Naples

Achievements for each user project

Nadav Shashar

The visit was very successful- due to the wonderful support by our hosts. We obtained both field and laboratory data, some of it first in its kind. We stated new projects and hopefully new collaboration.
The fact that a local student was assigned to help us was an amazing help.
We also made very good and useful connections
My one recommendation is to find a way to fund students' follow-up visitation to host and visiting laboratories.

Laure BONNAUD

In 5 days, the short term was considered. We learned the methods and applied it on several stages of development (25-27 to 29).
We evidenced NO superficially at late stages. This NO production concerns essentially sensorial structures of the skin (including chromatophores), suckers and olfactory lobes. We have not observed any signal in deep organs.
We have had fruitful discussion on the comparison between previous results on NADPH diaphorase obtained by Palumbo team and we have elaborated hypotheses to explain NO signaling. We have established working outlines for the second week of staying (of the other user).

John Parrington

Short -term objectives: TPC1 and 3 mRNAs were present at high levels in the sea urchin egg but declined exponentially during later developmental stages, suggesting these isoforms might only be present as maternal messages. In contrast TPC2 mRNA was present at low levels in the egg, but increased during development. TPC2 may thus have quite different functional roles compared to TPC1 and 3. Both NAADP or its precursor NAD+ applied to the outside of the starfish egg promoted intracellular calcium increases. Further experiments are needed to establish whether this increase is due to extracellular influx or release from intracellular stores.
Medium-term objectives: We generated probes for in situ RNA hybridization based on the TPC1, 2 and 3 ORFs. Immuno-localisation with anti-ARC and anti-TPC antibodies found differential patterns of localization for the different ARC and TPC isoforms during embryogenesis.
Long-term objectives: We generated RNA from TPC expression constructs to test in starfish oocytes. We designed and ordered morpholinos targeted against urchin TPCs. These morpholinos will be injected into urchin eggs and fertilization allowed to take place to observe effects on development.
Possible impacts: NAADP plays an important role in diverse physiological processes. Understanding the mechanism of action of NAADP and its target receptor may lead to greater understanding and treatment of conditions as diverse as heart disease, diabetes, obesity, and disorders of the immune system.
Difficulties encountered: The project was extremely successful. However, there were some problems in obtaining good quality starfish oocytes due to a limited number of animals available.

 

Julia Morales

During our stay, we were hosted in Dr. Ina Arnone's lab. Dr Arnone provided us with the necessary material and reagents, and shared her expertise in the manipulation of the animals, eggs and embryo culture. We were able to collect eggs and embryos at different developmental stages. Moreover, we treated the embryo cultures with various stress inducers, and observed that treatments triggered a cell cycle arrest. Samples were also collected from these treated embryos. The samples will be processed in our laboratory for western blot analysis of different translation factors, known to be modified following fertilization and in stress conditions, and for molecular screening of translated mRNAs. We have used the SZN Molecular Biology plateform for performing qPCR in order to monitor the expression of genes involved in translational regulation. We could also access the sea urchin maintenance facility, and got many advises on equipment and maintenance conditions for the S. purpuratus sea urchin.

Paul L.R. Andrews

NOTE: The data is currently being analysed so the following represents preliminary observations which require validation.
1. Obtained data which will inform the development of criteria for anaesthesia and nociception
2. Demonstrated nociceptive responses to both mechanical and chemical stimuli applied to the isolated arm. Obtained preliminary evidence for the presence of acid sensitive receptors and threshold to induce a response identified. Evidence also obtained for presence of osmoreceptors. Demonstrated feasibility of electromyographic recording of contractile activity in the isolated arm during withdrawal. Muscle contraction always occurs on the cephalic side of a noxious stimulus applied to the isolated arm-this has some similarities with the intestinal peristaltic reflex which may provide insights into how this should be studied as it is well characterized in mammals. Preliminary indications were found for a mucus secretory response to noxious chemical stimulation applied to an isolated arm tip.
3. Incidental observations identified the potential to study tissue healing by investigation of animals which had sustained a natural injury to an arm in the wild prior to capture.
When analysis of the above studies is completed and the results discussed with colleagues at SZN (Drs Graziano and Brown) it should be possible to provide an evidence base to inform the discussions of the revision to EU 86/609 involving cephalopods and to publish data to support application for funding to undertake additional behavioural and physiological studies.

Arne Nygren

Ischia was very good for our research, we stayed there for one week. Our one week in Naples were less productive. This was partly out of the institutions control as it was too windy to dredge for material. But even so, the facilities for collecting marine fauna is so much better at Ischia that I would strongly recommend to any other person with the same goal as us to go to Ischia instead. We applied for Ischia but for some reason Dr Brown told us that he thought we would have problems if we would have stayed as Ischia (I do not understand why). Thus it was decided that we were instead to stay at Naples. Just the week before our arrival at Naples, Lisa (from the adminstration) offered us the oppertunity to stay at Ischia one of the two weeks. A suggestion we gladly accepted. After our two weeks at Naples and Ischia I cannot understand why there should be a problem visiting Ischia for the whole period. The staff at Ischia was very helpful and the lab is perfectly suitable for marine taxonomic field work.Thanks to the week at Ischia we are very content with our trip.

Phillip Assmy

Both scanning (SEM) and transmission electron microscopy (TEM) as well as DNA extraction, amplification and sequencing of LOHAFEX diatom cultures for the construction of LSU phylogenies were performed to characterise the various cultures and check for intraspecific genetic and morphological variability. Access to the microscopy, molecular biology and phytoplankton taxonomy service at SZN was instrumental to achieve these objectives. For genetic characterisation of LOHAFEX diatom cultures and identification of phytoflagellates see ASSEMBLE report of Isabelle Schulz.
SEM/TEM investigation was performed on both culture and field material from LOHAFEX with the help of the electron microscopy service, Gandi Forlani and Drs. Marina Montresor and Diana Sarno. Cultures of Chaetoceros convolutes and Fragilariopsis kerguelensis were examined for life cycle stages (auxospores, gametes and gametangial cells),
small and big Corethron pennatum for their general morphology and particular the shape of their hooked spines and Pseudo-nitzschia cultures for their ultrastructural features. Unfortunately TEM preparations of Pseudo-nitzschia cultures, identified as potentially new species by phylogenetic analysis, were not successful due to the little material available and the delicate nature of their frustules. Theses preparations will need to be repeated with more material and a gentle cleaning and efficient concentration procedure. SEM/TEM examination of only dehydrated as well as acid cleaned field material was performed on samples taken at the start, during the middle and the end of LOHAFEX in order to obtain a good coverage of species succession during the iron-induced bloom. The main focus was on the identification of species that are difficult to identify during routine counting of field samples with LM (e.g. phytoflagellates, small Thalassiosira spp. and small pennate diatoms). Some species that could not be identified under LM (e.g. Lennoxia faveolata, Gyrodinium flagellare, Thalassiosira gracilis var expecta, Actinocyclus exiguus) could thereby be identified with certainty.