Achievements for each project

Georg Pohnert

The time of the ASSEMBLE-stay was used for basic training for a PhD-project on algae-virus interactions. Different strains of the coccolithophore Emiliania huxleyi were infected with specific viruses followed by daily monitoring of phytoplankton and viral abundance using flow cytometry. Gained skills in flow cytometry will be used for the establishment of this method at the home institution. When viral lysis reached a suitable abundance level, phytoplankton cells were filtered off to obtain viral stocks for future experiments. Some of the stocks were used for tries on cryo-fixation using different set-ups. Infectiousness will be compared in Roscoff following the stay. This aimed to find the least damaging treatment for long term storage without regular infection of host cultures.
Parallel metabolite extractions from phytoplankton cells and media using different extraction techniques were performed to serve as method development for larger experiments in metabolomic profiling. All extracts were taken back to the home institution to be measured and analyzed there. Phytoplankton cultures need to be axenic to avoid mix-up with bacterial metabolites and were therefore treated with antibiotics.
Basic skills on viral maintenance and different E. huxleyi cultures were provided and stocks for future experiments obtained. Difficulties encountered during the stay were due to reduced infectiousness of viruses after long preservation, which prevented the execution of planned experiments and some miss-communication previous to the stay. The anticipated meeting with the partners from Israel could not be realized due to several last minute schedule changes and time restrictions from the host lab.

Catarina Mota

An effective protein extraction methodology was obtained, suitable for 2D gel electrophoretic separation of fucoid algae adult vegetative tissue.
Fucus serratus individuals were collected in the proximity of the SBR at low tide, both severely desiccation-stressed and fully hydrated control samples, and protein extraction and 2D gel electrophoretic separation of both stressed and control samples was performed with the tested methodology.
Following comparative image analysis of the two experimental conditions for the identification of proteins involved in desiccation-resistance, some potentially differentially expressed spots were excised for posterior MS analysis using the available mass spectrometry facilities.
This recent development of proteomic tools for fucoid algae will allow our on-going research on the molecular basis of desiccation tolerance in Fucus to evolve from transcriptomics to proteomics, and this combined information will undoubtedly facilitate our identification of the molecular mechanisms underlying the remarkable desiccation/ rehydration resistance of these brown algae.

Christoph Bleidorn

Main aim of this research stay was to collect material for subsequent RNA- and In situ hybridisation studies. Divers of the biological station collected several Antedon bifida (Echinodermata, Crinoidea) for us, which are known to be hosts of the ectocommensal myzostomid species Myzostoma cirriferum. In the wet lab, we collected adult and juvenile individuals directly from the host and fixed them in RNAlater. Moreover, when separating large (>1mm) adult specimens into a petri dish, they are usually start to lay already fertilised eggs within hours. Within a few days these eggs develop into trochophora larvae with prominent chaetae. We were able to collect several hundred of these chaetae bearing larvae and fixed them for subsequent in situ hybridisation studies. Altogether, we obtained all the material we needed for our study during this trip. In the meantime, we constructed a cDNA library of part of this material and mRNA-sequencing with generating millions of short sequence reads using the Illumina platform has been conducted.
The ASSEMBLE-stay at the station was well organized. When we arrived, the divers had already collected the crinoid hosts and these animals were kept in a large seawater aquarium. The laboratories were well equipped for our studies and we got access to all the material we needed.

Florian Weinberger

The objective of this project was achieved and the hypothesis that wound activated hyroxylated fatty acids regulate LOX expression in G. chilensis was accepted:
Gracilaria chilensis was exposed for 0 h, 6 h, 12 h, 24 h to a dichloromethane extract produced from wounded G. chilensis (concentration: 10 mg l-1), as well as to a fraction rich in hydroxylated fatty acids that was produced from this extract. mRNA was extracted for 30 min, using the Plant RNeasy mini kit (Qiagen). Subsequent northern blotting after labeling the samples with the PCR DIG probe synthesis kit (Roche Applied Science) failed, due to general expression levels of LOX below the detection limit. As an alternative LOX expression was quantified by quantitative PCR (qPCR). In this approach RbcL (large subunit of ribulose-bis-phosphate carboxylase) and COX1 (subunit 1 of cytochrome oxidase) expression were used as controls. As key components of the cellular energy metabolism these two gene products were expected to be unaffected by the treatment.
The qPCR approach clearly demonstrated that the ratio of LOX expression and RbcL expression increased after 6 h of incubation with either the dichloromethane extract or with the purified fraction of this extract. The same increase in LOX expression was also observed when the ratio of LOX and COX was regarded.

Aschwin Engelen

Weather conditions during the stay made field work complicated and lack of reproductivity of the kelps has postponed some of the activities, namely the developmental studies on spores and gametophytes. Probably to unusual winter conditions, reproduction of the kelps is later in the year than normal. As a result some of our proposed activities namely temperature experiments on gametophyte stage development were performed in May. Demographic studies need longtime series before analyses make sense, so it is still too early to obtain any sense of the results. Clearing experiments made clear that this year there was strong recruitment, but different for the different kelp species and recovery time differed among kelp species. Possibilities to adapt to the circumstances were unclear as questions were not answered or extremely late by the local contact. Gametophyte development is strongly dependent on temperature and the temperature sensitivity differs between species. For my stay meals were suppose to be provided but were only occasionally available especially when it came to dinner. For Tânia her stay there were many things unclear even now, we have not received any confirmation that her stay was fully supported by Assemble.

Sam Dupont

I had easy access to the species of interest thanks to the help of the local team but, unfortunately, due to unexpected cold conditions, animals were not ready to spawn and I was unable to start cultures for Acrocnida bracchiatta.
However, plans were changed with very interesting outcomes including:
- Impact of OA on regeneration rates of the two populations (probably different species) of Acrocnida.
- Impact of OA on larval development of two sea urchin species: (i) Spharechinus granularis (thanks to Vlad Costache and Patrick Cormier) and (ii) Echinocardium cordatum (thanks to Sophie Martin).
- Impact of low pH on various aspect of the life cycle of the acoel worm Symsagittifera roscoffiensis (together with Xavier Bailly).
Moreover, I gave a seminar on my work and my presence in Roscoff.
In conclusion, this was a very nice and productive stay. In was amazed by the development and the quality of the facilities in Roscoff (e.g. the microscopy plateform). This stay gave me also the chance to talk to many interesting researchers and start some new collaborations (in particular with Xavier Bailly, Sophie Martin and Patrick Cormier).

Ted Hupp

Developed protocols for isolating heat shocked tissue and embryos following fertilization of Ciona intestinalis egg/sperm

Demonstrated the expression of stress proteins in the experiments that is forming a manuscript for publication

The successful data/manuscript forms data for continued analysis of Ciona as an evolutionary model that will form the basis for grant applications to continue to develop this research area in collaboration with the Roscoff station.

Bente Edvardsen

1.Eikrem took part in the Biomarks sampling, did EM-preparations and examined live material under the microsope. Egge and Edvardsen could not reach in time for the sampling due to the vulcano eruption.
2. Design of haptophyte pyrosequencing primers was discussed and agreed on by all involved partners.
3. Ploidy level was analysed and determined in ca 15 Chrysochromulina cultures brought from UiO to Roscoff (D. Marie & Edvardsen). This will be a part of a collaborative manuscript (in prep.).
4. Work was done on a collaborative manuscript that subsequently was submitted.

Adrianna Ianora

During the execution of the project we collected the calanoid copepod species Calanus helgolandicus from samples collected by the fishing service of Roscoff CNRS. These were divided in different tanks for feeding experiments using four different algae: the control diet dinoflagellate Prorocentrum minimum, the non toxic flagellate Rhodomonas baltica and two toxin producing diatom Skeletonema marinoi (Adriatic Sea and North Atlantic Ocean). We were interested in studying the stress response in the copepod C. helgolandicus belonging to the ingestion of the toxic oxylipin-producing diatom S. marinoi. It is known that several genera of diatoms produce toxic secondary metabolites (collectively termed oxylipins) which induce reproductive failure in zooplankton grazers (Miralto et al., 1999, Ianora et al., 2004, Romano et al., 2010). In addition we stored about 2g of each algae at the experiment time to confirm the metabolite composition of each algae with chemical analyses. Different tanks were used with about 15 C. helgolandicus specimens and these were fed with one of the four algae separately. To study the reproductive success of animals we collected eggs after the first, second, fourth and fifth day of feeding and observed the hatching success. The egg production and vitality according to this experiment did not show the expected results. After 12h, 24h, 48h and 128h of feeding we froze them in liquid nitrogen and stored at -80°C. To analyze different gene expression level in copepods fed the different algae we will extract in Naples RNA from each group of stored animals.

Jamie Stevens

During our stay at the Station Biologique de Roscoff (SBR), we spent two days of diving time (1 full day and 2 half days) and were able to collect 3 populations of Eunicella verrucosa and Alcyonium digitatum as follows:

1) 19.5.10 Rade de Brest - full day. SBR diving team dived at 3 sites to collect sufficient specimens of each species to constitute a sample for our population genetics research.
2) 20.5.10 Bay of Morlaix (Ile de Batz) - half day. SBR diving team dived to collect sufficient specimens for our research.
3) 21.5.10 Bay of Morlaix (East of Roscoff) - half day. SBR diving team dived to collect sufficient specimens for our research.

Discussions regarding possible future collaborations were held with Prof Frederique Viard (Director of Research, CNRS, SBR) and Dr Laurent Leveque (Chief of Scientific Diving team, CNRS, SBR).

The samples collected will provide important out-groups for both our population genetic and phylogeographic research. Having collected specimens from two areas in NW France, we intend to evaluate connectivity at differing scales on both the north and south coats of the Channel. We will remain in touch with Prof Viard and Dr Leveque and his team, and hope to consolidate collaboration once our markers are finalized and we have some preliminary data from the material collected.

We appreciate the excellent organization, support and hospitality of Dr. Laurent Leveque and his diving team, and Ms Gaielle Penault (SBR ASSEMBLE co-ordinator) who ensured our visit to SBR was productive, informative and enjoyable.

Christopher R. Bridges

During the stay in Roscoff juvenile (smaller than 80 mm carapace width) and adult life-stages (larger than 80 mm carapace width) of Cancer pagurus were collected. The oxygen consumption of the animals was measured for 24 hours using a specified respirometer. After taking a haemolymph sample, the pH was measured immediately. For further measurements of the baseline parameters, e.g. total CO2, Ca2+-, Cl-, Mg2+-concentration, buffer capacity and oxygen binding ability, the samples were taken to Düsseldorf. Experimental animals were also transported to Düsseldorf. A long term incubation (min. 2 months) at different CO2-levels in a special incubation-system will start soon. The oxygen consumption will be measured throughout the whole incubation to investigate the effect of an increasing CO2-level in the seawater on the oxygen consumption. After an incubation period of approximately two months a second haemolymph sample will be taken and measured.

Thorsten Stoeck

All goals set for the project funded by ASSEMBLE where achieved and no difficulties were encountered. These goals included: plankton sampling on one cruise using traditional sampling strategies and preservation of sample material for microscopy analyses, molecular analyses and chemical analyses. On the second cruise, a number of different sampling sand sample preservation strategies were applied in order to compare their performances. A third sampling cruise targeted benthos (sediment) samples to be analyzed in the same context as described above. For sampling we took advantage of the RV Neomysis of the SBR. In the following days, selected samples were analyzed microscopically and by flow-cytometer at the SBR under supervision of trained personnel from the SBR. These analyses allowed for initial species lists of planktonic organisms. Analyses of molecular samples will follow in the home lab to compare with morphodata.