Progress 01/01/22 to 12/30/24
Outputs
Target Audience:Over the course of this project, the target audience most impacted by the research products includes 1). members of the aquaculture industry, 2.) members of the animal disease research community 3.) Research trainees in the biochemical and agricultural sciences. Our research efforts focused on identifying small-molecule chemical agents that boosted the efficiency that copper can kill the oomycete pathogenSaprolegnia parasitica(Sp), determining the underlying mechanisms of such effects, investigating the required metal ions forSpgrowth and metabolic survival and establishing the lethal for ionophore and copper in catfish embryos. Toward this goal, we identified and characterized the dosage and mechanisms of several low to moderate-cost chemicals that could increase copper-mediated growth inhibition. We identified a selection of seven small chemical molecules with anti-saprolegnia activity, with many displaying copper-dependent activities. The Data from this research are essential in developing novel strategies to combat oomycete infectious diseases in an aquaculture setting. This research project also impacted the growth and development of the next generation of research scientists by providingexperiential learning opportunities to trainees spanning all higher education levels from undergraduate to post-doctoral. In total, this research project engaged and supported two undergraduates, two graduate, and one postdoctoral trainee(s). These research activities were performed at Texas State University, which strengthened the research environment at a resource-limited and one of the nation's largest Hispanic-serving Institutions. Engagement with the general public and research/aquaculture or the reporting period was achieved through the presentation of research findings at regional/national conferences and the publication of research materials in open-access journals. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?Training and Professional development opportunities were provided to all research mentees throughout the calendar academic year. Below lists the major activities undertaken by each participant. Postdoctoral researcher (Dr. Tomisin Ogunwa) - Peer mentoring training (through TX State University), Scholarship & Teaching Excellence Program (STEP); by the TX State University faculty development team), Student mentoring (Gracie Thornhill & Rahima Akter), one-on-one training on the preparation of scientific manuscripts. Graduate researcher (Ms. Rahima Akter) - Peer mentoring training and one-on-one training/mentoring in the areas of 1. scientific poster presentations, 2. Improvement of research skills: acquiring knowledge in the importation and interpretation of data from new equipment like ICP-MS, quantitative Real-time PCR, mRNA isolation, oxygen consumption studies, fluorescence microscope, etc.3. The preparation of personal statements for doctoral graduate school applications, 4. Long-term career planning and mentoring for the successful navigation of a MS thesis project. Graduate researcher (Mr. Dallas Lee) -A research experience and independent study for ~ 9 months while enrolled as a PhD student in the MSEC program. Due to personal challenges, he left the PhD program without completing the degree. Undergraduate researcher (Ms. Gracie Thornhill) - Peer mentoring training (through TX State University) and one-on-one training/mentoring in the areas of 1. scientific poster presentations, 2. Personal statements for graduate school applications, 3. Long-term career planning. How have the results been disseminated to communities of interest?The results of our studies have been disseminated to communities of interest in several forms over the project timeline. We have participated and presented our research findings at publicly accessible regional and national research conferences. These conferences include the TX State Summer Research Symposium and the 2023 CRWAD. Additionally, we have submitted one research report to the open-access server BioRxiv, which is currently under review for publication in an open-access peer-reviewed journal. One of the graduate students supported by this project will also submit an open-access MS thesis in the Spring 2025 semester detailing the metal nutritional requirements of S. parasitica and a manuscript detailing protocols for manipulating S. parasitica cellular metal levels. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
With respect to aim 1, we fully characterized the Cu, Fe, and Mn nutritional requirements needed to support the basic metabolism ofSaprolegnia parasitica(Sp). This included the development of a chemically defined media that could supportSpgrowth while allowing for opportunities to employ metal-specific chelators to alter metal bioavailability. Through this aim, we developed determined the impact of metal restriction and metal overload onSaprolegnia parasiticapropagation. Using developed new methods to determine total cellular metal levels using ICP-MS. We observed thatSaprolegnia parasiticahas an unusually low requirement for Cu-ions but relies more heavily on Fe and Mn for growth fitness. Using cytochrome c oxidase (CcO) and alternative oxidase (AOX) gene expression and activity as a readout of cellular metal status, we find that alteration in Cu-bioavailability diminishes the percentage of AOX respiration, and there becomes a preference to use CcO at the terminal oxygen for mitochondrial respiration. Under research objective 2, we identified and characterized the effects of copper and small molecule ionophore combinations on the ability to suppressSpgrowth. We identified a collection of seven chemical ionophores that could boost the anti-saprolegnia activity of copper sulfate. Our best ionophores displayed potent activity at sub-micromolar concentration ranges. Further investigation into the mechanism of action for our most promising ionophore, MPO, suggests that the mode of action does not alter cellular oxidative stress but may involve a more targeted copper-dependent mode of action. A manuscript overviewing this work is currently under review for publication in a peer-reviewed journal. Technical challenges were experienced, which prohibited us from accomplishing all of the original research goals pertaining to Aim 3. Were we unable to successfully isolate qualitySpmRNA from developing catfish embryos to assessSp's transcriptional response during the early stages of colonization. This was partially due to our accessibility to catfish embryos which was limited to approximately 5 weeks over the project period. Alternativity, we investigated the efficacy of the copper and ionophore combinations to inhibitSppropagation while maintaining catfish embryo health.?
Publications
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2023
Citation:
T. Ogunwa, M. Thornhill, D. Ledezma, and R. Peterson (2023). Modulation of Saprolegnia parasitica growth using copper and ionophores. Conference for Research Workers in Animal Diseases (CRWAD), Chicago. Poster presentation.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2023
Citation:
R. Peterson, T. Ogunwa, M. Thornhill, and D. Ledezma (2023). Boosting copper toxicity towards Saprolegnia parasitica. Conference for Research Workers in Animal Diseases (CRWAD), Chicago. Oral presentation.
- Type:
Other Journal Articles
Status:
Published
Year Published:
2024
Citation:
Modulation of Saprolegnia parasitica growth with copper and ionophores
Tomisin Happy Ogunwa, Madison Grace Thornhill, Daniel Ledezma, Ryan Loren Peterson
bioRxiv 2024.06.17.599375; doi: https://doi.org/10.1101/2024.06.17.599375
- Type:
Other Journal Articles
Status:
Submitted
Year Published:
2024
Citation:
Enhancing copper toxicity against Saprolegnia parasitica, a devastating oomycete pathogen in aquaculture
Tomisin Happy Ogunwa, Madison Grace Thornhill, Daniel Ledezma, Ryan Loren Peterson
UNDER REVIEW
|
Progress 01/01/23 to 12/31/23
Outputs
Target Audience:Overthe 2023calendar year, the target audience most impacted by the research products include1). members of the aquaculture industry, 2.) members of the animal disease research community 3.) Student research trainees Our research efforts focused on identifying small-molecule chemical agents that boosted the efficiency that copper can kill the oomycete pathogenSaprolegnia parasitica(Sp),determining the underlying mechanisms of such effects, investigating the required metal ions for Sp growth and metabolic survival and establishing the lethal for ionophore and copper in catfish embryos.Toward this goal, we identified and characterized the dosage and mechanisms of several low to moderate-cost chemicals that could increase copper-mediated growth inhibition.Our data from this research are essential in developing new strategies to combat oomycete infectious diseases in an aquaculture setting.Our project progress andresearch findings were communicated at theconference of research workers in animal diseases (CRWAD) in Chicago in January of 2023. Throughout this funding period, this research project also impacted the growth and development of the next generation of research scientists. These activities were performed at Texas State University, which is one of the nation's largest Hispanic-serving Institutions. This project supports the research efforts of one postdoctoral research trainee and one graduate student. Laboratory training and scientific presentation opportunities were provided to all participants. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?Grantsmanship professional development opportunites have been given to graduate student Ms. Rahima Anker. She prepared a successful internal fellowship through the TX State University Graduate Collegeto support her research efforts. Professional networking and development opportunities were given topost-doctoral researcher Dr. Tomisin Ogunwa by presenting a poster at the 2023 CRWAD conference as well as mentoringin the preparation ofscientific manuscripts. How have the results been disseminated to communities of interest?In part at the 2023 CRWAD conference. What do you plan to do during the next reporting period to accomplish the goals?A no-cost extension has been requested and awarded for this project to complete the unfinished portions of proposed research objectives 1 and 3. This includes RNA-Seq studies onS. parasiticagrown under metal-stress conditions and in the early stages of catfish embryo colonization.
Impacts
What was accomplished under these goals?
Over the course of the reporting period, we made progress on all three proposed research objectives. However, our primary focus was set on objectives 1 and 3. The progress we made towards research objective 1 was primarily aimed at describing the basic metal metabolic requirements ofS. parasiticaand establishing methodologies for isolating high-quality RNA to be used for transcriptional studies. We developed protocols for quantifying cellular copper, iron, and manganese levels inS. parasiticagrown under different culture conditions.In addition, we piloted RNA extraction protocols fromS. parasiticato be used for transcriptomic studies.We are modifying the tested protocols for RNA isolation to produce high-quality RNA devoid of contamination and degradation for analysis.Our efforts during the next funding period will focus on using these validated tools/protocols to describe basic cellular processes and gene expression patterns inS. parasiticacultured under metal-stress growth conditions. We completed many of the original research objectives described in Aim 2 and are in the final stages of manuscript preparation to report these results to the broader research community. We worked on developing reproducible methods and protocols to generateS. parasiticaspores to evaluate the toxicity of ionophores/copper combinations on differentS. parasiticalife stages. Our research progress towards aim 3 primarily focused on developing controlledSaprolegnia parasiticainfection protocols for catfish embryos and investigating embryo tolerance levels of copper/ionophore mixtures. We determined the toxicity and lethal doses of a selection of ionophores, copper and BCS on the catfish embryos using microscopy to monitor embryo development and investigated the ability of metal chelators to reverse copper toxicity. Future experiments will be directed at RNA isolation from the embryos under infection and various chemical treatments to identifyS. parasiticagenes needed for early colonization on catfish embryos and specific molecular pathways altered upon Cu/ionophore treatment.
Publications
- Type:
Conference Papers and Presentations
Status:
Accepted
Year Published:
2023
Citation:
T. Ogunwa, M. Thornhill, D. Ledezma, and R. Peterson (2023). Modulation of Saprolegnia parasitica growth using copper and ionophores. Conference for Research Workers in Animal Diseases (CRWAD), Chicago. Poster presentation.
- Type:
Conference Papers and Presentations
Status:
Accepted
Year Published:
2023
Citation:
R. Peterson, T. Ogunwa, M. Thornhill, and D. Ledezma (2023). Boosting copper toxicity towards Saprolegnia parasitica. Conference for Research Workers in Animal Diseases (CRWAD), Chicago. Oral presentation.
|
Progress 01/01/22 to 12/31/22
Outputs
Target Audience: Over the course of the 2022 calendar year, the target audience most impacted by the research products includes 1). Members of the Aquaculture farming industry and 2.) Student research trainees. Our research efforts focused on identifying small-molecule chemical agents that boosted the efficiency that copper can kill the oomycete pathogenSaprolegnia parasitica(Sp). Towards this goal, we identified several low to moderate-cost chemicals that could increase copper-mediated growth inhibition. Ultimately, these preliminary experiments will help build the body of research needed to develop new strategies to combat oomycete infectious diseases in an aquaculture setting. Over the course of this funding period, this research project also impacted the growth and development of the next generation of research scientists. These activities were performed at Texas State University, which is one of the nation's largest Hispanic Serving Institutions. This project supports the research efforts of one full-time postdoctoral research trainee and one graduate student. Two undergraduate research training opportunities were provided over the summer academic break. Laboratory training and scientific presentation opportunities were provided to all participants. Changes/Problems: There have been no major changes in the approach. We have experienced some technical hurdles with respect to Aims 1 and 3. With respect to Aim 1, we had difficulties identifying suitable antibodies to be used as lysate-loading controls for western blots. We have tried several commercial antibodies with high homology toSaprolegniaproteins and reported cross-species reactivity to other microbes. However, we have been unsuccessful in detecting these protein targets inSaprolegnialysates. We have started to investigate the generation of species-specific antibodies for our applications. As our group is new to the aquaculture research community, we did experience some technical challenges with the disinfection of catfish embryos from our local hatchery. We reached out to LSU professor John Hawke and researcher Cristine Darnall for technical assistance in this area. They were helpful in sharing protocols and troubleshooting. We anticipate we now have the required equipment and technical training to tackle the infection studies this upcoming spring. What opportunities for training and professional development has the project provided?Training and professional development opportunities were provided to all research mentees over the course of the calendar academic year. Below lists the major activities undertaken by each participant. Postdoctoral researcher ( Dr. Tomisin Ogunwa ) - Peer mentoring training (through TX State University), Scholarship & Teaching Excellence Program ((STEP); by the TX State University faculty development team), Student mentoring (Gracie Thornhill & Rahima Anker), one-on-one training on the preparation of scientific manuscripts. Undergraduate researcher ( Ms. Gracie Thornhill ) - Peer mentoring training (through TX State University) and one-on-one training/mentoring in the areas of 1. scientific poster presentations, 2. Personal statements for graduate school applications, 3. Long-term career planning. How have the results been disseminated to communities of interest?During the current reporting period, Ms.Gracie Thornhill presented our first formal research presentation on this project at the TX State University Summer Research Program Symposium. This multidisciplinary symposium spans all STEM disciplines and had ~ 100 participants. This forum allowed our research to reach a broad audience while simultaneously allowing for networking opportunities with other researchers on our campus. What do you plan to do during the next reporting period to accomplish the goals?The major research goals we would like to complete during the '23 calendar year include: Regarding Aim 1, we will execute the RNA-Seq investigations into genes that are responsive to selective Cu, Fe, and Mn metal ion withholding and overload growth conditions. The goal is to identify and validate biomarkers that are characteristic of the metal-ion stress response and determine the basic cellular metal concentrations forSp. With respect to aim 3, we will finish the design of the primer library to be used to complete the targetedSptranscriptional response studies during the early stages of catfish embryo colonization. Prior to the infection studies performed on catfish embryos, we will validate our microbial decontamination protocols on sunshine bass embryos which are produced at the AE Wood Hatcher prior to catfish production. This will allow us to make any needed modifications to our protocols prior to working with catfish embryos. With respect to both of the aforementioned research aims, we will work closely with our collaborator Dr. Yuan Lu to set up a bioinformatics pipeline for identifying differentially expressed genes.
Impacts
What was accomplished under these goals?
Over the course of the reporting period, we made progress on all three proposed research objectives. However, our primary focus was set on objectives 2 and 3. The progress we made towards research objective 1 was aimed at generating and establishing biochemical tools that are necessary to study the basic cell biology ofSaprolegnia parasitica. We tested and generated custom antibodies that can be used to monitor cellular processes inS. parasitica. Our efforts during the next funding period will focus on using these validated tools to describe basic cellular processes and gene expression patterns inS. parasiticadue to alterations in metal ion stress conditions. We completed many of the original research objectives described in aim 2. We have identified a collection of ~ 6 small molecules that are efficient at eliminatingS. parasiticaproliferation in a laboratory setting. We have determined the half-maximal inhibitory concentration (IC50) for the small molecules alone and in the presence of added copper(II) ions. Through a series of experiments, we propose that these small molecules function in one of three distinct mechanisms to lead toS. parasiticadeath. Future research directions in the next funding period will focus on determining the speed andS. parasiticalife stage the identified small molecules are most efficient at killing. Our research progress towards aim 3 primarily focused on establishing protocols for the disinfection of catfish embryos from the AE Wood Hatchery (San Marcos TX) to be used for controlled infection studies withSaprolegnia parasitica. Over the ~ 6-week 2021 spawning window, we developed laboratory protocols toward the goal of sterilizing the embryos of other potential microbial pathogens. These protocols will be necessary for us to achieve our ultimate research goal which aims to understand metabolic processes and secreted proteins that assistS. parasiticatoadhere and proliferate on catfish embryo substrates. This research project is focused on understanding the basic metal cell biology of the oomycete pathogenSaprolgenica parasiticaand investigating if disruptions in metal-ion homeostasis can be used as an alternative treatment strategy to combatSaprolegniasisdisease.Over the past reporting period and as described above, we made significant progress towards completing our original three proposed research objectives.
Publications
|
|