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Projects / Programmes source: ARRS

Establishment of bee cell lines and standardization of in vitro assays for evaluation of harmful effects caused by toxins and pathogens in bees

Research activity

Code Science Field Subfield
4.02.00  Biotechnical sciences  Animal production   

Code Science Field
B400  Biomedical sciences  Zootechny, animal husbandry, breeding 

Code Science Field
4.02  Agricultural and Veterinary Sciences  Animal and Dairy science 
Keywords
bees, bee cell line, chemicals, methabolic disturbance, gene expression
Evaluation (rules)
source: COBISS
Researchers (16)
no. Code Name and surname Research area Role Period No. of publications
1.  07914  PhD Dušan Benčina  Veterinarian medicine  Researcher  2014 - 2016  223 
2.  33147  PhD Luka Bolha  Biochemistry and molecular biology  Researcher  2016  24 
3.  11169  PhD Janko Božič  Biology  Researcher  2013 - 2016  316 
4.  29430  PhD Ivanka Cizelj  Biotechnology  Researcher  2013 - 2015  51 
5.  34506  PhD Manuela Čitar  Microbiology and immunology  Researcher  2016  19 
6.  05098  PhD Peter Dovč  Biotechnology  Researcher  2013 - 2016  908 
7.  25530  PhD Petra Draškovič  Biochemistry and molecular biology  Researcher  2013 - 2014  59 
8.  18324  PhD Gordana Glavan  Neurobiology  Researcher  2013 - 2016  140 
9.  24769  PhD Gregor Gorjanc  Animal production  Researcher  2013 - 2016  473 
10.  10448  PhD Aleš Gregorc  Animal production  Researcher  2014 - 2016  489 
11.  06537  PhD Dragomir Kompan  Animal production  Researcher  2013 - 2016  740 
12.  24503  PhD Peter Kozmus  Biology  Researcher  2013  312 
13.  05008  PhD Mojca Narat  Biotechnology  Principal Researcher  2013 - 2016  675 
14.  24298  PhD Irena Oven  Animal production  Researcher  2013 - 2016  81 
15.  27547  PhD Zala Prevoršek  Animal production  Researcher  2013 - 2016  40 
16.  19626  Rok Štravs  Biotechnology  Researcher  2015 - 2016  17 
Organisations (3)
no. Code Research organisation City Registration number No. of publications
1.  0158  BIA, Laboratory and process equipment company, Ltd.  Ljubljana  5327601  17 
2.  0401  Agricultural institute of Slovenia  Ljubljana  5055431  19,438 
3.  0481  University of Ljubljana, Biotechnical Faculty  Ljubljana  1626914  64,492 
Abstract
Background: In vitro cultivation of cells is an essential technology, which enables learning about mechanisms that are present in vivo. Numerous cytotoxicity assays have been developed using cell cultures, allowing cheaper, faster and more standardized testing, which complies with the principle of 3Rs. Slovenia is encountering a similar problem of unexplained bee deaths as the rest of the world. Possible explanations take into account the impact of bee pathogens and chemicals: protective agricultural agents (FFS), synthetic pesticides, and environmental pollutants. Bees have a crucial role in the ecosystem since they participate in pollination, an important factor in the production of food. In order to solve the “bee problem” successfully and in time, we first need to understand the causes. Disturbances in metabolic pathways and the molecular mechanisms of activity of chemicals and pathogens on a cellular and genetic level need to be studied with an appropriate methodology. Hence, establishing bee cell lines is the first step in research and designing standardized in vitro assays. Problem: Intensive agriculture depends heavily on the use of FFS that can in certain conditions have negative effects even on non-target organisms, including bees. There is no information available about the effects of long-term exposure of bees to low doses of presumably harmless chemicals, the effects of successive or simultaneous exposure to multiple chemicals. The European committee encourages all EU members to perform extensive laboratory research in the field of bee protection and conservation. European researchers have recognized the problem of lack of transformed cell lines that would enable performing repeatable experiments in controlled conditions. During this project, we will systematically study the possibilities for establishing cell cultures from different bee organs. The novelty of our approach lies in the use of phenotype microarrays for the optimization of culturing media, which has so far been the limiting factor in bee cell cultivation. Continuous cell lines will be established using lentivirus vector for transduction of cells with oncogenes. The effects of certain chemicals on the cellular level and on the levels of metabolism and gene expression will be tested in vitro using cell lines. Similar approach will be used to test the effects of combinations of chemicals that occur in the nature and to evaluate the cumulative effects. Goals: a) establish cell lines from different bee tissues b) optimize the growth media; c) characterize the changes in gene expression that are triggered by exposure to chemicals and/or pathogenes; d) characterize the mechanisms that lead to cell death after exposure to chemicals e) identify the changes in metabolic pathways after exposure to individual chemicals, pathogens and combinations of these; f) identify relevant changes (marker genes, metabolic pathways) and utilize them for developing an in vitro toxicity test; g) application: design a special phenotype microarray that is specific for bees and develop a test for evaluating the harmful risks of chemicals. Relevance: Establishing bee cell lines is in concordance with the directives of the EU and will contribute to solving the problem of bee killings in an international scale. The project enables collaborations with other international projects (COLOSS network, IBRA, etc.). The research will contribute to the understanding of how chemicals affect bees and enable a more realistic evaluation of the danger of using insecticides in different conditions and combinations. Qualifications: Several members of the team are already involved in studying bee killings, but for most members and the institution applying for the project, this is a new chapter in their research. However, we find this topic of essential importance and we believe that we are qualified to perform it. For all methods proposed, we have the equipment and the references.
Significance for science
We have established several types of continuous honeybee cell lines, which is in concordance with the EU guidelines and directives and represents a significant contribution in the research field as well as contributes to the solution of the problems of bee killings. These solutions are offered on a (sub)cellular level with the use of cell cultures. We have established standardized protocols for isolation and preparation of bee cell cultures from various tissues and developmental stages and therefore systematically approached to the establishment of bee cell lines from different tissues and medium optimization. We have in preparation an article for publication in an international peer reviewed journal, which will enable establishment of bee cell cultures to other research groups. Presentation of our results at the international scientific meeting EurBee 2016 has gained a lot of interest since many groups have been searching for an appropriate in vitro model system for years now, and based on this we have already established some new international collaborations. Even though we did not yet succeed to establish a continuous cell culture, we have succeeded to establish a long-term bee cell culture. This also enables a good start in implementing in vitro tests for evaluation of harmful effects of pesticides and pathogen infections on bee health, as well as enables further research on gene expression in “normal state” as well as after various treatments with chemicals and/or pathogen infections in controlled and standardized laboratory conditions, which is the key to validated tests. We have also optimized growth media for cell cultures from various tissues, which is currently the biggest obstacle in case of successful cultivation of bee cells, and therefore contributed new basic knowledge in cultivation of bee cells. With a realistic evaluation of chemicals and/or pathogen infection effects on the expression of genes in each developmental stage of bees, we have also contributed to new basic knowledge. Our results have been published in an international peer reviewed journal. With the understanding of the mechanisms underlying the changes in gene and molecular pathways expressions after treatment with chemicals and/or infections, we could implement new standards and policies in the protection of bee health. Considering that there are only a few data on how chemicals and pathogens affect bees, and that these data are obtained in different ways, non-comparable and widely dispersed, we assume that a systematic approach is of key importance and will enable a significant progress in finding a solution for bee killings. The possibility of standardized research methods of synergistic effects of chemicals, used in agriculture, bee-protective agents, and other chemicals present in the environment at the cellular level will give a significant advantage. Namely, with the simulations of controlled environment we could study the combinatorial effect of influential factors. In this aspect, we hope to achieve progress in scope of researching various influences that are important for the understanding of how this occurs in the natural environment. Additionally, evaluation of synergistic effects of multiple chemicals is very important for understanding of bee health – honeybee can fly up to 7 km in search of food where is potentially exposed to more than one pesticide. Results of our study enable a global insight into environmental impact on honeybees. The understanding of the events that occur at genetic and cellular levels is a necessary basis to plan further interventions in nature. In summary, our study has contributed new basic knowledge that is relevant both for international as well as domestic professional public. At the same time, we are now a step closer to the possibility of implementing new methods for testing potentially harmful effects of chemicals on bee health and application of basic knowledge to practice.
Significance for the country
Our group is the first one in Slovenia that succeeded in establishing long-term honeybee cell cultures and other Slovenian research groups working on bee health have already shown interest in our cell lines. Our cell cultures can be used for establishment of in vitro tests to evaluate the risk of phytopharmaceutical agents, especially in the scope of evaluating the synergistic effects between chemicals already existing in the environment and new chemicals that manufacturers wish to place on the market, which should be of interest to both our government as well as the economy. The direct impact of this project would also enable testing of new products for plant protection (insecticides, pesticides and other chemicals) at the level of cell cultures and in this way give faster results and answers and also enable the government (MKO, FURS, VURS) to either approve or deny the use of these products. This will eventually lead to the reduction of risks in this particular area. Understanding of the mechanisms of how chemicals affect bee health is the basis for solving the problem of bee killings. The long tradition of apiculture with the Carniolan bee, which has an irreplaceable role in assuring food supply for a large portion of population, we are obliged to preserve this important genetic potential in Slovenia. Carniolan bee and the apiculture also represent an important part of our natural and cultural heritage and biotic variability. Carniolan bee (Apis Mellifera Carnica) is an autochthon and protected bee species in Slovenia. We estimate the results of this project will contribute significantly to taking these measurements that will be based on concrete scientific results. At the governmental level Ministry of agriculture and the environment (MKO) and its Council for Animal husbandry, as well as a well-known bee-breeding association – Slovenian Beekeepers’ Association are interested in this research. We will report the results of the project to all parties involved, including Phytosanitary Administration of RS (FURS), because we believe that it would be of key importance to implement in the routine additional tests to evaluate the risk of chemicals (FFS). As members of the Commission for Genetic Resources for Food and Agriculture (CGRFA) and European Regional Focal Point for Genetic Resources (ERFP) we are especially involved in the activities for preservation and protection of autochthonous animal (sub)species. The results of our study have been published in international peer reviewed journals and have been presented at international scientific meetings (EUR-BEE, COLOSS), which contributes significantly to the recognition of our researchers, our research group as well as worldwide promotion of our country. Finally yet importantly, since we are included in the teaching process at Biotechnical faculty, the results of this study are also presented in seminars and in student laboratory work as well as in lectures.
Most important scientific results Annual report 2014, 2015, final report
Most important socioeconomically and culturally relevant results Annual report 2015, final report
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