesus, Gonçalo Calado, João Serôdio, Anders Meibom, Cédric Hubas, and Michael Kühl are providing new insights into the biological significance of kleptoplasty. Cruz talks about their pioneering work on kleptoplasty

Your latest work vary according to the algal food source. The What have been your key findings so far? looks at kleptoplasty. first step here is to answer that question for Are there any that you are particularly Can you explain the in the algae. Then one must pleased with? this animal-plant verify if the same processes are working I am particularly keen on the investigation interaction? in the kleptoplast inside the sea slug and of the fate of photosynthetically fixed carbon for how long will these mechanisms be and nitrogen, along with showing that My current research supported. This is relevant because it is photosynthesis is indeed relevant for the Doctor Sónia Cruz focus on both known that many of these photoprotective sea slug E. viridis’ performance. We have understanding processes require synthesis of components unequivocally proven that photosynthesis the algae physiology and the particular coded by genes that are located in the algal is used to provide metabolites to the interaction where the animal (host), nucleus. Hence, if the animal digests the host and we are currently investigating instead of living symbiotically with the algae nucleus and does not incorporate other sea slug . We also have whole microalgae, digests the algae cellular algal nuclear genes into its genome, how enough information to advance a possible components except for the chloroplasts. can the animal sustain some of these function of these metabolites in the host Because they are ‘stolen’ plastids, they photoprotective mechanisms and repair the metabolism, which is a huge step forward were named ‘kleptoplasts’. In the animal photosynthetic machinery? This is one of in this field. Additionally, we are introducing kingdom, kleptoplasty is only known to the research lines we are following. new methodologies in this field of research take place in some sea slugs of the order (lipidomic tools, hyperspectral reflectance Sacoglossa (molluscs). In this case, the We were very surprised when we discovered analysis, NanoSIMS studies). chloroplasts are incorporated and kept that ’ main food source, the functional inside cells of the sea slug’s algae tomentosum, does not Furthermore, we have found strong digestive diverticula for some days, weeks or have a functional xanthophyll cycle for evidence for a link between kleptoplasty several months. instance. The xanthophyll cycle is one of and the sea slug’s behaviour in response to the main photoprotective mechanisms in light. As yet we have not been able to show Chloroplasts are known to be very sensitive algae and plants. C. tomentosum, as other how the kleptoplasts communicate with to oxidative stress. What mechanisms Bryosidales as we have recently shown, the host, but our results in this field so far protect these organelles upon their does not possess this capacity. Instead, they strongly imply this communication. We have incorporation by the sea slug? might rely on a more efficient repair of the also made interesting findings regarding photosynthetic machinery or depend to a novel photoprotection mechanisms in the In principle, the same mechanisms that large extent on chloroplast movement inside algae food sources but this is still on-going protect the chloroplasts inside the algae the algae. We are currently working on this work that can take many directions. could protect the kleptoplast inside the sea topic to understand which mechanisms slug. These mechanisms will depend on and protect the chloroplast from excessive light.

56 www.impact.pub Autotrophy driving animal fitness

In the HULK project, scientists from the universities of Aveiro, Lisbon and Lusófona in Portugal are collaborating with academic groups in Denmark, Switzerland and France to combine biochemical, molecular and state-of-the art imaging techniques

Understanding more about the processes is made up of researchers, Sónia Cruz, STATE-OF-THE-ART ANALYSIS of why and how an animal feeds itself both Paulo Cartaxana, Bruno Jesus, Gonçalo Based on previous observations on the from the sun’s energy and from other Calado, João Serôdio, Anders Meibom, physiology of kleptoplastic sea slugs, Cruz organisms is driving a research project Cédric Hubas, and Michael Kühl, who explains that the researchers participating underway in Portugal called ‘Functional have a strong background in photobiology in the HULK project hypothesised that Chloroplasts inside Animal cells: cracking studies in algae and symbiotic associations. the ‘autotrophic nutritional component the puzzle’ (HULK). Project Coordinator provided by the kleptoplasts is relevant Dr Sónia Cruz says the team is employing SOLAR-POWERED SEA SLUGS for their metabolism, thereby turning their ‘experimental procedures that have so far Kleptoplasty (from the Greek kléptes, model organism into a mixotroph, and that not been used in an attempt to explore the meaning thief) is the sequestration of algal the chloroplast’s inherent photoprotective only known case of kleptoplasty in the animal chloroplasts and their maintenance in a capacity plays an important role in kingdom: that of sacoglossan sea slugs’. functional status by host organisms such maintaining kleptoplasts photosynthetically as ciliates, dinoflagellates or foraminifera. active in the long term’. Their aim is to learn more about the Cruz points out that within metazoans balance of autotrophic and heterotrophic this phenomenon is only present among The first hypothesis is being investigated components in these animals. ‘We want certain sea slugs in the order Sacoglossa. by combining NanoSIMS and fatty acid to know to what extent the autotrophic ‘As opposed to other sea animals such profiles, which enables the scientists to component is relevant for the animal as corals, which establish a symbiotic address the fate of photosynthesis-derived survival and fitness, as well as how the interaction with the whole microalgae, carbon and nitrogen inside host cells. ‘In animal (host) is capable of retaining kleptoplastic sea slugs digest the rest of order to address the second question, a functional chloroplasts after ‘stealing’ algal components and keep the ‘stolen’ combination of methodologies commonly them,’ she explains. chloroplasts inside their digestive employed to investigate photoprotective diverticula, where they remain functional’, mechanisms in algae and plants, but never HULK is a 36 month research project, she says. applied to the study of kleptoplasts so far, which commenced in June 2016 and is used’, notes Cruz. has the main goals of understanding Intriguingly, although many of the both the fate of photosynthesis-derived mechanisms preventing chloroplasts The photoprotective component of non- carbon and nitrogen inside the animal degradation require the synthesis of photochemical processes is quantified host and the impact of photoprotection new proteins encoded in the nucleus of through chlorophyll fluorescence analysis mechanisms on kleptoplast longevity. This the microalgae, some slug species can techniques. ‘In turn, energy dissipation is a multidisciplinary collaboration with top keep functional kleptoplasts over several capacities are assessed through High- researchers in areas such as photobiology, months. ‘Much as photosynthesis by performance liquid chromatography gastropoda , animal biology, kleptoplasts alone is not sufficient to (HPLC) pigment analysis of the xanthophyll NanoSIMS technology and lipidomic sustain host growth, previous studies point cycle combined with the quantification analysis. to an essential role of kleptoplasty’, Cruz of accumulating reactive oxygen species observes. Yet, the results on the relevance (ROS)’, she says. ‘Finally, the repair There are three Portuguese institutions of this process for sea slug survival have so capacity of the photosynthetic apparatus is involved: University of Aveiro; University of far remained controversial. evaluated by quantifying the accumulation Lisboa; and, University Lusófona. The team of D1 protein.’

www.impact.pub 57 The unravelling of the underlying mechanisms could Project Insights

lead to practical biotechnological applications FUNDING R&D project (PTDC/BIA- ANM/4622/2014 – POCI-01-0145- FEDER-016754) and CESAM support (UID/AMB/50017 – POCI-01-0145- FEDER-007638) through Fundação para Cruz points out that they have some during the development of the HULK a Ciência e Tecnologia (FCT, Portugal), experiments planned which will analyse project and would not have been possible if Programa Operacional Regional de kleptoplast gene expression levels Dr Cruz could not count on a team of ‘great Lisboa (POR Lisboa), and Fundo Europeu de Desenvolvimento Regional and kleptoplast lipidome during food people that have always found time for (FEDER), within the Portugal 2020 deprivation to better understand which discussion and planning, including meeting Partnership Agreement and COMPETE components are responsible for long- in Portugal’. In such a multidisciplinary 2020 (European Union). term failure of photosynthetic activity. project covering diverse areas of expertise, Importantly, the inclusion of different sea the integration of the different results into PARTICIPATING INSTITUTIONS slug and algal species will allow for the a single story was not obvious at first, but Department of Biology & CESAM understanding of the evolutionary gradient all the data are fortunately starting to make University of Aveiro • Department of Life Sciences University Lusófona • Faculty of explaining the different timeframes in which sense together. Given the innovative nature Sciences University of Lisbon • Institute kleptoplasts are maintained functional. of the project, the scientists are constantly of Chemical Sciences and Engineering required to use all their creativity in order to Ecole Polytechnique Fédérale de

SOLID RESULTS adapt techniques and equipment normally Lausanne • Muséum National d’Histoire In line with the proposed role for kleptoplast applied in plant research or geosciences to Naturelle de Paris • Marine Biological photosynthesis in providing additional the use of an animal model. Section, University of Copenhagen nutrients when the animals are deprived CONTACT from their food source, recent work within POSITIVE IMPACT Sónia Cruz Evaluating the contribution of kleptoplast the HULK project shows that, at least in Principal Investigator the species E. viridis, the presence of light photosynthesis to the host metabolism is advantageous in terms of keeping size is fundamental to help better understand T: +351 234247092 and weight during starvation episodes. and classify this intriguing plant-animal E: [email protected] The combination of lipidomic tools, association, unique within the animal W: http://www.cesam.ua.pt/soniacruz hyperspectral reflection analysis and kingdom to sacoglossan sea slugs. ‘This NanoSIMS studies has unequivocally association provides a unique opportunity PROJECT COORDINATOR BIO proved the contribution of photosynthesis to study how genetic and biochemical Dr Sónia Cruz specialised in to supply metabolites, whose translocation components have evolved to form a ecophysiology of microalgae with a degree in Biology (University of Aveiro) and function within the host cell are functional photosynthetic unit and the and a PhD in Plant Physiology at the currently being investigated. ‘Strong potential for the evolution of permanent University of Sheffield, in collaboration evidence for a link between kleptoplasty and photosynthesis in an animal. The with the University of Leipzig. She sea slug behaviour has also been found, unravelling of the underlying mechanisms continued this specialisation as since E. viridis and E. timida respond to could lead to practical biotechnological a postdoctoral researcher at the light depending on their acclimation status applications’, says Cruz. University of Cambridge. In 2011 thus avoiding excessive light that would Cruz returned to Portugal, still as a postdoctoral researcher, to start quickly degrade kleptoplasts, thereby Towards the end of the project, the working with photosynthetic sea slugs. drastically reducing photosynthetic activity’, HULK consortium plans to carry out a Meanwhile, she worked at the University expounds Cruz. dissemination work comprising seminars, of Copenhagen, allowing establishing hands-on workbenches and photographic crucial international collaborations for In this respect, these results imply a exhibitions in collaboration with Fábrica current projects. Two years ago Cruz communication between kleptoplasts da Ciência, a centre resulting from the became an independent researcher and the host, possibly through oxidative partnership between the University of at the University of Aveiro, Portugal (Department of Biology & CESAM & stress signalling. The researchers are also Aveiro and the National Agency for Science ECOMARE). interested in the chloroplast photoprotective and Technology of Portugal. Alternatively, measures employed by the algae and the group of Cruz submits each accepted whether they are present in the sea slug. manuscript to the University of Aveiro Cruz says that to their great surprise, newsletter, whose information services Codium tomentosum, the main food then send a press release to different source of E. viridis, was found to lack a communication media for the dissemination functional xanthophyll cycle and may rely on of the research to the general public. other mechanisms such as more efficient machinery repair systems or chloroplast The HULK project is starting to deliver movement within the cell. positive results and will hopefully not only shed light on the chloroplast-sea slug The coordination between groups from interaction, but also contribute to open new different institutions and countries has challenging and exciting research fields. been one of the most recurrent challenges

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