TNA projects

This TNA project was carried out by Menekşe Didem Demircan (Associate Professor at the Istanbul University, Faculty of Aquatic Sciences) at the University of Stirling (UoS) with the assistance of Manfred Weidmann (University of Stirling, Institute of Aquaculture). 

The project attempted to culture and sequence Lymphocystivirus (LCDV) from Turkish sea bream samples. Menekşe said that the TNA program provided to opportunity to learn important techniques about cell culture and Virology. It also gave Menekşe the chance to meet new researchers in her field and experience the life, culture and food of Stirling and Scotland.

For more information contact Menekşe Didem Demircan - dican@istanbul.edu.tr or Manfred Weidmann  - m.w.weidmann@stir.ac.uk

This TNA project was carried out by Isabel Forner-Piquer at IATS-CSIC (Spain). For more information about this work contact Isabel at isabel.forner.piquer@gmail.com 

Summary: Bisphenol A (BPA) and Di-isononylphthalate (DiNP) are two Endocrine Disrupting Chemicals (EDCs) found in common plastic items that have been reported for their negative health effects. In this project, we proposed to study the effects of such EDCs in the expression of key genes involved in lipid metabolism and reproduction. To accomplish that, we administered via diet both EDCs to two-year-old Sparus aurata (Gilthead sea bream) during the first sex reversal stage.

This project took advantage of the gilthead sea bream transcriptome database from the Nutrigenomics and Fish Endocrinology Group (IATS-CSIC) from where the sequences of target genes were selected (http://www.nutrigroup-iats.org/seabreamdb/). The project aimed to determine how BPA and DiNP are linked with metabolic alterations and infertility/subfertility in different classes of vertebrates. 

The TNA, by providing gene expression data by qPCR, improved the knowledge of the effects of BPA and DiNP in a new ecotoxicological model, Sparus aurata. Target genes were studied in liver, gonads and brain confirming the detrimental effects of BPA and DiNP. Additionally, the novelty of the project was the fish model, gilthead sea bream, a very interesting species due to sex reversal process during the first years of life, as well as one of the most farmed fish in the Mediterranean region and highly appreciated by consumers.

Isabel said "the TNA was part of my PhD studies providing a valuable set of data for successfully concluding my PhD thesis hold at the Universita Politecnica delle Marche (Ancona, Italy)".

Picture of the group, from left to right: Dr. Josep Calduch (TNA referent), technician Maria
Angeles Gonzalez, Prof. Jaume Perez (team leader) and Isabel Forner

Results: The TNA and its results have been spread in some websites:

• https://sciencetrends.com/beyond-the-plastics/ (Article written by Isabel at Science Trends, a divulgation science web).
• http://nutrigroup-iats.org/welcome/single_new/79
• http://iats.csic.es/accesos-transnacionales-de-aquaexcel2020-publicacion-de-alto-impacto/
• http://www.ipacuicultura.com/noticias/en_portada/63105/estudio_en_el_marco_de_aquaexcel_evidencia_los_efectos_negativos_de_una_alimentacion_expuesta_a_bisphenol_a_y_dinp_en_dorada_.html
• https://aquaexcel2020.eu/sites/default/files/inline-files/AquaExcel2020_NL4_final_web.pdf

1. Forner-Piquer et al. Endocrine disruptors in the diet of male Sparus aurata: Modulation of the endocannabinoid system at the hepatic and central level by di-isononyl phthalate and bisphenol A (https://www.sciencedirect.com/science/article/pii/S0160412018303787). 
2. Forner-Piquer et al. Effects of di-isononyl phthalate (DiNP) on the endocannabinoid and reproductive systems of male gilthead seabream (Sparus aurata) during the spawning season (https://link.springer.com/article/10.1007%2Fs00204-018-2378-6).
3. Oral presentation at the 29th conference of European comparative endocrinologists, 19th-22nd August 2018, Glasgow (UK). 
4. Poster presentation at SETAC (Society of Environmental Toxicology and Chemistry) Europe 29th Annual Meeting, 26-30th May 2019, Helsinki (FI). (Abstract book:  http://helsinki.setac.org/wp-content/uploads/2019/05/SETAC-Helsinki-Abstract-Book-2019.pdf).
5. Forner-Piquer et al. Effects of dietary bisphenol A on the reproductive function of gilthead sea bream (Sparus aurata) testes (https://www.mdpi.com/1422-0067/20/20/5003)

Other: You can read more about the DISRUPBREAM project here: https://www.aquaexcel2020.eu/news/disrupting-effects-plasticizers-male-gilthead-sea-bream

Rosa Inés Strem, a PhD student under the supervision of Dr. Galit Sharon, from Israel participated in the AQUAEXCEL²⁰²⁰ TNA program with the project  “Characterization of an unidentified pathogen (virus) in grey mullet (MugilRosa Inés Strem, a PhD student from Ben Gurion University under the supervision of Dr. Galit Sharon from the National Center for Mariculture - IOLR, from Israel, participated in the AQUAEXCEL²⁰²⁰TNA program with the project “Characterization of an unidentified pathogen (virus) in grey mullet (Mugil cephalus) causing a significant threat to aquaculture farms in Israel”. The project was carried out from September to November of 2016 at the Institute of Aquaculture from the University of Stirling (UoS - IoA), under the supervision of Prof. Mamfred Weidmann.

Summary: 

During the last years in Israel, grey mullet (Mugil cephalus) breeding stocks and larvae have been showing neurological signs such as uncoordinated circular swimming as well as oral hemorrhages. A few days after the onset of the clinical signs, the fish are found dead. Nineteen tissue samples from fish presenting clinical signs were collected and carried to the IoA – UoS. During the three-month period, all the samples were processed and inoculated in 12 well plates with SSN-1 cell line until the fifth passage, however, only three samples showed some cytopathic effect during the fourth and fifth passage. RNA from all the tissue and supernatant samples were extracted, followed by a reverse transcriptase PCR for Nodavirus. Results from the PCR were negative for all the tissue and supernatant samples. Simultaneously, the RNA from the tissue samples were processed following the TRAPD based whole genome sequencing protocol. The results from MiSeq revealed that, there were no similar sequences using both: a novo and a reference based approach. After running a RT-qPCR from all the samples, the results showed high Ct values of the vRNA for Nodavirus in most of the tissue samples, which means there are less than 10⁴ copies of the vRNA, which is the sensitive threshold for NGS. Up until now, there was no success in identifying the pathogen. To date, Rosa and colleagues are continuing their analyses, focusing on both, DNA and RNA viruses using the techniques learned during the training period at the University of Stirling, and focusing in other possible pathological agents.

Rosa said that  the AQUAEXCEL²⁰²⁰ TNA program help her to learn important techniques not only for her research, but also to implement cell line culture and viruses study in her lab in Israel.

MSc. Mohammadmehdi Saberioon, Ph.D. of  the University of South Bohemia in České Budějovice carried out his TNA research at the IFREMER Palavas Experimental Aquaculture Research Station infrastructure (IFREMER-PEARS)

Summary: Fish size and mass information during different growth stages is important for precise feeding regime management, oxygen consumption calculations, antibiotic prescription and improving fish welfare, but also to facilitate decisions on grading, harvesting and time to harvest. The main purpose of this study was to develop a method to measure fish mass using dorsal information in real-time. It aims to provide information as a non-invasive and remotely sampling approach and automatically fish size and mass estimation in recirculating aquaculture system(RAS) using infrared reflectance system (IREF). Through this study, researchers developed an automatic system to estimate fish mass using fish dorsal geometrical features and machine learning algorithms.  The introduced system can provide an inexpensive, stress-free and accurate approach for fish farmers and researchers to accurately and continuously monitor fish growth rate in tanks. Photo credit - Reference: (Saberioon and Cisar, 2018: https://doi.org/10.1016/j.compag.2018.05.025); copyright owner is Elsevier.

Mohammadmehdi said  "The TNA project permits us to access to The Institut français de recherche pour l'exploitation de la mer (IFREMER) infrastructure and its experts to help complete this experiment".

Results of this research have been already published, interested parties can find it as below : Saberioon, M., Cisar, P. (2018) Automated within tank fish mass estimation using infrared reflectance system. Computers and Electronics in Agriculture. 150, 484-492. https://doi.org/10.1016/j.compag.2018.05.025

For more information about this project contact MSc. Mohammadmehdi Saberioon, Ph.D. at msaberioon@frov.jcu.cz

Conducted at ULPGC-FITU by Dr. Claudia Torno (c.torno@gma-buesum.de) and Florian Daum

Summary: The constant need to improve fish nutrition to assure good fish product quality and simultaneously reduce the reliance on finite marine resources is the motivation behind this TNA project. Marine fish like the gilthead sea bream (Sparus aurata) have a high need for marine derived omega-3 long chain polyunsaturated fatty acids (LC-PUFAs) like eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA). Marine fish are not able to endogenously synthetize EPA and DHA from precursor fatty acids to prevent physiological impairment when these fatty acids lack in the diet. Nevertheless, it is generally accepted that the genetic capacity to perform the fatty acid synthesis is present but dormant in marine fish like sea bream. A stimulation of this synthesis beyond its natural capacity might be realized by using bioactive polyphenols. The findings of the TNA project indicate that only minor but significant improvements of the fatty acid composition of sea bream fillets could be achieved with one of the polyphenols (genistein). Furthermore, an interaction of the polyphenols with underlying molecular mechanisms could be proven. Thus, this approach can aid to further reduce the aquaculture’s reliance on LC-PUFAs from fish oil and fish meal by indicating an alternative to ensure high lipid quality in fish farmed with more vegetable diets.

Claudia said "This TNA project gave me the opportunity to conduct part of my PhD research in a renowned research facility with expertise in the field of fish nutrition and fatty acids. I got the opportunity to enrich my work with knowledge about a species foreign to myself. Furthermore, the TNA project enabled me to make new contacts with international researchers and to profit from their networks".

Public results:

Peer-Reviewed Publication:

Torno, C., Staats, S., Michl, S.C., Izquierdo, M., de Pascual-Teresa, S., Rimbach, G., Schulz, C., (2018): Fatty acid composition and selected gene-expression in gilthead sea bream (Sparus aurata) is affected by low-fish oil diets, dietary resveratrol, and holding temperature. Marine Drugs, 16, 379. doi:10.3390/md16100379

Torno, C., Staats, S., Fickler, A., de Pascual-Teresa, S., Izquierdo, M., Rimbach, G., Schulz, C., (2018): Combined effects of nutritional, biochemical and environmental stimuli on growth performance and fatty acid composition of gilthead sea bream (Sparus aurata). Submitted to peer-reviewed Journal

Dissertation: Torno, Claudia: Potential of resveratrol and genistein to influence marine and freshwater fish fatty acid metabolism and performance. urn:nbn:de:gbv:8-diss-238213, https://macau.uni-kiel.de/receive/dissertation_diss_00023821

Presentation: Torno, Claudia: Does the bioactive resveratrol modulate the fatty acid composition of gilthead sea bream (Sparus aurata)?, 18^th International Symposium on Fish Nutrition and Feeding (ISFNF) 2018, Las Palmas de Gran Canaria, Spain, 4^th June 2018

Michela Massimo carried her TNA research at the University of Stirlings Institute of aquaculture (UoS - IoA)

Summary: The research investigated the defence mechanisms adopted by European sea bass (ESB) against the protozoan parasite Amyloodinium ocellatum (AO). To achieve this, an expression fluorescent in situ hybridization (FISH) protocol was developed at the Institute of Aquaculture of the University of Stirling. Two riboprobes were designed for Chemokine CC1 and Hepcidin2 transcripts. To support FISH results, qPCR was previously performed to evaluate the expression levels of the two transcripts in ESB gill tissue from uninfected and infected fish. mRNA in situ hybridization contributed to localise, in the gills, the tissue-spatial distribution of leukocytes expressing Chemokine CC1 emphasising the different localisation of the transcript between control and infected fish and, in the latter, highlighting the host-parasite interactions. Instead, no signal was observed on any of the gill slides incubated with Hepcidin2 probe.

In addition, confocal investigations were carried out to deepen the understanding on the host parasite relationship. Gill tissue samples from AO infected ESB were stained with different fluorescent stains (Calcofluor white; Propidium iodide; DAPI; TRITC-Phalloidin; WGA and WGA+Rhodamine lectins) and visualized under a confocal laser scanning microscope. The objective of the study was to better detail the anatomy of A. ocellatum trophonts (the parasitic stage of the protozoan) through an alternative approach.

Michela said of she was very satisfied with her TNA experience "At the Institute of Aquaculture, the intellectual environment was extremely stimulating, scientific and technical support were excellent and all the laboratories were well-organised. I could learn very interesting and innovative techniques that helped me in better understanding the mechanisms underlying Amyloodinium ocellatum and host relationship, which is the central core of my PhD thesis. Therefore, the results obtained by this project contributed to implement my doctoral thesis."

For more information  contact Michela at massimo.michela@spes.uniud.it

Dr Jovanka Lukic, Institute of Molecular Genetics and Genetic Engineering (IMGGE), University of Belgrade (UB), Serbia led this TNA project which was a continuation of previous TNA with further optimization and refinement of analyses. National Agricultural Research and Innovation Centre, Research Institute for Fisheries and Aquaculture (NAIK-HAKI), Hungary, was the host Institute.

Summary: Research performed in this TNA project aimed to alleviate growth retardation associated with the process of rapid weaning in pike-perch intensive culture by application of probiotic bacteria. Probiotics were used for modification of nutritive value of live food and commercial dry feed, with emphasis on amino acid and fatty acid availability. Administration of probiotic-modified feed prevented the growth of potentially pathogenic bacteria and improved digestion efficacy and skeletal differentiation in rapidly weaned larvae. We expect this research to be accompanied by further studies aiming to facilitate the rapid weaning process in pike-perch and achieve maximal economic sustainability of intensive wide-scale pike-perch production.

Jovanka said "This TNA project, along with the previous TNA project, which was realized during 2016 (LAPPAQ, AE010001), directed my research from basic to applicative science. Furthermore, access to NAIK-HAKI, Hungary, has enabled me to do a kind of research that I would not have the opportunity to perform in my country".

There were two events where the project was promoted to wider civic society:

• TV series “Science to the Economy”, by Institut za usmeravanje komunikacija (INUKOM) (transl. Institute for direction of communications), Serbia. We filmed a short narrative and demonstrative presentation of our research in the Institute of molecular genetics and genetic engineering (IMGGE) in Belgrade, Serbia (from 15:36 to 23:41 minute at https://www.youtube.com/watch?v=hzbyiDeUO3w), which was presented on national TV channel). This TV series is intended primarily for entrepreneurs who aim to improve their business by introducing innovations from science. It was filmed only on Serbian language, but, given the opportunity next spring, there are plans  to film another presentation in NAIKHAKI in English.
• Participation at the Researchers’ Night 2018, Serbia presenting this project in a way that is understandable and interesting to ordinary people, especially children. There was also one video conference during manifestation where the project was presented to citizens in Slovenia. See photos showing people watching the demonstration of probiotic application to fish.  Photos were shot by SCIMFONICOM 20182019.

Three manuscripts with results of this project have been submitted to international journals. 

Lukic, J., Vukotic, G., Stanisavljevic, N., Kosanovic, D., Molnar, Z., Begovic, J., Terzic-Vidojevic, A., Jeney, G., Ljubobratovic, U. (2019). Solid state treatment with Lactobacillus paracasei subsp. paracasei BGHN14 and Lactobacillus rhamnosus BGT10 improves nutrient bioavailability in granular fish feed. PLoS ONE 14(7): e0219558. https://doi.org/10.1371/journal.pone.0219558

Ljubobratovic, U., Kosanovic, D., Demény, F.Z., Krajcsovics, A., Vukotic, G., Stanisavljevic, N., Golic, N., Jeney, G., Lukic, J. (2020). The effect of live and inert feed treatment with lactobacilli on weaning success in intensively reared pike-perch larvae. Aquaculture 516:734608. https://doi.org/10.1016/j.aquaculture.2019.734608

Lukic, J., Stanisavljevic, N., Vukotic, G., Kosanovic, D., Terzic-Vidojevic, A., Begovic, J., Golic, N., Jeney, G., Ljubobratovic, U. (2020). Lactobacillus salivarius BGHO1 and Lactobacillus reuteri BGGO6-55 modify nutritive profile of Artemia franciscana nauplii in a strain ratio, dose and application timing-dependent manner. Anim. Feed Sci. Tech 2259:114356. https://doi.org/10.1016/j.anifeedsci.2019.114356

Ljubobratović, U., Fazekas, G, Koljukaj, A., Ristović, T., Vass, V., Ardó, L., Stanisavljević, N., Vukotić, G., Pešić, M., Milinčić, D., Kostić, A., Lukić, J., (2021). Pike-perch larvae growth in response to administration of lactobacilli-enriched inert feed during first feeding. Aquaculture 542: 736901. https://doi.org/10.1016/j.aquaculture.2021.736901

Additional publications Dr. Jovanka Lukic has been involved in through the AQUAEXCEL²⁰²⁰ TNA projects:

Ljubobratovic U, Kosanovic D, Vukotic G, Molnar Z, Stanisavljevic N, Ristovic T, Peter G, Lukic J, Jeney G (2017). Supplementation of lactobacilli improves growth, regulates microbiota composition and suppresses skeletal anomalies in juvenile pike-perch (Sander lucioperca) reared in recirculating aquaculture system (RAS): a pilot study. Research in Veterinary Science. 115: 451-462. DOI: https://doi.org/10.1016/j.rvsc.2017.07.018

For more information contact Dr Jovanka Lukic, PhD, IMGGE, UB, Serbia - lukicjovanka@imgge.bg.ac.rs

Jinfeng Pan carried out this TNA research at JU-IAPW 

Summary: Carps enriched in n-3 HUFA produced in the Czech Republic have showed benefits on human plasma lipids improvement. Fish muscle freshness, denoted by K value, the ratio between the sum of inosine and hypoxanthine to the sum of all other ATP breakdown products, is important for consumers. If the healthy n-3 HUFA will affect the freshness and quality stability of carp is of interest. This project aims to give a comprehensive consideration for this healthy fatty acids’ effect on fish quality.

To date the results have still not been published as there is still analyses are taking place. 

Jinfeng wrote this about his experience of the TNA program "Thanks to the support by TNA, October 2018, I got the chance to work for 4 weeks at JU-IAPW, South Bohemia University, Czech, the only country keeping large scale freshwater fish farming and processing, and learn a lot about their traditional carp food. China is the biggest producer for fresh water fish, the information and knowledge about carp obtained in the Czech Republic broadened my horizons. More important, this project helped us find our common interests on fresh water fish. It provides great potential opportunities for the further collaboration on fish quality, product development and fish benefits for human being between my working unit in China and JU-IAPW".

For more information about this project contact Jinfeng Pan at pjf613@163.com or Jan Mraz of the JU-IAPW, South Bohemia University at jmraz@frov.jcu.cz

Douglas Tocher with the help of colleagues at the University of Stirling (Monica Betancor, Matthew Sprague and Gong Xu) led a TNA project at The Institute of Marine Research, Norway, Environmental lab installation (IMR-ELI). 

Summary: Omega-3 are both beneficial and essential in our diet and we can only get them from fish and seafood. Nowadays more than half of all fish and seafood is actually farmed. The ENDOPUFA project has told us how much of this beneficial omega- 3 can be made by Atlantic salmon when they are farmed on sustainable feeds that do not contain any marine-derived ingredients. Samples from this research are still being analysed and data are still being accumulated.

Douglas said "The TNA enabled us to collaborate with researchers in Norway also interested in omega-3 and take advantage of their facilities".

For more information contact Douglas at d.r.tocher@stir.ac.uk

Francesc Piferrer from CSIC carried out this TNA project in co-operation with INRA-IFREMER . The project was a study of the integration of genomic and environmental information by epigenetic mechanisms to bring about the sexual phenotype.

The results of this project were published in a journal article which can be found here - Dafni Anastasiadi, Marc Vandeputte, Núria Sánchez-Baizán, François Allal & Francesc Piferrer (2018): Dynamic epimarks in sex-related genes predict gonad phenotype in the European sea bass, a fish with mixed genetic and environmental sex determination, Epigenetics, DOI: 10.1080/15592294.2018.1529504

Francesc said that the "TNA  allowed the cooperation between two labs with different but complementary expertise. Our lab, at the Institute of Marine Sciences (CSIC) in Barcelona, with expertise on epigenetics, and the lab at Palavas-les-Flots (INRA-IFREMER) with expertise on genetics, working together to better understand how sex ratios are established in the European sea bass and to devise methods to bring them under our control for the benefit of the aquaculture industry".

For more information contact:

Francesc Piferrer -  piferrer@icm.csic.es

Marc Vandeputte - marc.vandeputte@inra.fr

Marta Ribeiro Carvalho carried out this TNA project at University of Las Palmas de Gran Canaria, Feed Ingredients and Additives Testing Unit (ULPGC-FITU)

Summary: Meagre (Argyrosomus regius) is a promising new aquaculture species, but there are still some bottlenecks to high-scale commercial production. One of the constraints is the scarce information about its  nutritional requirements, which means the production of optimized diets is difficult and can lead to pathological alterations such as hepatic steatosis and hepatic granulomatosis.  Essential fatty acids are nutrients required to sustain growth and vital functions along with maintaining immune status and disease resistance. In this project, Matha evaluated the depletion of dietary n-3 LC-PUFA levels by increasing the inclusion of VO in diets, and how this affected liver health status of meagre juveniles. The results of the project described a higher and severe hepatic steatosis and a higher incidence of hepatic granulomas in Meagre fed the lowest n-3 LC-PUFA diets. These achievements suggest that the inclusion of vegetable oils in diets for Meagre juveniles should be taken with careful, guaranteeing that 2.0 % DM of n-3 LC-PUFA is dietary available, in order to maintain the normal hepatic function in Meagre.

Matha wrote of the TNA program "The possibility to carry out this TNA project gave me a great understanding of fatty acids metabolism and hepatic function and pathology of teleost fish.  Furthermore, it also gave me a wide variety of transferable work skills, such as autonomy, responsibility, accuracy, organization and team, very important for my future. Finally, this TNA project opened a job opportunity for me".

The results of this TNA project were presented at 18^th International Symposium of Fish Nutrition and Feeding (June 2018) and were recently submitted to an international scientific journal for their public publication, which is under review at this time.

For more information contact Marta at martaribeirocarvalho27@gmail.com

Fazel Abdolahpur Monikh carried out this TNA project at The Institute of complex systems (ICS), part of the University of South Bohemia (JU-ICS)

Summary: Aquaculture is a rapidly growing industry with a requirement for sustainable and novel feed ingredients to decrease or substitute fish meal and fish oil applications. Selenium (Se) ENPs are promising smart materials as nutritional supplements. The use of Se ENPs as nutritional supplements in fish diet is however limited due to their unknown toxicity profile. The objective of this project was to investigate the influence of Se ENPs on swimming behavior of zebrafish (Danio rerio) upon food exposure when long term exposure occurs using a 3D monitoring system for fish detection and trajectory analysis. The aim was to provide data and novel approaches to support application of Se ENPs as nutritional supplements in fish diet at safe concentrations and size in order to substitute fish meal and fish oil applications

Fazel said the TNA not only provided him with an opportunity to increase and integrate his knowledge in nanoscience, environmental science and aquaculture, but also to develop his network and other transferable skills. Most of all, he said  "the project allowed me to develop my idea and test it practically in collaboration with two relevant infrastructures". This provided the opportunity for Fazel and his team to use the JU-ICS infrastructures knowledge, experience  and tools to take their research a step further.

The results will be available in 2019

Fazel would like to thank the Research Group of Molecular Biology and Nanomedicine, Department of Chemistry and Biochemistry, Mendel University in Brno, Czech Republic for the photo provided here.

For more information contact Fazel at f.a.monikh@gmail.com

Dr Jovanka Lukic, Institute of Molecular Genetics and Genetic Engineering (IMGGE), University of Belgrade (UB), Serbia led this TNA project. National Agricultural Research and Innovation Centre, Research Institute for Fisheries and Aquaculture (NAIK-HAKI), Hungary, was the host Institute

Summary: A growing concern faced by pike-perch breeders is high stress sensitivity of pike-perch to human handling and crowding. This is of special importance during early weaning, due to poor development of the digestive tract in larval fish, which complicates the acceptance of a formulated diet. This, in concert with low stress resistance, deleteriously affects fish survival, growth, skeletal development and immune defence, the latter leading to overgrowth of potentially pathogenic bacteria on fish mucosal surfaces. Lactobacilli are beneficial bacteria widely used as probiotics for humans and other animals, and although there are numerous reports of their successful use in aquaculture, no such beneficial application has been previously reported for pike-perch larviculture.

The TNA programme allowed the set up of a new project, in collaboration between NAIK-HAKI, the Institute of Molecular Genetics and Genetic Engineering (IMGGE) at the University of Belgrade (UOB) and the Institute for Virology, Vaccine and Sera “Torlak” in Serbia. This study concludes that the addition of lactic acid bacteria (LAB) significantly improved skeletal development and protein utilisation efficacy in pike-perch, the latter being an indicator of improved fish growth and improved nutritive quality of reared fish. Additionally, when lactobacilli were supplemented via Artemia, fish growth was also boosted. The supplementation via a formulated diet on the other hand reduced the level of pathogenic microbiota, which might substantially lower the incidence of infection outbreaks later in life.

As a continuation to this study, further optimisation of the lactobacilli supplementation regime has been performed through another TNA

Related publication: Ljubobratovic U, Kosanovic D, Vukotic G, Molnar Z, Stanisavljevic N, Ristovic T, Peter G, Lukic J, Jeney G (2017). Supplementation of lactobacilli improves growth, regulates microbiota composition and suppresses skeletal anomalies in juvenile pike-perch (Sander lucioperca) reared in recirculating aquaculture system (RAS): A pilot study. Research in Veterinary Science, 115, 451-462. DOI:10.1016/j. rvsc.2017.07.018. The audio presentation featuring the results of this is available at the following link: bit.ly/2jIN4Se2

For more information contact Dr Jovanka Lukic, PhD, IMGGE, UB, Serbia - lukicjovanka@imgge.bg.ac.rs

During 2017, the analytical platform of the Spanish National Research Council (CSIC) at the Institute of Aquaculture Torre de la Sal (IATS-ANA) hosted three AQUAEXCEL2020 Transnational Access (TNA) projects. They covered different topics of interest for three important farmed fish: Atlantic salmon, gilthead sea bream and European sea bass. The IATSANA platform, managed by the Nutrigenomics and Fish
Endocrinology Group of IATS (www.nutrigroup-iats.org), provides simultaneous gene expression profiling of candidate genes and takes advantage of reliable and curated genetic information for the three fish species.

PhD student Mar Marcos-López (Galway-Mayo Institute of Technology, Ireland) used the platform to assess the effects of experimental and natural infections with Neoparamoeba perurans, a primary cause of amoebic gill disease (AGD) in the gills of Atlantic salmon. The study highlighted an expression increase of genes related to mucus production and a decreased expression of those genes related to immune responses in the gills of AGD-affected fish. The same analysis demonstrated the effectiveness of freshwater treatment against the parasite, with a restoration of the host’s immune, mucosal, structural and anti-oxidant status.

PhD student Isabel Forner-Piquer (Marche Polytechnic University, Ancona, Italy) assessed the effects of two contaminants from plastic manufacturing, Bisphenol A and Diisononyl-phthalate (DiNp), on the metabolism of gilthead sea bream. Her research examined genes associated with obesity, reproductive capacity and central nervous system functioning to evaluate the effects of these contaminants in gonads, the brain and the liver. Initial results have highlighted the adverse effects of these contaminants on the brain’s regulation of appetite, and a stimulation of gene expression on central nervous system components that increased lipid accumulation in the livers of gilthead sea bream.

PhD student Renato Ferraz (CIIMAR, Portugal) addressed the effects of dietary seaweed (Gracilaria sp.) supplementation on the response of European sea bass to a bacterial challenge involving lipid metabolism, mitochondrial function, oxidative stress and immune response, amongst other processes.

Results on immune tissues (head kidney and spleen) evidenced the activation of the immune system in Gracilaria supplemented feeds and a better immune profile when facing a bacterial infection. Additionally, dietary Gracilaria induced adaptive responses in the liver which reduced the risk of oxidative stress. The results support the use of seaweeds as promising fish feed additives. The IATS-ANA platform has thus been revealed as a versatile molecular tool for the assessment of fish health and welfare in a wide range of experimental models and processes for European farmed fish.

Catarina Moreira, a PhD student from Normandy University, France used IATS-CSIC facilities in her Transnational Access (TNA) research. She presented a poster on the results of her TNA Project at the 31st Congress of the European Society of Comparative Physiology and Biochemistry held on the 9-12 September 2018 in Porto, Portugal.

Summary: The study entitled “Effects of Xenoestrogen exposure during immune system ontogenesis of the European sea bass, Dicentrarchus labrax” is a collaborative and multidisciplinary work involving several research groups from IATS (Fish Pathology group and Live preys in aquaculture, larviculture and ecotoxicology group), Centro de s. Ciências do Mar, Universidade do Algarve (Portugal) and the Department of Microbiology and Ecology, University of Valencia (Spain). This proved a challenging TNA project, since it involved the use of different IATS units and required fish being reared from very early stages until they reached the adequate size for the bacterial challenge. However, it was also a great opportunity for collaboration between the groups with expertise available including pathology, microbiology, immunology, toxicology, ontogeny, etc.

Oestrogens, such as Oestradiol, are well-known endocrine disruptors present in natural forms in fish farms because non-degraded hormones are released by the fish into the water body. Oestradiol is involved in the regulation of numerous physiological functions, including the modulation of immune system function and performance. As a result, this project explored the long-term consequences of exposure to environmentally relevant concentrations of oestradiol on fish immunocompetence.

European sea bass of 47 and 60 days of age (post-hatch) were exposed to 20 ng/L of oestradiol for 7 days and one month, respectively, in the ecotoxicology facilities of the IATS. Following each exposure, fish were sampled to assess the impacts of oestradiol on the ontogenesis of the thymus, the primary lymphoid organ responsible for lymphocyte T cell differentiation important in immunity. After a depuration phase, fish were transferred to the fish pathology unit of IATS and intracoelomically injected with Vibrio harveyi, and the cumulative mortality was recorded. This pathogen test was performed to determine if the previous exposure to oestradiol at different windows of the development of the fish was affecting their capacity to fight bacterial infection.

From the results of the bacterial test, it seems that oestradiol has immunostimulating or immunosuppressive effects depending on the critical window of development. The relationship between these immunomodulatory effects, thymus development and associated peripheral T cells is currently being further investigated by gene expression and immunohistochemistry.

Host infrastructure: NAIK-SDC, Hungarian University of Agricultural and Life Sciences (MATE), Hungary

Users of infrastructure: Jovanka Lukić, lead researcher (Institute of Molecular Genetics and Genetic Engineering (IMGGE), University of Belgrade, Serbia) & Alan Koljukaj (“Srbijašume”, Serbia)

TNA project PROFEE (2019-2020)

Successful as usual, the pike-perch team produced great results again, after our previous two TNA projects, LAPPAQ and LABRAWEAN. This trial was the first of its kind to test the effect of probiotic supplemented inert feed on the growth of first feeding pike perch larvae. In comparison to our previous studies, which were based on use of live probiotics, we now coated inert larval feed with lysed probiotics. We demonstrated that lysed probiotics can act as a source of Glycine, which is an appetite stimulating and growth promoting amino acid. Lysed probiotics have also showed the ability to reduce diffusion of water soluble proteins, presumably due to electrostatic binding with lipids from fragmented probiotic membranes. Availability of protein bound (hydroxyl-)Pro from feed proteins was also increased in feed coated with probiotic homogenates, most probably because of the activation of trypsin by lipid bilayers from fragmented probiotic membranes.

These in vitro findings were correlated with fish performance in large scale in vivo trial performed in recirculation facilities of MATE. This study additionally demonstrated that first feeding pike perch larvae can be successfully weaned with the aid of probiotics, which overcome the amino acid shortage present in inert feed. Due to the presence of appetite stimulating amino acids in probiotic coated feed, feed consumption was increased in this experimental group, which suppressed trypsin activity in larval digestive tract, presumably facilitating nutrient absorption via cholecystokinin (CCK) pathway. This interesting finding opened the door for additional research on the involvement of CCK pathway in beneficial effects of probiotics. Aside from further knowledge development in the area of fish nutrition and larval development, these results paved the way for industrial application of probiotics in pike-perch larviculture. However, in order to make our results industrially applicable, further elaboration of feed coating procedure, as well as testing in industrially relevant environment, is needed. Our latest TNA project (PROBASSIC), which has just begun, is expected to make this connection with industry, building on the results of PROFEE and our previous TNAs.

Photo 1. PROFEE team (Tijana Ristović, Vivien Vass and Alan Koljukaj, from left to right), working in research facilities of MATE (by Uros Ljubobratovic)

Two manuscripts in peer-reviewed journals, featuring above presented results, have been published:

1. Lukić, J., Stanisavljević, N., Vukotić, G., Terzić-Vidojević, A., Begović, J., Golić, N., Ljubobratović, U., 2021. Enrichment of larval fish feed with free amino acids and proteins by coating with Lactobacillus paracasei subsp. paracasei BGHN14 homogenate. Turk. J. Fish. & Aquat. Sci. 21: 569-573. http://doi.org/10.4194/1303-2712-v21_11_05

2. Ljubobratović, U., Fazekas, G, Koljukaj, A., Ristović, T., Vass, V., Ardó, L., Stanisavljević, N., Vukotić, G., Pešić, M., Milinčić, D., Kostić, A., Lukić, J., 2021. Pike-perch larvae growth in response to administration of lactobacilli-enriched inert feed during first feeding. Aquaculture 542: 736901. https://doi.org/10.1016/j.aquaculture.2021.736901