Progress 10/01/12 to 09/30/17
Outputs
Target Audience:The target audience reached during this reporting period were fellow scientists involved in this field of research and novel collaborators (through scientific publications and conference presentations), K12 students and teachers (through the EnvironMentors program), the general public (through seminars and internet communications), aquaculture producers, NGOs (invasive species issue), and government organizations (in particular Cal Fish and Wildlife and US Fish and Wildlife Service). Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?The project has provided training for 6 graduate students and 6 undergraduate students in the PIs laboratory. Since funds from this prject are very limited these students have been supported from other sources. Two of the graduate students were woman from an underrepresented ethnic minority (hispanic/ latino). How have the results been disseminated to communities of interest?Tilapia are economically important as aquaculture species and invasive species. Therefore, it is critical to know how these fish deal with salinity and other environmental stress, what consequences it has on growth, longevity, reproductive success, fertility, and fecundity, and how such stress can be minimized in fish in general. This knowledge is of interest to multiple stakeholders. Results were disseminated in the form of scientific publications, via press releases, during seminars, during outreach activities (e.g. EnvironMentors), through conversations with producers and CE specialists. Results of this project are also disseminated via a SQL database that is accesible via the internet (https://kueltzlab.ucdavis.edu/CAMP/camp.cfm). What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
In this project we have taken advantage of proteomics tools developed in another project (multi-state project) that enable efficient proteomics analysis of tilapia tissues. Such tools include analytical separation strategies, biological mass spectrometry approaches, and proteomics/ bioinformatics pipleines, which form the basis of this project. The project focuses on tilapia because they are extremely stress-hardy fishes. Tilapia are also an established model for ecophysiology, evolutionary biology, toxicology, and molecular and cellular biology, in particular in relation to biological mechanisms of environmental stress adaptation. Knowledge of the physiological mechanisms of stress resistance and the biochemical networks underlying such physiological mechanisms is a prerequisite of being able to predict and minimize consequences of environmental stress. Proteomics is a field that generates such knowledge at a very fast rate and we have applied sophisticated, recently developed network modeling approaches to salinity stress responses of euryhaline fish. As part of this project we were able to accomplish development of a data-independent acquisition (DIA, SWATH) mass spectrometry assay using carefully curated target and trasnition lists for 1000 - 2000 proteins per tissue. These cDIA assays were set up for gill, brain, liver, and kidney tissue of tilapia. These cDIA assays represent consistent and standradized matrices for protein networks in which every node is measured in every sample in exactly the same way. Network analyses were done in collaboration with Professor Eivind Aalmaas' lab at the Norwegian University of Science and Technology. In addition, we have initiated collaborations with network experts (Mthemathicians) at UC Davis. We have further developed approaches for quantitative assessment of the stoichiometry of 9 different types of posttranslational protein modification (PTM) in several tilapia tissues. These PTMs are phosphorylation, acetylation, methylation, dimethylation, hydroxylation, dihydroxylation, formylation, ethylation, and ubiquitination. It will take continued work to be able to integrate these PTMs into protein regulatory networks because it is ncessary to reliably quantify both modified and unmodified forms of the corresponding peptide to be able to quantify PTM stoichiometry. Tilapia that were acclimated to different salinities have been analyzed for the stoichiometry of these PTMs in a tissue-specific manner to identfy posttranslational mechanisms of salinity stress responses. Moroever, we were able to identify single amino acid variants that represent allelic variation in tilapia populations and are now beginning to apply this novel tecnhique to identify protein isoforms with superior function during salinity stress and other types of stress. In addition, we were able to compare salinity and growth responses of euryhaline Mozasmbique tilapia with a stenohaline strain of Nile tilapia and stenohaline Astatotilapia. Acclimation experiments that enable such comparisons have been designed and conducted to enable identification of protein network nodes and sub-networks that are associated with rapid growth and stress tolerance. These comparisons have been done in collabopration with Drs. Jean-Francios Baroiller and Helene D'Cotta at CIRAD (Montpellier, France) and with Dr. Avner Cnaani at Volcani Center in Bet-Dagan (Israel).
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2017
Citation:
Osmolality/salinity-responsive enhancers (OSREs) control induction of osmoprotective genes in euryhaline fish
X Wang, D K�ltz
Proceedings of the National Academy of Sciences, 201614712
- Type:
Journal Articles
Status:
Published
Year Published:
2017
Citation:
Tgm1-like transglutaminases in tilapia (Oreochromis mossambicus)
SIR Cruz, MA Phillips, D K�ltz, RH Rice
PloS one 12 (5), e0177016
- Type:
Journal Articles
Status:
Published
Year Published:
2017
Citation:
Skeletal stiffening in an amphibious fish out of water is a response to increased body weight
AJ Turko, D K�ltz, D Fudge, RP Croll, FM Smith, MR Stoyek, PA Wright
Journal of Experimental Biology 220 (20), 3621-3631
|
Progress 10/01/15 to 09/30/16
Outputs
Target Audience:The target audience reached during this reporting period were fellow scientists involved in this field of research and novel collaborators (through scientific publications and conference presentations), K12 students and teachers (through the EnvironMentors program), the general public (through seminars and internet communications), aquaculture producers, NGOs (invasive species issue), and government organizations (in particular Cal Fish and Wildlife and US Fish and Wildlife Service). Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?The project has provided training for 6 graduate students and 3 undergraduate students in the PIs laboratory. Since funds from NIFA are very limited these students have been supported from other sources. Two of the graduate students were woman from an underrepresented ethnic minority (hispanic/ latino) How have the results been disseminated to communities of interest?Tilapia are economically important as aquaculture species and invasive species. Therefore, it is important to know how these fish deal with salinity and other environmental stress, what consequences it has on growth, longevity, reproductive success, fertility, and fecundity, and how such stress can be minimized in fish in general. This knowledge is of interest to multiple stakeholders. Results were disseminated in the form of scientific publications, via press releases, during seminars, during outreach activities (e.g. EnvironMentors), through conversations with producers and CE specialists. Results of this project are also disseminated via a SQL database that is accesible via the internet (https://kueltzlab.ucdavis.edu/CAMP_dda.cfm). What do you plan to do during the next reporting period to accomplish the goals?We are continuing to explore comparisons of different euryhaline fish species and different tilapia hybrids and natural populations that have different stress tolerance. Top that end we have established in this reporting period collaboprations with Drs. Jean-Francios Baroiller and Helene D'Cotta at CIRAD (Montpellier, France) and with Dr. Avner Cnaani at Volcani Center in Bet-Dagan (Israel). In addition, one of the PIs graduate students, Mr. Larken Root, was in Africa for 3 months during the summer to establish collaborations with researchers in Zambia, Burkina Faso, and Tanzania. These collaborators have excellent access and experience to differently stress-tolerant tilapia strains. We also plan on continuing comparisons of salinity stress responses of Salton Sea tilapia with an aquaculture population of tilapia grown in freshwater at Pacific Aquafarms in Niland, CA. Proteomics workflows and approaches developed in another project (Multistate project) will be utilized for these comparisons. In addition to assessing salinity effects on protein abundances and PTMs and modeling dynamic regulation of the corresponding networks, these comparisons will also focus on identifying specific single amino acid variants of allelic protein isoforms that differ most in frequency among the most stress-tolerant and more stress-sensitive tilapia strains. Identification of such protein coding sequence variation will be validated using genomic DNA sequencing of a suitable number of individuals to enable future analyses directed at assessing the adaptive value for salinity stress tolerance in selected proteins. These laborious studioes are already under way and will be continued for the next period.
Impacts
What was accomplished under these goals?
In this project we have taken advantage of proteomics tools developed in another project (multi-state project) that enable efficient proteomics analysis of teleost tissues, mostly from tilapia but also from other euryhaline fish such as sticklebacks. By comparing multiple species it is possible to dissect evolutionarily conserved mechanisms of osmoregulation and osmosensing. The project focuses on tilapia because they are extremely stress-hardy fishes but sticklebacks are also very euryhlaine and environmentally stress-tolerant. Both species complexes are established models for ecophysiology, evolutionary biology, toxicology, and molecular and cellular biology, in particular in relation to biological mechanisms of environmental stress adaptation. Knowledge of the physiological mechanisms of stress resistance and the biochemical networks underlying such physiological mechanisms is a prerequisite of being able to predict and minimize consequences of environmental stress. Proteomics is a field that generates such knowledge at a very fast rate and we have applied sophisticated, recently developed network modeling approaches to salinity stress responses of euryhaline fish. This was done in collaboration with Professor Eivind Aalmaas' lab at the Norwegian University of Science and Technology. We had started this project already last year but have since expanded its scope and utility greatly. For instance, the spectral library for tilapia gill tissue was used to generate a transition list containing more than 15000 transitions for highly accurate quantitation of more than 1000 branchial tilapia proteins. This transition list was manually curated by selecting three proteotypic peptides and 5 (of 10 initially evaluated) transitions for each peptide that give the greatest signal during SWATH-MS. We have also continued our work on identification and quantitation of posttranslational modifications. Preliminary transition lists containing modified and unmodified variants of particular peptides representing branchial proteins have been generated for tilapia gills. In addition, we have identified more single amino acid variants of tilapia and stickleback proteins using proteomics verified by DNA sequencing.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2016
Citation:
Population-specific renal proteomes of marine and freshwater three-spined sticklebacks
D K�ltz, J Li, D Paguio, T Pham, M Eidsaa, E Almaas
Journal of proteomics 135, 112-131
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2016
Citation:
Development of a reporter assay for identifying osmotically responsive enhancers in euryhaline fish
X Wang, D K�ltz
The FASEB Journal 30 (1 Supplement), 977.3-977.3
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2016
Citation:
Effects of Hyperosmotic Stress on Cellular Proteome and Phenotype of Tilapia (Oreochromis mossambicus) OmB Cells
TN Wiegers, D K�ltz
The FASEB Journal 30 (1 Supplement), 760.30-760.30
|
Progress 10/01/14 to 09/30/15
Outputs
Target Audience:The target audience reached during this reporting period were fellow scientists involved in this field of research and novel collaborators (through scientific publications and conference presentations), K12 students and teachers (through the EnvironMentors program), the general public (through seminars and internet communications), aquaculture producers, NGOs (invasive species issue), and government organizations (in particular Cal Fish and Wildlife and US Fish and Wildlife Service). Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?The project has provided training for 5 graduate students and 3 undergraduate students in the PIs laboratory. Since funds from NIFA are very limited these students have been supported from other sources. How have the results been disseminated to communities of interest?Tilapia are economically important as aquaculture species and invasive species. Therefore, it is important to know how these fish deal with salinity and other environmental stress, what consequences it has on growth, longevity, reproductive success, fertility, and fecundity, and how such stress can be minimized in fish in general. This knowledge is of interest to multiple stakeholders. Results were disseminated in the form of scientific publications, via press releases, during seminars, during outreach activities (e.g. EnvironMentors), through conversations with producers and CE specialists. Results of this project are also disseminated via a SQL database that is accesible via the internet (https://kueltzlab.ucdavis.edu/CAMP/camp.cfm). What do you plan to do during the next reporting period to accomplish the goals?We are exploring comparisons of different tilapia hybrids and natural populations that have different stress tolerance. These comparisons will be done in collabopration with Drs. Jean-Francios Baroiller and Helene D'Cotta at CIRAD (Montpellier, France) and with Dr. Avner Cnaani at Volcani Center in Bet-Dagan (Israel). These collaborators have excellent access and experience to differently stress-tolerant tilapia strains. In addition, we plan on comparing salinity stress responses of Salton Sea tilapia, which have evolved in an extremely stressful hypersaline (multistressor) environment for many decades with an aquaculture population of tilapia grown in freshwater at Pacific Aquafarms in Niland, CA. Proteomics workflows and approaches developed in another project (Multistate project) will be utilized for these comparisons. In addition, to assessing salinity effects on protein abundances and PTMs and modeling dynamic regulation of the corresponding networks, these comparisons will also focus on identifying specific single amino acid variants of allelic protein isoforms that differ most in frequency among the most stress-tolerant and more stress-sensitive tilapia strains. Identification of such protein coding sequence variation will enable future analyses directed at assessing the adaptive value for salinity stress tolerance in selected proteins.
Impacts
What was accomplished under these goals?
In this project we have taken advantage of proteomics tools developed in another project (multi-state project) that enable efficient proteomics analysis of tilapia tissues. Such tools include analytical separation strategies and bioinformatic proteomics systems, which form the basis of this project. The project focuses on tilapia because they are extremely stress-hardy fishes. They are also an established model for ecophysiology, evolutionary biology, toxicology, and molecular and cellular biology, in particular in relation to biological mechanisms of environmental stress adaptation. Knowledge of the physiological mechanisms of stress resistance and the biochemical networks underlying such physiological mechanisms is a prerequisite of being able to predict and minimize consequences of environmental stress. Proteomics is a field that generates such knowledge at a very fast rate and we have applied sophisticated, recently developed network modeling approaches to salinity stress responses of euryhaline fish. This was done in collaboration with Professor Eivind Aalmaas' lab at the Norwegian University of Science and Technology. We have further developed approaches for quantitative assessment of the stoichiometry of 9 different types of posttranslational protein modification (PTM) in several tilapia tissues. These PTMs are phosphorylation, acetylation, methylation, dimethylation, hydroxylation, dihydroxylation, formylation, ethylation, and ubiquitination. Tilapia that were acclimated to different salinities have been analyzed for the stoichiometry of these PTMs in a tissue-specific manner to identfy posttranslational mechanisms of salinity stress responses. Moroever, we were able to identify single amino acid variants that represent allelic variation in tilapia populations and are now beginning to apply this novel tecnhique to identify protein isoforms with superior function during salinity stress and other types of stress.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2015
Citation:
Physiological mechanisms used by fish to cope with salinity stress
D K�ltz
Journal of Experimental Biology 218 (12), 1907-1914
- Type:
Journal Articles
Status:
Published
Year Published:
2015
Citation:
Direct Ionic Regulation of the Activity of Myo-Inositol Biosynthesis Enzymes in Mozambique Tilapia
FD Villarreal, D K�ltz
PloS one 10 (6), e0123212
- Type:
Journal Articles
Status:
Published
Year Published:
2015
Citation:
Population-specific renal proteomes of marine and freshwater three-spined sticklebacks
D K�ltz, J Li, D Paguio, T Pham, M Eidsaa, E Almaas
Journal of proteomics
- Type:
Journal Articles
Status:
Published
Year Published:
2015
Citation:
Population?specific plasma proteomes of marine and freshwater three?spined sticklebacks (Gasterosteus aculeatus)
D K�ltz, J Li, X Zhang, F Villarreal, T Pham, D Paguio
Proteomics 15 (23-24), 3980-3992
|
Progress 10/01/13 to 09/30/14
Outputs
Target Audience: The target audience reached during this reporting period were fellow scientists involved in this field of research (through scientific publications and conference preentations), K12 students and teachers (through the AggieMentors program), the general public (through seminars and internet communications), aquaculture producers, NGOs (invasive species issue), and government organizations (in particular Cal Fish and Wildlife and US Fish and Wildlife Service). Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided? The project has provided training for 4 graduate students and 4 undergraduate students in the PIs laboratory. Since funds from NIFA are very limited these students have been supported from other sources. How have the results been disseminated to communities of interest? We have shared novel tilapia cell lines generated in the PIs laboratory with other researchers around the world. For instance, our tilapia cell lines have been shipped to research labs in Singapore and Israel and are used there to improve tilapia aquaculture by mechanistically understanding advantageous phenotypes.Results were also disseminated in the form of scientific publications, via press releases, during seminars, during outreach activities (e.g. AggieMentors), through conversations with producers and CE specialists. Results of this project are also disseminated via a SQL database that is accesible via the internet (https://kueltzlab.ucdavis.edu/CAMP/camp.cfm). What do you plan to do during the next reporting period to accomplish the goals? We plan on utilizing reverse genetic and protein purification techniques to study direct biochemical regulation of the enzymes involved in myo-inositol biosynthesis pathway. The goal is to identify whether and how these enzymes are regulated rapidly by mechanisms that do not require changes in abundance. In addition, we will start deciphering the osmosensory signal transduction pathway that leads to osmotic induction of the enzymes responsible for myo-inositol biosynthesis. This will be done by utilizing our new cell lines for enhancer trap reporter assays followed by affinity purification of transcription factor and identification via proteomics. In addition, we will utilize our label-free quantitative proteomics workflow for obtaining mechanistic insight into the biochemistry of stress responses and growth of tilapia and tilapia cell lines.
Impacts
What was accomplished under these goals?
The accomplishment for the reporting period were that environmental effects on tilapia stress tolerance, growth, health, and performance were identified and documented as planned per original project outline in the publications listed above. Please refer to details in the publicly available abstracts for those publications. The funds provided by the AES-Hatch project were essential but of supplemental nature for achieving these accomplishments. Briefly, the main accomplishment during the reporting period was that we have documented the effects of copper nanoparticles on tilapia. The dependence of such effects on the specific environmental context that tilapia are cultured in has been emphasized. In addition, we have utilized novel tilapia cell lines generated in the PIs lab to study mechanisms that underly stress responses. Quantitative proteomics was used to characterize these cell lines and their tissue-specific biomarkers. The comprehensive results of this research were made publicly available in a newly designed sql database. Mechanistic aspects underlying the induction of the myo-inositol synthesis pathway and its implications for stress responses were studied in tilapia larvae and cell lines. We found that this pathway is critical during salinity stress of tilapia and that it provides a robust platform for deciphering osmosensory signal transduction pathways in tilapia.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2014
Citation:
FD Villarreal, GK Das, A Abid, IM Kennedy, D K�ltz (2014) Sublethal effects of CuO nanoparticles on Mozambique tilapia (Oreochromis mossambicus) are modulated by environmental salinity. PloS one 9 (2), e88723.
- Type:
Journal Articles
Status:
Published
Year Published:
2014
Citation:
AM Gardell, Q Qin, RH Rice, J Li, D K�ltz (2014) Derivation and Osmotolerance Characterization of Three Immortalized Tilapia (Oreochromis mossambicus) Cell Lines. PloS one 9 (5), e95919.
- Type:
Journal Articles
Status:
Published
Year Published:
2014
Citation:
R Sacchi, AM Gardell, N Chang, D K�ltz (2014) Osmotic regulation and tissue localization of the myo?inositol biosynthesis pathway in tilapia (Oreochromis mossambicus) larvae. Journal of Experimental Zoology Part A: Ecological Genetics and Physiology 321 (8), 457-466.
|
Progress 01/01/13 to 09/30/13
Outputs
Target Audience: The target audience reached during this reporting period were fellow scientists involved in this field of research (through scientific publications and conference preentations), K12 students and teachers (through the AggieMentors program), the general public (through seminars and internet communications), aquaculture producers, NGOs (invasive species issue), and government organizations (in particular Cal Fish and Wildlife and US Fish and Wildlife Service). Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided? The limited amount of funds provided by this particular project were sufficient to pay for a research assistentship (and associated laboratory/ experimental costs) and provide an opportunity for one undergraduate student (through internship) to participate in our tilapia stress response project. How have the results been disseminated to communities of interest? The target audience reached during this reporting period were fellow scientists involved in this field of research (through scientific publications and conference preentations), K12 students and teachers (through the AggieMentors program), the general public (through seminars and internet communications), aquaculture producers, NGOs (invasive species issue), and government organizations (in particular Cal Fish and Wildlife and US Fish and Wildlife Service). What do you plan to do during the next reporting period to accomplish the goals? During the next reporting period we are planning to provide an opportunity for one undergraduate student to participate in our tilapia stress response project to advance to goals of this AES-Hatch project. Specifically, this student will work with other members of the Kueltz Lab to maintain tilapia cell lines and conduct osmotic stress response experiments with those cell lines and with whole animals to determine which molecular salinity stress responses of tilapia can be reproduced with the immortalized cell lines. This work will represent the basis for validating these cell lines as physiologically relevant models for mechanistically studying tilapia salinity stress responses (e.g. by emplying reverse genetics and high throughput quantitative molecular phenotyping by proteomics).
Impacts
What was accomplished under these goals?
The accomplishment for the reporting period were that salinity effects on tilapia were identified and documented as planned per original project outline in the publications listed above. Please refer to details in the publicly available abstracts for those publications. The funds provided by the AES-Hatch project were essential but of supplemental nature for achieving these accomplishments. Briefly, the main accomplishment during the reporting period was that we have established, documented, and validated, a system for mechanistically studying the basis for tilapia stress (salinity) tolerance. This system is described in the publications listed above. Its main components include enzymes of the myo-inositol biosynthesis pathway that are highly induced at mRNA and protein levels and the corresponding metabolite, newly immortalized tilapia cell lines, and recombinant tagged proteins that can be expressed in those cell lines. These components will be used to study how tilapia sense, transduce, and respond to environmental (salinity) stress and to model the underlying mechanisms and the logic of information flow.
Publications
- Type:
Book Chapters
Status:
Published
Year Published:
2013
Citation:
D K�ltz (2013) Osmosensing. In: Euryhaline Fishes (Fish Physiology Series, eds. McCormick,S.D., Farrell, A.P., Brauner, C.J.), vol 32, pp. 45-68, Elsevier / Academic Press (Amsterdam).
- Type:
Journal Articles
Status:
Published
Year Published:
2013
Citation:
D K�ltz, J Li, A Gardell, R Sacchi (2013) Quantitative molecular phenotyping of gill remodeling in a cichlid fish responding to salinity stress. Molecular & Cellular Proteomics 12 (12), 3962-3975.
- Type:
Journal Articles
Status:
Published
Year Published:
2013
Citation:
R Sacchi, J Li, F Villarreal, AM Gardell, D K�ltz (2013) Salinity-induced regulation of the myo-inositol biosynthesis pathway in tilapia gill epithelium. The Journal of experimental biology 216 (24), 4626-4638
- Type:
Journal Articles
Status:
Published
Year Published:
2013
Citation:
AM Gardell, J Yang, R Sacchi, NA Fangue, BD Hammock, D K�ltz (2013) Tilapia (Oreochromis mossambicus) brain cells respond to hyperosmotic challenge by inducing myo-inositol biosynthesis. The Journal of experimental biology 216 (24), 4615-4625.
|
Progress 01/01/12 to 12/31/12
Outputs
OUTPUTS: The PI presented invited talks about this project at several national and international conferences in 2012 and 2013 (including in Aarhus, Denmark, and San Diego, CA). We have deposited the first batch of proteomics data generated with our newly developed workflow into the PRIDE proteomics database (European Bioniformatics Institute, EMBL). These data include metadata, identifications, spectra, and label-free quantitation data. To enable this we have worked with the PRIDE team (Attilla Csordas) and Bruker Daltonics (Peter Hufnagel) to convert our data formats to PRIDE xml. These files are fully compliant with MIAPE guidelines for publishing proteomics data in all major journals. Therefore, we have achieved a critical milestone in this project. The PI continued to actively support two K12 education outreach programs at Davis and Sacramento high-schools in which several students participated. Outreach to the general public was provided via our lab website. PARTICIPANTS: Not relevant to this project. TARGET AUDIENCES: Scientists using tilapia as a model to study fundamental biological questions - Aquaculturists using tilapia as a culture species - Comparative Genomicists and Evolutionary Biologists analyzing the evolution of proteomes from agriculturally important and domesticated animals. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
We have performed a complete set of experiments in which tilapia have been acclimated to 34, 70 and 90 ppt salinity by gradual transfer from freshwater. Several tissues have been harvested and processed for LCMS quantitative proteomics. The corresponding data have been analyzed and submitted to the PRIDE proteomics database. They are currently available for access by reviewers of the corresponding manuscript (which is in preparation). This manuscript documents the proteomics and bioinformatics workflows we have developed over the past years to perform label-free quantitation of many hundreds of proteins simultaneously. It will also report significant new insights into molecular mechanisms used by tilapia to overcome and cope with salinity stress. We have also continued to develop workflows for analysis of posttranslational modifications. This work included purification of select proteins by recombinant expression of His tagged surrogates for determining the stoichiometry of PTM. This work is laborious and the corresponding bioinformatics not yet fully optimized and we are still actively working on this front to enable identification, characterization and quantitation of posttranslational protein modifications and their regulation in response to salinity stress. We have also applied our proteomics workflows to collaborative projects on Daphnia and cow milk.
Publications
- The combinatorial nature of osmosensing in fishes. Kultz D. Physiology (Bethesda). 2012 Aug;27(4):259-75. doi: 10.1152/physiol.00014.2012. Review. PMID: 22875456
- Controlling proteome degradation in Daphnia pulex. Kemp CJ, Kultz D. J Exp Zool A Ecol Genet Physiol. 2012 Dec;317(10):645-51. doi: 10.1002/jez.1766. Epub 2012 Oct 1. PMID: 23027649
- Consumption of lysozyme-rich milk can alter microbial fecal populations. Maga EA, Desai PT, Weimer BC, Dao N, Kultz D, Murray JD. Appl Environ Microbiol. 2012 Sep;78(17):6153-60. doi: 10.1128/AEM.00956-12. Epub 2012 Jun 29. PMID: 22752159
|
Progress 01/01/11 to 12/31/11
Outputs
OUTPUTS: The PI presented invited talks about this project at several national and international conferences in 2011 and 2012 (including in Montpellier, France, Washington DC, and San Diego). Because the tilapia genome has been completed and released in March 2011 by the BROAD Institute we needed to re-write our software to accommodate inclusion of this new tool. In addition, we have ported the software development from MySQL to a MSSQL server to have a more stable and extendable platform for future use. We continue to use JAVA and Coldfusion as a software platform. The PI continued to actively support two K12 education outreach programs at Davis and Sacramento high-schools in which several students participated. Outreach to the general public was provided via our lab website. PARTICIPANTS: A graduate student (Alison Gardell), an undergraduate student (Jonathon Li), and a highscool student (Gillian Donald) were trained and participated in this project last year. TARGET AUDIENCES: - Scientists using tilapia as a model to study fundamental biological questions - Aquaculturists using tilapia as a culture species - Comparative Genomicists and Evolutionary Biologists analyzing the evolution of proteomes from agriculturally important and domesticated animals PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
We have used proteomics and conventional biochemistry and physiology methods to continue characterizing the salinity stress response of euryhaline tilapia. In particular, we have identified and quantified proteins from 5 different tilapia tissues after exposing the fish to different types of osmotic stress to determine what responses are triggered at which levels of salinity stress. The tisues that were analyzed are heart, liver, gill epithelium, brain, and kidney. This was done using a liquid chromatography mass spectrometry based protoemics approach. The workflow for this approach including bioinformatics was adjusted by the pI during the reporting period to allow inclusion of the annotated tilapia proteome, which became available in March 2011. Inclusion of this greatly valuable resource necessitated re-writing large parts of the software and workflow, which took a major portion of the time spend on this project during the past year. During the past year we have extended the scope of experimentally tractable tilapia proteins to over a thousand. The focus this past year was on label-free quantitation but we have also started using heavy-istope labeled peptides and started developing targeted proteomics approaches for selected proteins of interest. In addition, we have started developing specific manageable and reproducible workflows for mapping posttranslational modifications resulting from stress in tilapia tissues. In particular, we have focused on hydroxylation, phosphorylation, and acetylation. These efforts will be continued in the future. It is significant to characterize proteome responses to stress because the proteome represents the molecular phenotype that selection acts on. Thus, we will continue to study how the molecular phenotype is altered during salinity and other types of stress.
Publications
- Kultz, D. (2011) Osmosensing. In: Encyclopedia of Fish Physiology: From Genome to Environment. Ed.: Farrell, A.P., Academic Press (San Diego) 2: 1373-1380.
- Fiol,D.F., Sanmarti,E., Lim,A.H., Kultz,D. (2011) A novel GRAIL E3 ubiquitin ligase promotes environmental salinity tolerance in euryhaline tilapia. Biochim. Biophys. Acta 1810: 439-445.
- Gabert,B.J., Kultz,D. (2011) Osmoprotective proteome adjustments in mouse kidney inner medulla. Biochim. Biophys. Acta 1814: 435-448.
- Colbourne,J. et al. (2011) The ecoresponsive genome of Daphnia pulex. Science 331(6017): 555-561.
- Serafini,L.. Hann,J.B, Kultz,D., Tomanek,L. (2011) The proteomic response of sea squirts (genus Ciona) to acute heat stress: a global perspective on the thermal stability of proteins. Comp.Biochem.Physiol.D 6: 322-334.
- Zhang,Y., Doroshov,S., Famula,T., Conte, F., Kultz, D., Linares-Casenave,J., Van Eenennaam,J., Struffenegger,P., Beer,K., Murata,K. (2011) Egg quality and plasma testosterone (T) and estradiol-17 beta (E(2)) in white sturgeon (Acipenser transmontanus) farmed for caviar . J. Appl. Ichthyol. 27: 558-564.
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Progress 01/01/10 to 12/31/10
Outputs
OUTPUTS: The PI presented invited talks about this project at several national and international conferences in 2010 and 2011 (including in Barcelona, Spain, Muenster, Germany, and London, England). Because the completion of the tilapia genome by the BROAD Institute has been delayed for quite some time now and a completion date is uncertain the PI decided to pursue software development for unambiguous assignment of protein hits when using non-species specific protein database as a reference for searching mass spectrometry data. The development of this software using MySQL, JAVA, and Coldfusion represented a major effort and will be pursued further to make it available to the scientific community. The PI continued to actively support two K12 education outreach programs at Davis and Sacramento high-schools in which several students participated. Outreach to the general public was provided bia our lab website. PARTICIPANTS: A graduate student (Alison Gardell) and two undergraduate students (Jonathon Li and Jeffrey Woo) were trained and participated in this project. TARGET AUDIENCES: -Scientists using tilapia as a model to study fundamental biological questions -Aquaculturists using tilapia as a culture species -Comparative Genomicists and Evolutionary Biologists analyzing the evolution of proteomes from agriculturally important and domesticated animals PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
We have used proteomics and conventional biochemistry and physiology methods to continue characterizing the salinity stress response of euryhaline tilapia. In particular, we performed studies in which tilapia have been exposed to different severities of osmotic stress to determine what responses are triggered at which levels of salinity stress. This was done using a liquid chromatography mass spectrometry based protoemics approach. The workflow for this approach including bioinformatics was developed by the pI during the reporting period. This was a huge endeavor that took a great amount of time because of the many parameters that needed to be optimized. However, we have now collected data using this workflow and are able to monitor many hundreds of proteins simultaneously for changes in expression and posttranslational modifications resulting from stress in tilapia tissues. Targeted proteomics assays for these proteins based on labeled proteotypic peptide standards can now be developed for quantitative proteomics. This effort is significant because the proteome represents the molecular phenotype that selection acts on. Thus, we are now in a position to better characterize how the molecuylar phenotype is altered during salinity and other types of stress.
Publications
- Fiol,D.F., Sanmarti,E., Lim,A.H., Kultz,D. (2011) A novel GRAIL E3 ubiquitin ligase promotes environmental salinity tolerance in euryhaline tilapia. Biochim. Biophys. Acta in press: -.
- Kammerer BD, Cech JJ Jr, Kultz D. (2010) Rapid changes in plasma cortisol, osmolality, and respiration in response to salinity stress in tilapia (Oreochromis mossambicus). Comp. Biochem. Physiol. A 157(3): 260-265.
- Kammerer,B.D., Sardella,B.A. & Kultz,D. (2009) Salinity stress results in rapid cell cycle changes of tilapia (Oreochromis mossambicus) gill epithelial cells. J. Exp. Zool. A 311(2): 80-90.
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Progress 01/01/09 to 12/31/09
Outputs
OUTPUTS: The PI presented an invited talk about this project at the Aquaculture Symposium at the PAG conference in San Diego (Jan 2010). At this conference he met with Dr. Tom Kocher and other collaborators to discuss how best to advance genomic and proteomic resource generation for tilapia and related cichlids. In addition, the PI presented a talk on this project at the Desert Fishes Council meeting in Nov 2009. The PI met repeatedly with Aquaculture producers of California (Stolt Sea Farms, etc.) to share results from this project as well as educate producers about the tilapia genome project. In addition, the PI actively supported two K12 education outreach programs at Davis and Sacramento high-schools in which several students participated. Collaboration with Drs. Keith Miles and Doug Barnum was established that provides a link to Salton Sea Environmental concerns and Salton Sea Aquaculture farms involving tilapia. The PI also acquired new instrumentation and software that allowed setting up a Proteinscape proteomics server that hosts all data generated by this project and iis publicly accessible at the internet. PARTICIPANTS: A graduate student (Brittany Kammerer), a Davis High school intern (Mario Salvagno), and a postdoctoral fellow (Dr. Brian Sardella) were trained and participated in this project. TARGET AUDIENCES: -Scientists using tilapia as a model to study fundamental biological questions -Aquaculturists using tilapia as a culture species -Comparative Genomicists and Evolutionary Biologists analyzing the evolution of proteomes from agriculturally important and domesticated animals PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
We have used proteomics and conventional biochemistry and physiology methods to continue characterizing the salinity stress response of euryhaline tilapia. In addition, we have broadened our tilapia findings to other euryhaline fish species, including green sturgeon and leopard sharks, to test whether the pathways we have identified are phylogenetically conserved in teleosts and elasmobranchs. One of the major conserved features of osmotic stress signaling in tilapia that was identified as highly phylogenetically conserved during this analysis is the central involvement of cytokines. In particular, tumor necrosis factor alpha (TNFa) plays a critical role for orchestrating osmosensory signal transduction networks. In parallel to these analyses we have also established primary cultures of several tilapia tissues in the lab and obtained an endothelial tilapia cell line and optimized the culture conditions for that cell line. These tools will be invaluable for performing functional studies that rely on reverse genetics approaches. Such approaches include overexpression of tilapia osmotic stress genes and suppression of those genes. We have done overexpression studies of tilapia osmotic stress genes in a herterologous cell culture system (HEK293 cells) but the new tilapia cell cultures will allow us to get to a new level with such experiments.
Publications
- Allen, P.J., Cech. J.J., Jr., Kultz, D. (2009) Mechanisms of seawater acclimation in a primitive, anadromous fish, the green sturgeon. J. Comp. Physiol. B 179: 903-920.
- Dowd,W.W., Harris,B.N., Cech,J.J.,Jr. & Kultz,D. (2009) Proteomic and physiological responses of leopard sharks (Triakis semifasciata) to salinity change. J. Exp. Biol. 213(Pt 2):210-24.
- Fiol,D.F., Sanmarti,E., Sacchi,R. & Kultz,D. (2009) A novel tilapia prolactin receptor is functionally distinct from its paralog. J. Exp. Biol. 212: 2007-2015.
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Progress 01/01/08 to 12/31/08
Outputs
OUTPUTS: The PI participated and distributed information resulting from this project at PAG in San Diego (Jan 2009). In addition, the PI participated as an invited speaker at three national and international conferences (Experimental Biology, San Diego, April 2008; ICEF, Calgary, Canada, Jun 2008; AFS Physiology (Portland, Sep 2008). The PI met repeatedly with Aquaculture producers of California (Stolt Sea Farms, etc.) to share results from this project as well as educate producers about the tilapia genome project. In addition, the PI hosted a high-school intern as part of the Davis High school biotechnology program who participated in this project. The PI sponsored a poster presentation of this student at Davis High school and communicated results of this project at the Davis High school Biotechnology symposium in coordination with Ann Moriarty (Biology teacher at Davis High School). The PI met and discussed collaborative opportunities in the context of this project with Mark Drawbridge (Hubbs Seaworld). PARTICIPANTS: A graduate student (Brittany Kammerer), a Davis High school intern (Mario Salvagno), and a postdoctoral fellow (Dr. Brian Sardella) were trained and participated in this project. TARGET AUDIENCES: -Scientists using tilapia as a model to study fundamental biological questions -Aquaculturists using tilapia as a culture species -Comparative Genomicists and Evolutionary Biologists analyzing the evolution of proteomes from agriculturally important and domesticated animals PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
Using proteomics and transcriptomics approaches we have made major progress in identifying the biochemical signaling network that controls high environmental stress resistance and immune-tolerance in fish. By comparing the activation of this network in response to different types of stress and in other fish species beyond just tilapia we have shown that this stress signaling network is highly conserved in fishes and likely in other vertebrates. This finding has major implications for many agriculturally important and domesticated species beyond just tilapia. An important discovery made from identifying this stress signaling network is that key molecular pathways mediating immunosuppression are triggered by abiotic environmental stress explaining why animals are more susceptible to diseases when grown under suboptimal conditions (i.e. in suboptimal environments). Identification of the stress signaling network will allow more efficient and accurate health monitoring and establishment of optimal culture conditions as well as provide a mechanistic link for manipulating immunotolerance and stress resistance of tilapia and other farmed animals in circumstances when this is necessary or desirable (e.g. during disease outbreaks, droughts/ water shortage, etc.).
Publications
- Kammerer BD, Kultz D. (2009) Prolonged apoptosis in mitochondria-rich cells of tilapia (Oreochromis mossambicus) exposed to elevated salinity. J Comp Physiol [B]. 2009 Jan 11. [Epub ahead of print] PMID: 19137443
- Kammerer BD, Sardella BA, Kultz D. (2008) Salinity stress results in rapid cell cycle changes of tilapia (Oreochromis mossambicus) gill epithelial cells. J Exp Zool Part A Ecol Genet Physiol. 2009 Feb 1;311A(2):80-90. PMID: 18785193
- Sardella BA, Kultz D. (2009) Osmo- and ionoregulatory responses of green sturgeon (Acipenser medirostris) to salinity acclimation. J Comp Physiol [B]. 2008 Dec 10. [Epub ahead of print] PMID: 19066909
- Sardella BA, Sanmarti E, Kultz D. (2008) The acute temperature tolerance of green sturgeon (Acipenser medirostris) and the effect of environmental salinity. J Exp Zool Part A Ecol Genet Physiol. 2008 Oct 1;309(8):477-83. PMID: 18615462
- Sardella BA, Kultz D, Cech JJ Jr, Brauner CJ. Salinity-dependent changes in Na(+)/K (+)-ATPase content of mitochondria-rich cells contribute to differences in thermal tolerance of Mozambique tilapia. J Comp Physiol [B]. 2008 Mar;178(3):249-56. Epub 2007 Dec 18. PMID: 18087703
- Dowd,W.W.; Wood,C.M.; Kajimura,M.; Walsh,P.J. & Kultz,D. (2008) Natural feeding influences protein expression in the dogfish shark rectal gland: A proteomic analysis. Comp. Biochem. Physiol. D 3: 118-127.
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