Approved STSMs 2016 & 2017
Approved STSMs 2016 & 2017
The COST Action FA1301 – CephsInAction – during 2016 already granted 2 STSMs, belonging to the extended 2nd year of our Action, and 8 STSMs for the 3rd year.
Short 2016 & 2017 STSM Summary Reports
Ms. Giulia Di Cristina: Acquisition of electrophysiological methods for the study of nociception in Octopus vulgaris
Period from 06/03/2016 to 08/04/2016
Full name: Giulia Di Cristina
Home Institution: Stazione Zoologica Anton Dohrn, Napoli (IT)
Host Institution: Universität zu Köln, Cologne (DE)
STSM Summary Report
Research Interest: Use of electrophysiological methods to study nociceptive pathways in invertebrates.
Summary of research completed during STSM: During my STSM at the University of Cologne in Prof. Dr. Büschges laboratory I firstly reviewed several papers on extra- and intracellular recording in invertebrates, fish and mammals, some of which were focused on the study of polymodal sensory neurons in invertebrates. I also reviewed literature on the neural basis of elementary behavior and the anatomy of stick insects and I practiced dissections on the stick insects Carausius morosus and Sipyloidea sipylus, in order to expose the nervous system and apply extracellular electrodes on the nerves running out from the thoracic ganglia. I practiced electrophysiological methods to record the neural extracellular activity evoked by the application of different kinds of stimuli on the nerves of the insect. After the experiments I performed the analysis of the recordings in order to study the responding units thresholds and to measure the conduction velocity of the signal trough the fibers. All this under the supervision of the extremely skilled and helpful staff of Dr. Büschges lab. I also had the opportunity to visit Prof. Dr. Gary Lewin research group at the Max Delbrück Center for Molecular Medicine in Berlin, where electrophysiological and behavioral assay aimed to study pain and sensory perception in mice and humans are conducted.
Synthesis and application of research: All the knowledge and practical experience I gained during my STSM will be applied during my PhD project at the Stazione Zoologica in Naples: I am planning to use extracellular recording to study sensory perception and nociception in Octopus vulgaris, in order to investigate the physiological features of the fibers running along the axial nerve cord of the arm and to identify any high-threshold responding unit, or units responding only to chemical noxious stimuli.
Wider benefit of the STSM to the participant: The STSM at the University of Cologne, where extra- and intracellular recording techniques are used to study the neural control of active locomotion in stick insects and Drosophila, helped me to learn and practice extracellular registration techniques, such as the registration of electrical activity evoked by different kinds of stimuli on invertebrates and the analysis of the recorded activity. I gained confidence with each component of the electrophysiological set-up and the Spike 2.0 Software and I am looking forward to use this newly learned techniques for the purpose of my PhD project.
Mr. Raphaël Lenoble: Exploring the application of the ‘Grimace Scale’ concept to cephalopods based on behavioral signs/indicators of their health and welfare status
Period from 28/03/2016 to 22/04/2016
Full name: Raphaël Lenoble
Home Institution: Groupe Mémoire et Plasticité comportementale, Université de Caen Normandie, Caen (FR)
Host Institution: Stazione Zoologica Anton Dohrn, Napoli (IT)
STSM Summary Report
Research Interest: Exploring the application of the ‘Grimace Scale’ concept to cephalopods based on behavioral signs/indicators of their health and welfare status.
Summary of research completed during STSM: This STSM occurred in three steps. Firstly, the analysis of the “grimace scale” concept use in rodents, this concept consists of use some facial expression to allow the pain and suffering of mice or rats during and after experiments (like surgeries or chemical injections). This noninvasive concept is useful to improve the efficiency of experimentation. With this scale, researchers are able to understand quickly and easily when their subjects feel pain during experiments or after some surgeries for example, and can react quickly to improve, if possible, the animal’s status.
The second step was to explore the possible application of these concept to cephalopods species. To be able to apply it some conditions are necessary, the existence of some indicators of pain and suffering, and also the existence of way to induce the behaviors and body patterns expected to highlight a possible pain. For the first condition, cephalopods present interesting abilities which can allow use to understand their status, indeed they express situation-dependent body patterns studied by many authors in literature. For the second condition, some stimulation have previously been highlighted as noxious stimuli for some cephalopods.
The last step was to propose some protocols which can be used to apply the “grimace scale” concept in cuttlefish, squids and octopus.
Synthesis and application of research: The grimace scale concept is originally use in rodents, and consists of use some facial expression to allow the pain and suffering of mice or rats during and after experiments (like surgeries or chemical injections). This noninvasive concept is useful to improve the efficiency of experimentation. An application in cephalopods species seems possible, in view of some interesting particularity which can allow experimenter to understand to understand their status. To apply it, this STSM propose several protocols, with the use of different noxious stimulation.
Wider benefit of the STSM to the participant: This STSM allowed me to learn more about cephalopods species, particularly on octopus, with which I worked. My project was very rewarding and convinced me of the importance of animal welfare, which despite the additional difficulties to set up studies, allows finally to obtain more convincing and more relevant results. I also had the opportunity to meet researchers working on several interesting projects around this model of study and to assist to several scientific conferences. This experience was very rewarding for me and confirmed my goal of exercising the profession of researcher. In addition, it allowed me to discover another culture and improve my English.
Key output: This STSM achieved the setup of several possible protocols to apply the “grimace scale” concept to cephalopods species.
Ms. Sarah Long: Development of a non-invasive health screening technique for the common octopus Octopus vulgaris held under laboratory conditions and assessment of the health effects of exposure to a bioactive therapeutic sulfate galactan
Period from 06/06/2016 to 01/07/2016
Full name: Sarah Long
Home Institution: University College Cork (UCC), Cork (IE)
Host Institution: Institute of Marine Research (IIM-CSIC), Vigo (ES)
STSM Summary Report
Research Interest: Improving cephalopod welfare and condition in captivity using novel techniques and natural therapeutics.
Summary of research completed during STSM: The growing popularity of culturing and holding the common octopus Octopus vulgaris in captivity raises significant animal welfare concerns. ‘Superinfections’ regularly occur, with Aggregata spp. and Vibrio spp. being two of the most common and dangerous pathogens. Non-invasive health screening of captive O. vulgaris and treatment with therapeutics are required to minimise infections and to comply with EU Directive 2010/63/EU regulations.
Non-invasive sampling of faeces and filtered seawater, and pathogen DNA extraction methods for the molecular screening of pathogens were assessed and optimised in this study. Four techniques were tested to open resistant Aggregata spp. sporocysts prior to DNA extraction: (1) hot/cold cycles; (2) acid pepsin digestion; (3) acid pepsin digestion and hot/cold cycles; (4) acid pepsin digestion with varying incubation length. A Chelex/Proteinase K DNA extraction method was then tested. A non-invasive method of visualising Aggregata spp. sporocysts in the faeces of O. vulgaris was also tested and optimised as a possible alternative to the currently used method of lethal sampling of the caecum of animals for disease diagnosis.
Several laboratory trials were conducted exposing O. vulgaris, infected with Aggregata spp. and Vibrio lentus, to the bioactive compound (sulfate galactan) to evaluate pathogen development and O. vulgaris performance. Faeces and filtered seawater samples were collected every sampling day for the duration of the trials and PCR was carried out to detect the DNA of both pathogens. Humoral and cellular immune parameters (lysozyme activity and haemocyte counts) were also assessed.
Both Aggregata spp. and Vibrio spp. DNA was detected in faecal and filtered seawater samples by PCR, however, a significantly higher detection of pathogens was observed when screening the filtered seawater samples. All sulfate galactan exposed animals showed short-term spikes in immunocompetence, although not statistically significant to control individuals, however, Aggregata spp. sporocyst numbers did decrease significantly over time in the Vibrio lentus trial.
This study found filtered seawater to be an effective, inexpensive, and easily repeatable method of non-invasive health screening of O. vulgaris in captivity, and the tested DNA extraction methods were successful. The analysis of faeces at light microscopy was also found to be an effective method of detecting Aggregata spp. presence in O. vulgaris, and could be used as an effective non-invasive method for disease diagnosis. Sulfate galactan showed promise as a potential naturally derived therapeutic, although more research is needed to evaluate its usefulness in large-scale culture and aquarium settings over longer periods of time.
Synthesis and application of research: This work aimed to find a non-invasive method of detecting Aggregata spp. and Vibrio spp. by PCR using filtered seawater and faeces, and a non-invasive method of visualising infection with Aggregata spp. to replace lethal sampling (light microscopy evaluation of faeces), in order to help comply with recent regulations (EU Directive (2010/63/EU)). Filter paper was significantly more effective than faeces at detecting pathogens and the visualisation of Aggregata spp. sporocysts in the faeces of O. vulgaris was successful. Modified DNA extraction techniques were also successful. The effectiveness of a naturally derived potential therapeutic, sulfate galactan, showed promise, with short-term increases in haemocyte count and lysozyme activity.
Wider benefit of the STSM to the participant: The research conducted at CSIC-IIM will be used to complete the research component of my Masters in Marine Biology in University College Cork. The research conducted helped me to learn and develop laboratory skills that will be useful in my future academic pursuits. I gained confidence in general in the laboratory setting, and in particular with DNA extraction and amplification, microscope techniques, and welfare of octopus in research facilities. I hope to publish the findings of this research, in particular the use of filter paper as a non-invasive health screening mechanism, and the effectiveness of sulfate galactan as a naturally derived therapeutic for use in improving the defence of O. vulgaris in aquaculture or research facilities.
Key output: Development of viable non-invasive health screening techniques for O. vulgaris in aquaculture/research facilities. Testing the short-term effectiveness of an immunostimulant, sulfate galactan, as a possible naturally-derived therapeutic for O. vulgaris held in aquaculture/research facilities.
Short quote: I thoroughly enjoyed my time spent working with Camino and everyone at CSIC-IIM and learnt so much during my stay. Thanks to everyone at COST and CSIC-IIM for this wonderful opportunity!
Ms. Astrid Deryckere: Towards developing Octopus vulgaris induced pluripotent stem cells as alternative model for molecular study of neural development
Period from 09/07/2016 to 23/07/2016
Full name: Astrid Deryckere
Home Institution: Katholieke Universiteit Leuven, Leuven (BG)
Host Institution: Stazione Zoologica Anton Dohrn, Napoli (IT)
STSM Summary Report
Research Interest: Considering the highly variable genetic background of the Octopus vulgaris, we want to establish a versatile and well-characterized cellular system to allow molecular studies on various organ systems (including the nervous system).
Summary of research completed during STSM: Despite the short, but intense schedule, I was exposed to a glance of the main aspects of scientific research with the octopus. This covered care of the animals, techniques and principles for tissue sampling and preparation for further studies.
I have been introduced to the present status of EU legislation and ethics involving cephalopods and to the existence of guidelines for the care and welfare of cephalopods in research. Principles of project preparation and application to National Competent Authorities have also been discussed.
I also benefited of short interactions with other students of the host lab involved in other projects that provided a wider overview of possible research to be carried out with cephalopods. I was also introduced to the basic techniques of stimulus presentation to octopus.
Finally, I have been instructed on the concept of “procedure” in the use of live animals in compliance with the Directive 2010/63/EU as applied to cephalopods.
I also learned how to deal with anesthesia of the octopus, and isolated cells from hemolymph (the octopus blood) and white bodies (the octopus hematopoietic system) and tested different culture conditions. I was taught how to isolate an Octopus brain and studied its general anatomy and morphology with the aim to build up a preliminary personal atlas in different plains of the octopus brain for neuroanatomical study purposes.
Synthesis and application of research: The main purpose of this STSM was to provide an initial training to set-up a solid basis for future experimental work on the cephalopod mollusc Octopus vulgaris at the home institution (Lab of Developmental Neurobiology, KU Leuven, Belgium).
I therefore received a general training on Octopus vulgaris husbandry and on methods for appropriate sampling from O. vulgaris in compliance with Directive 2010/63/EU. I also performed a first attempt to isolate and culture cells as a first step to establish induced pluripotent stem cells from O. vulgaris, aiming to reduce the number of animals needed for scientific purposes in the future.
Wider benefit of the STSM to the participant: Research on the Octopus vulgaris is completely new to the Host institution and in Belgium in general. Therefore, exchange of knowledge from an institution with long-established experience with these animals will help us to build our own installation. The Stazione Zoologica will in the future also be our first contact for the many questions that will still rise.
Key output: First of all, I gathered know-how on Octopus vulgaris husbandry and was trained to appropriately take samples from O. vulgaris in compliance with Directive 2010/63/EU. I also made a first rough design of an animal facility at the home institution.
Second, I studied the morphology of the octopus brain and performed a first attempt to isolate and culture primary cells from both hemolymph and white bodies.
Short quote: (After a long day in the lab) – “When you are ready with the PCR, you can start to dye/die”, a quote from a lab member, tutoring me.
Dr. Jorge Hernández Urcera: Octopus vulgaris genetics: for the development of a comprehensive molecular toolbox
Period from 09/08/2016 to 10/10/2016
Full name: Jorge Hernández Urcera
Home Institution: Aquaculture Department, Oceanographic Center of Vigo, Spanish Institute of Oceanography, Vigo (ES)
Host Institution: Fish Muscle Research Group, Scottish Oceans Institute, University of St Andrews, St Andrews (UK)
STSM Summary Report
Research Interest: Development of a comprehensive molecular toolbox for Octopus vulgaris.
Summary of research completed during STSM: Although the sequencing of the Octopus bimaculoides genome represents a landmark for the development of genetic resources for cephalopods, the genome of Octopus vulgaris has not been sequenced yet. However, there are other resources available (e.g. the Short Read Archive from NCBI) that can be exploited. The aim of the proposed STSM in collaboration with the University of St Andrews was to expand the existent genetic resources for the O. vulgaris and to establish a research network for the implementation of future projects on cephalopod genetics. The STSM have had four main objectives: (a) assembly and annotation of different de novo transcriptomes from O. vulgaris using the data available in the Short Read Archives (SRA) for this species; (b) data mining for the identification of genes of interest related with growth, metabolism and stress; (c) evaluation of the degree of similarity between O. vulgaris and O. bimaculoides sequences to determine the utility of the O. bimaculoides genome in future studies; (d) establishment of a research network with the University of St Andrews to explore future research projects using the tools obtained from the above objectives.
Next Generation Sequencing libraries for Octopus vulgaris were obtained from the SRA. A total of two libraries from 454 platforms and two from Illumina were downloaded and de novo assembled using Newbler 2.8 and Velvet 1.2.10 software respectively. Contigs generated from the de novo assembly were blasted (Blastx) against the annotated proteomes from Zebrafish (Danio rerio) and Fruitfly (Drosophyla melanogaster) as a vertebrates and invertebrate models respectively, using BioEdit free software. Data mining from the annotated transcriptome was used to identify genes of interest. The list of genes retrieved was defined by literature search and were included stress related genes, metabolism related genes, protein synthesis and degradation and muscle growth related genes. Sequences retrieved were cured to only contain the coding regions, eliminating UTR regions and miss-assembly regions. Data mining was also done in the O. bimaculoides genome in order to complete possible holes in the pathways and systems analysed. The degree of similarity between O. vulgaris and O. bimaculoides was established by blasting (Blastn) the de novo transcriptomes against the O. bimaculoides predicted genes from the EnsemblMetazoa database using BioEdit. In addition, maximum likelihood phylogenetic trees were constructed to test if vertebrate annotation could be valid to apply to octopus orthologues.
Synthesis and application of research: The main result of this STSM has been the characterization of 123 genes (based on vertebrate nomenclature) that could be involved in metabolic processes related to muscle development and stress in O. vulgaris. However, our results demonstrate that the gene nomenclature applied to the invertebrates orthologues based exclusively on BLAST results is a misleading approach and not supported by any evolutionary relationship. Our results also demonstrate that the identity between O. vulgaris and O. bimaculoides is very high, with a minimal identity at nucleotide level of the 78% and with the great majority of data with over 90% identity.
Wider benefit of the STSM to the participant: The STSM conducted at the University of St Andrews and supervised by Dr. García de la serrana has helped me enormously to learn and master the necessary skills to analyze de novo transcriptomes for non-model species. In additon, during my STSM I had the opportunity to attend to the course “Introduction to Unix for Bioinformatics and Genomic Analysis” organised by the St Andrews Bioinformatics Unit (StABU) and focused in the methodology that I had been learning. The results obtained in this STSM allow us to think about future projects in order to go deeper in the transcriptome analyses to improve the knowledge in the physiology of the muscle development of the common octopus.
Key output: Despite the primary objective of the visit was not the generation of enough data to generate a manuscript, if more time is invested in phylogenetic analysis, the work developed in the present project would represent an interesting evolutionary study. We will study the actual evolutionary relationships between vertebrates and octopus orthologues for genes involved in muscle development and regulation in order to identify genes family expansion through gene duplication and signs of convergent evolution of the molecular networks involved.
Dr. Pamela Imperadore: Label-free multiphoton microscopy as a tool for the investigation of nerve regeneration in the cephalopod mollusc Octopus vulgaris
Period from 28/11/2016 to 09/12/2016
Full name: Pamela Imperadore
Home Institution: Association for Cephalopod Research – CephRes (IT)
Host Institution: Universitätsklinikum Carl Gustav Carus Dresden (DE)
STSM Summary Report
Research Interest: Mechanisms involved in peripheral nerve regeneration taking advantage of the cephalopod mollusc Octopus vulgaris.
Summary of research completed during STSM: Coherent anti Stokes Raman scattering (CARS) microscopy is a non-linear variant of Raman spectroscopy that provides intensity information about single molecular vibration modes at submicrometer resolution. Multi-photon microscopy, among other applications, is extremely helpful for the imaging and study of axon injury after spinal cord contusion or lesion. Indeed, CARS allows visualization of lipid content, endogenous two-photon excited fluorescence (TPEF) is used for endogenous fluorescent molecules and second harmonic generation (SHG) mainly for collagen.
My visit at Prof. Kirsch’s Laboratory aimed to obtain training on the CARS system and, for the first time to test it on Octopus vulgaris samples. Regenerating and control pallial nerves were indeed imaged through this label-free technique to evaluate the possibility of imaging cephalopod tissues without the aid of markers or antibodies of any kind.
CARS, TPEF and SHG were combined in one unique image, each signal allowing detection of different structures or phenomena. CARS allowed visualization of axons inside the pallial nerve, of neurons and neuropil inside the stellate ganglion. Muscles around the nerve were also visible with CARS. These structures did not appear to give any signal in SHG, while only neurons gave a detectable and strong signal in TPEF. Blood vessels running inside the nerve and hemocytes were also visualized through CARS. This helped also the identification of the contribution of hemocytes to the formation of the cicatricial tissue and their infiltration inside the lesioned areas. Remarkably, the degenerating fibers of the peripheral stump were highlighted at all-time points investigated with a stronger signal in CARS. They also showed a strong signal in TPEF, not detectable in intact axons. Degenerating fibers were indeed perfectly recognizable and distinguishable from intact or regenerating fibers. This signal was also detected two weeks post lesion.
At this time point peculiar structures were detected through CARS and TPEF signal. I indeed visualized many drop-shaped structures surrounded by connective tissue filled with round granules. They were identified in the central stump and into the area of lesion, where the two stumps meet. They are probably hemocytes that have phagocytized waste material resulting from axon degeneration. Both stumps also showed very small lipidic granules diffused into the lesioned nerve, well organized in rows.
Synthesis and application of research: The work carried out at the Universitätsklinikum Carl Gustav Carus Dresden will allow the imaging of cephalopod tissues through Coherent anti Stokes Raman scattering (CARS) microscopy, a non-linear variant of Raman spectroscopy that provides intensity information about single molecular vibration modes at submicrometer resolution. This label-free technique results extremely helpful in the identification of cells, structures and events involved in the regenerative processes occurring after nerve lesion in Octopus vulgaris. However these promising results open the way to new potential applications beside regenerative studies.
Wider benefit of the STSM to the participant: This STSM represented a great opportunity to work side by side with researchers that utilize CARS microscopy on a daily basis for several applications. This allowed an intense and complete theoretical and practical training on the technique and an overview of possible applications testable on cephalopods.
Key output: Analysis of the imaging obtained through CARS microscopy on regenerating Octopus samples allowed the identification of structures and cells involved in the process. Additional testing will be required with the aim of publishing a first paper on the use of the technique on cephalopod tissues.
Ms. Elena Baldascino: Development of non-invasive methods for “cephalopods endocrinology”
Period from 07/07/2016 to 29/07/2016
Full name: Elena Baldascino
Home Institution: Stazione Zoologica Anton Dohrn, Napoli (IT)
Host Institution: Cretaquarium, Thalassocosmos Hellenic Centre for Marine Research (HCMR) Crete (GR)
STSM Summary Report
Research Interest: My major interest is applying organismal biology to the study of growth and welfare of cephalopods for increasing opportunities for aquaculture for these species.
Summary of research completed during STSM:
Non-invasive approaches to detect the ‘endocrinological’ status of cephalopods have never been developed. The sole known exception is represented by the study of Larson & Anderson (2010) that detected corticosterone in fecal samples of the giant pacific octopus Enteroctopus dofleini. The use of these approaches in several organisms is grown during the last years and nowadays it is possible to measure concentrations of androgens, estrogens, glucocorticoids and steroidal compounds active as pheromones (Scott & Ellis, 2007). These methods include measurement of steroid hormones in fish from water samples taken from tank-holding water, thus providing a valid and reliable non-invasive alternative to bleeding.
Description of the work carried out: All the procedures applied to live animals during this STSM have been carried out as part of the routine veterinary practice undertaken for the purposes of animal husbandry at the Host Institute, and therefore these have to be considered not for experimental purposes, and thus outside of the scope of Directive 2010/63/EU. All the interventions provided to the live animals were applied with the best veterinary practice.
Octopus vulgaris (N=12; body weight: 0.3 – 1.4 kg) have been daily monitored for 18 days in the quarantine area of the Cretaquarium, (Greece) as part of the current holding practice. Animals were randomly assigned to two groups (N=6) fed with different diets, i.e. monodiet (Boops boops) and mixed diet (Boops boops, shrimps, mussels and crabs). During daily routine, the position of the animal in the tank, its general appearance and the following samples were taken: faeces (every day), seawater from the tank and mucus from each animal (every three days). ELISA assays (n=388 samples: water, mucus, faeces and haemolymph) for all hormones studied (i.e. corticosterone, testosterone, estradiol and progesterone) provided data and revealed inter-individual variations between animals. I also noticed trends of changes in the concentration of different hormones in time and it appears correlated between different samples.
Synthesis and application of research: This STSM project was aimed to develop a method to assess stress and sexual hormonal status of individual Octopus vulgaris while holding in tanks by using non-invasive approaches such as: seawater samples and faeces.
In addition, a novel approach to collect mucus from octopuses that does not include any scraping of the animal’s skin or body has been developed and aimed to significantly minimize their handling.
Wider benefit of the STSM to the participant: I have had the great opportunity to enjoy Cretaquarium part of the Thalassocosmos Hellenic Centre for Marine Research (HCMR) in Crete and its facilities as host for my STSM. The work under the supervision of Dr. Panagiotis Grigoriou was challenging and very fruitful. I am convinced that the project, the atmosphere and the knowledge of Dr. Grigoriou provided an important ground to increase my knowledge of cephalopod biology and my capability to proper handling the animals.
As an added value I had the opportunity to network with other students and professionals from a different country, which is an exceptional opportunity for direct exchange of experience. This STSM allowed me to help in developing methods and techniques for the study of animal behavior and welfare.
Key output: A scientific publication of the work is planned to be submitted to a peer reviewed scientific journal. The collaboration achieved during this STSM has founded the ground for a future collaboration on this subject in the future.
Mr. Pablo García Fernández: Transcriptomic study of Octopus vulgaris paralarvae: identification of potential biomarkers of welfare, gene expression regulation and novel gene annotation
Period from 10/01/2017 to 10/03/2017
Full name: Pablo García Fernández
Home Institution: Instituto de Investigaciones Marinas (IIM), Agencia Estatal del Consejo Superior de Investigaciones Científicas (CSIC), Vigo (ES)
Host Institution: University of St Andrews, St Andrews (UK)
STSM Summary Report
Research Interest: Develop a paralarvae transcriptomic landscape to provide a basis for further molecular analysis.
Summary of research completed during STSM: Paralarvae mortality string the success of the culture of Octopus vulgaris and as consequence its introduction in aquaculture industry. Molecular tools, like a biomarkers panel, would help in order to find the best culture conditions to improve the survival of the reared paralarvae.
In our study of the impact of culture conditions over paralarvae welfare, we performed a culture of O. vulgaris paralarvae in the IEO (Vigo, Spain) facilities. We have tested two diets: Artemia sp (control) and spider crab zoea (treatment), also complemented with Artemia sp; The effect of these two different diets were tested at two different temperatures: 14±1oC and 21±1oC. We also captured wild paralarvae to be used as reference group. All the paralarvaes which form part of the study were 10 days post-hatching old.
After individual RNA extraction, we performed a transcriptomic analysis by RNAseq to achieve a broad comparison of the effect of these different culture conditions at transcript expression level. Three individuals per group (four culture conditions plus wild individuals) were individually and sequenced (Illumina Hi-seq 3000 platform) in order to obtain 15 cDNA libraries. A total of 700,000,000 high quality pair-end reads (150 bp of length) was obtained.
Our first result was the assembly of a de novo transcriptome of the O. vulgaris paralarvae. This transcriptome was the result of assembly our 15 cDNA libraries using Trinity after a reduction of the contig redundancy using EvidentialGene. The constructed gene database has a strong potential and amply the scope of future studies around octopus paralarvae. Secondly, we performed a differential gene expression analysis at transcript level of our culture groups, using also wild individuals as reference. Kallisto was used for the quantification of gene expression and DESeq2 for the digital gene expression analysis.
The culture group “Artemia sp. & 14±1oC” showed the lowest differences against wild individuals, 66 transcripts differentially expressed between them. In the opposite extreme “Artemia sp. & 21±1oC” showed 6509 transcripts differentially expressed. For a diet based on Artemia sp. the temperature showed a strong effect over the gene expression pattern, however when we compare wild individuals against paralarvae feed with a diet based on spider crab zoea the effect of the temperature is much lower (14±1oC = 401 transcripts differentially expressed and 21±1oC = 542 transcripts differentially expressed), that suggest an interaction between diet and temperature.
These differences will be analyzed in detail in the next months in order to stablish a set of genes to be used as welfare biomarkers. These biomarkers will be evaluated under other different culture conditions and different paralarvae ages.
Synthesis and application of research: The gene database generated will be useful as complement of several metabolic studies and will help to clarify the requirements for a proper development of a paralarvae of O. vulgaris. The develop of a panel of welfare biomarkers will help to improve the evaluation of the impact of different culture conditions over the reared paralarvae.
Together, these data will help to improve the success of the paralarvae cultures not only in terms of growth, but also to overcome the mortality bottleneck.
Wider benefit of the STSM to the participant: That was my first time in a long term stage in a foreign country and this experience give to me the opportunity to improve my biologic expertise and also to learn about a different life style. As a person who lives in a no native English speaker country, these stage in St Andrews brings to me the opportunity to improve my English.
Talking about my PhD project, the STSM has open a collaboration window which will improve the quality my job taking advantage of his experience using this technique.
Key output: Paralarvae transcriptome and differential gene expression between five groups of parlarvae including culture and wild individuals.
Mr. Manuel Nande: Octopus nutrigenomics: the role of docosahexaenoic acid (DHA) and phosphatidylcholine (PC) in the enzymatic expression pathway’s in the neuronal development of Octopus vulgaris paralarvae
Period from 15/12/2016 to 15/03/2017
Full name: Manuel Nande
Home Institution: Instituto Español de Oceanografía, Vigo (ES)
Host Institution: Interdisciplinary Centre of Marine and Environmental Research (CIIMAR), Porto (PT)
STSM Summary Report
Research Interest: Nutrigenomic analysis on the relationship between docosahexaenoic acid (DHA), the biosynthesis of phosphatidylcholine (PC) and the arm synthesis/development of Octopus paralarvae.
Summary of research completed during STSM: During my short term scientific mission on the CIIMAR Interdisciplinary Center of Marine and Environmental Research of the University of Porto supervised by Dr. Filipe Castro allowed me increasing my knowledge related to the identification and analysis of enzymes involved to lipid metabolisms. Especially about polyunsaturated fatty acids (PUFAs), and the role of DHA, as well as a high proportion of phosphatidylcholine (PC), due to are essential for a good growth and development of the paralarvae. The main bottleneck of rearing Octopus vulgaris paralarvae occurs during the planktonic stage and is caused by several factors, among which nutritional deficiencies are believed to play a key role. The arm growth is a key factor for the change from a planktonic to benthonic life strategy or what is the same, to the next stage: juveniles. In paralarvae, the development of the arm also comprises protein and neuronal synthesis. The genesis of neurons during development is significantly influenced by the proportion of uridine, choline, and the omega-3 fatty acids docosahexaenoic acid (DHA, EPA and ALA) and phosphatidylcholine (PC) in the diet. The final reaction of the Kennedy pathway is the condensation of CDP-choline with diacylglycerol (DAG from DHA) to form Ptd-Cho in a reaction catalyzed by CDP-choline: 1,2-diacylglycerol choline phosphotransferase (CPT).
For this purpose, we used samples of paralarvae of Octopus vulgaris grown under different diets as the main variable that has an effect on differential expression. It was used as a control group paralarvae fed with Artemia (Artemia franciscana) that was compared with the study group of paralarvae fed with crabs zoeae (Maja brachydactyla). Differential expression of genes involved in the biosynthesis in the metabolic pathways of docosahexaenoic acid (DHA; 22: 6n-3) as fatty acyl desaturase with Δ5 activity (Fad) and EVOL (2 and 5) and Stearoyl-CoA Desaturase with Δ9 activity (Scd) were analysed. Ubiquitin expression was used as the reference gene. The enzyme CDP-choline: 1,2-diacylglycerol cholinephosphotransferase (CPT) was characterized using Sequence Read Archive (SRA) obtain in NCBI database (http://ncbi.nlm.nih.gov), performing BLAST between them and the homolog sequence of enzyme annotated in the genome of Octopus bimaculoides thus obtaining the CPT-enzyme sequence for O. vulgaris allowing the design of PCR primers. Different gene expression carried out using RT-PCR.
Synthesis and application of research: The results of this study will contribute to a better understanding of the role of DHA as diacylglycerol in the biosynthesis of phosphatidylcholine (PC) and its differential effect in different structures of the paralarvae. One of the improvements in this study was for the first time the nutritional effect on the differential expression of isolated structures such as mantle, head and arm was evaluated. The digestive gland was compared between the different groups but separately from the other structures because it is the organ performs the absorption and assimilation in the first instance of the food.
Wider benefit of the STSM to the participant: During my STSM at CIIMAR in Portugal under the supervision of Filipe Castro I learned new tools in the use of transcriptome analyses in order to obtain sequences of key genes in metabolism. The use of Sequence Read Archive (SRA) located in NCBI database (http://ncbi.nlm.nih.gov), will allow the analysis of conserved sequences allowing the design of PCR primers. In addition to the synergistic advance in the conjunction of molecular techniques with high-quality biologic samples. This STSM positively increased my experience in the use of new techniques that expand my knowledge and training, as well as to obtain a greater capacity of multidisciplinary analysis. In addition, during my time at CIIMAR I was able to attend different seminars given by the researchers of the center as well as the workshop (AQUAIMPROVE WORKSHOP – 2nd Edition Date: 17th of March 2017).
Key output: This study will be presented at congresses related to the area as well as its possible publication in scientific journals. A short introduction was made on this studio during the meeting held in Crete, (COST-CIAC Cretaquarium meeting March, 28-30 2017).
Mr. Ricardo Tur Estrada: Cloning and characterization of the putative corticosteroid, serotonin and dopamine receptors in the common octopus
Period from 22/03/2017 to 30/04/2017
Full name: Ricardo Tur Estrada
Home Institution: Oceanografic Centre of Canary Island (IEO), Santa Cruz de Tenerife (ES)
Host Institution: Interdisciplinary Centre of Marine and Environmental Research of the University of Porto, Matosinhos (PT)
STSM Summary Report
Research Interest: Cloning and characterization of the putative corticosteroid, serotonin and dopamine receptors in the common octopus Octopus vulgaris.
Summary of research completed during STSM: The molecular cloning and characterization of the putative corticosteroid (CR), serotonin (HTR1A) and dopamine receptors (DRD1, DRD2, DRD3, DRD4, DRD5) is being performed in Octopus vulgaris during the STSM. Additionally, the previously obtained data from the different stress-related amines by HPLC will allow to select key enzymes associated to the serotonin and dopamine metabolism pathways. The obtained sequences for serotonin (HTR) and dopamine (DRD) receptors from the common octopus using public gene data bases and O. vulgaris paralarvae transcriptome (Dr. C. Gestal, IIM-CSIC) obtained under the project OCTOWELF were aligned with homologous genes sequences from Octopus bimaculoides. Specific primers were designed and validated reference genes from O. vulgaris (Sirakov et al. 2009) will be used for qPCR assays. Gene expression data from stressed and undisturbed paralarvae will be compared to the obtained HPLC results and could be used as a potential stress biomarkers in the first stages of the common octopus life.
All these activities are being supervised by Dr. Benjamín Costas and Dr. Filipe Castro, host researchers at CIIMAR, Interdisciplinary Centre of Marine and Environmental Research of the University of Porto (Matosinhos, Portugal).
Synthesis and application of research: The molecular cloning and characterisations of the putative corticosteroid (CR), serotonin (HTR) and dopamine receptors (DRD) as well as key enzymes of serotonin and dopamine metabolic pathways will provide useful information in the stress physiology of O. vulgaris paralarvae and could be used to identify potential stress biomarkers in the first stages of common octopus life, helping to improve the rearing and welfare of the octopus culture.
Wider benefit of the STSM to the participant: During my stay at CIIMAR (Matosinhos, PT) I gained insights into the molecular biology and different laboratory methodologies to clone and characterize relevant genes involved in the of the common octopus paralarvae stress, which is one of the main objectives of my PhD thesis. In addition, I have had the opportunity to work under supervision of Dr. Benjamín Costas and Dr. L. Filipe Castro and the invaluable help of all members of this department.
Key output: The results obtained in this STSM are very useful to gain a deeper knowledge in the stress physiology of the octopus paralarvae, helping to identify new stress biomarkers in O. vulgaris paralarvae in order to improve the culture technology of this species.
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