Final Report
TITLE
Understanding the role of solar radiation and sea surface temperature on the allocation of resources into immune defense, growth and reproduction of Acropora cervicornis.
NAME AND AFFILIATION OF RESEARCHERS
Dr. Carlos Toledo-Hernandez, Socidedad Ambiente Marino (SAM)
Dr. Claudia Patricia Ruiz-Diaz, Sociedad Ambiente Marino (SAM)
Dr. Juan Sebastián Ramírez-Lugo, University of Puerto Rico, Río Piedras
RESULTS AND FINDINGS
FPs and PO analyses
On going analyses show that FP increase during seasons were temperature and solar radiation increase. That is, the emission intensity of FPs was relatively higher in fragments placed on farms at 5m water depth, than at 8m and 12m during spring and summer. Temperature and solar radiation were higher during these seasons at all depths when compared to the other seasons i.e. fall and winter. Yet, comparing clones placed at different water depths, FPs emission intensities were similar. Overall, these findings suggest that FPs might have a photoprotective function in corals
On the other hand, PO activity was higher in shallow waters (i.e. 5 m) during spring as compared to the others depth, and them PO activity in fragments placed at 12m depth overpass those fragments placed at 5 and 8m depths. No differences have been observed in PO activity when comparing clones placed at different depths.
Growth analyses
Coral fragments exhibited higher growth rate and higher rate of branching in farms placed at 5m water depths than at any other depth. Interestingly, growth rate and branching peaked during JanMar, the coldest water temperature periods. These finding were consistent among clones. That is, clones fragments placed at farms in shallower waters showed the highest growth rate and branching when compared to the fragments placed in deeper areas. Furthermore, fragments collected from La Ahoga, exhibited lowest growth rates and branching when compared to the other collection sites.
Mortality of fragments
Coral mortality was low at all farms, as 23 fragments died during the study period. Mortality was significantly higher at the shallowest farms when compared to the deeper farms. For instance, fragment mortality at the shallower farms were twice as high than at mid depth farms and four times higher than the deepest farms. As in growth and branching measurements, mortality was higher on fragments collected at La Ahoga.
METHODOLOGY
A total of 150 colonies from Luis Pena, Tamarindo Chico and La Ahoga reefs were collected (50 per reef), tagged and placed in temporary farms in Punta Soldado Reef at a depth of 5m for two weeks for acclimation. Them, fragments were further cut (hereafter clones), re-tagged and distributed in nine farms installed at 5, 8 and 12m of water depth (three farms per depth). At three-month intervals, twenty out of the 150 colonies tagged (20 colonies per depth) were collected and used for biochemical analyses of Fluoresce Proteins (FPs) concentration and Phenoloxidase (PO) activity (Specific Aims 1). Sixty un-manipulated tagged colonies (30 per depth) were used for growth rate analyses (Specific Aims 2). The remaining 90 tagged-colonies were used to measured somatic growth and for the reciprocal transplant experiment (Specific Aims 4).
To measure FPs concentration, 30µl of extracted samples were placed in a 96-well plate and along with buffer-only controls. Each well was excited using a BioTek ELx808 Absorbance Microplate Reader at 280nm and the emission spectra measured from 400nm to 600nm at 5nm intervals. Relative fluorescence and absorbance of each sample were normalized to total protein concentration determined using Bradford Reagent and measuring absorbance at 595nm utilizing the same protocol described above. A repeated measurement ANOVA will be performed to compare FPs concentration as measured through seasons against depths. Additionally, the relationship between FPs concentration and temperature and FPs with light will be analyzed performing a linear regression ANOVA analysis.
To quantify the PO enzymatic activity, as PO is an enzymatic precursor of melanin (Palmer et al. 2011). we processed tissue portion obtained for FPs concentration as explained by Palmer et al (2011) with slight modifications as described above. PO activity was determined by adding 20µl of the extracted coral sample to a 96-well plate, in addition to 40µl of 50mmol l-1 phosphate buffer, pH 7.8 and 25µl of double distilled water (ddH2O). After incubating at room temperature for 20min 30µl of a 10mmol l-1 dopamine hydrochloride solution was added as a substrate and the change in absorbance of the reaction was monitored every 5min for 45min using a BioTek ELx808 Absorbance Microplate Reader at 490nm. The change in absorbance for the linear portion of the reaction curve will be used to determine PO activity. Control reactions under the same condition but with 70µl of 50 mmol l-1 phosphate buffer, pH 7.8, and no substrate will be run in parallel. PO activity will be normalized to total protein concentration in each sample. Total protein concentration of the extracted coral sample will be determined using Bradford Reagent and measuring absorbance at 595nm utilizing the same protocol described above. A repeated measurement ANOVA will be performed comparing PO activity measured through seasons against depths. Additionally, the relationship between PO activity and temperature and PO activity with light will be analyzed performing a linear regression ANOVA analysis.
The growth rate of 60 tagged colony (30 per depth) that were not previously manipulated, were estimated at three months intervals for two years following Mercado-Molina et al (2015). Briefly, branches from each of the tagged colonies were photographed from different angles to ensure that all branches can be appreciated in their full extension. From these photographs we estimated the linear length of branches using the software Coral Point Count with Excel extension (CPe). These estimates were converted to growth rates (final length of tissue – initial length of tissue/ total number of days between photographs) and expressed in cm/day. Growth
rates of colonies from each depth were compared using an analysis of variance with colony depth as the independent variable and growth rates as the dependent. A repeated measurement ANOVA was performed to compare Growth rate as measured through seasons against depths. Additionally, the relationship between growth rate and temperature and growth rate with light was analyzed performing a linear regression ANOVA analysis.
Environmental assessment
To measure water temperature and solar radiation at each depth, a Hobo-pendant temperature/light data loggers devices were installed. These devices were replaced at monthly intervals, for two years.
OBJECTIVES
Specific Aim 1. Measure the Seasonal (Spring, Summer, Fall and Winter) and Depth (5m vs. 15 m of Depth) Variations in the Concentration of FPs And Melanin in A. cervicornis Tissue
90% completed. All tissue samples have been collected, however, data analysis will be completed in the next two weeks. Hurricanes Irma and Maria destroyed all farms killing most of the experimental fragments. Therefore, we were to start the project all over again, with fragments that survived the disturbance causing a delay in the data analyses.
Specific Aim 2. Measure Temporal Variability in Somatic Growth of A. cervicornis Colonies Naturally Inhabiting Reef Zones with Contrasting Temperature and Light Regimes (5m vs. 15m).
100% completed
Specific Aim 3. Compare the Reproductive Effort (Gametes Produced per Polyp) of A. cervicornis Colonies Naturally Inhabiting Reef Zones with Contrasting Temperature and Light Regimes (5m vs. 15m).
0% completed
Hurricanes Irma and Maria destroyed all farms killing most of the experimental fragments. Therefore, we were to start the project all over again. In addition, the lab facility to be used for the histological analyses at the UPR-PR were seriously damaged and these rendered our capacity to fulfill Aim 3. Thereby this aim was withdrawn from the proposal.
Specific Aim 4. Perform a Reciprocal Transplant Field Experiment to Test the Capacity of A. cervicornis Colonies to Adjust the Allocation of Resources in Immune Constituents vs. Somatic Growth and Reproduction by Transplanting Colonies at 5m to 15m and vice versa.
100% completed
LIST PI’S SUPPORTED
(Provide a breakdown of time and effort attributed to PI’s, Co-PI’s and associates. Use a man-month measure for time dedicated to the project and the amount paid from both SG and Match during the report period). See attachment
LIST STUDENTS AND OTHERS SUPPORTED
Eight undergraduate students were participating, on the field-data collection. These students are: Pedro Carmona (pjcarmona421@gmail.com), Juan Luis Sánchez (got.juan.13@gmail.com), Eric Velez (Eric.velez2@ upr.edu), Jose Méndez (joymendezrojas@gmail.com), Gustavo Acevedo (gustavoacevedo@gmail.com), Sergio Ayala (Sergio.ayala@upr.edu), Frances García (fa.garciarobles@gmail.com), and Nicolas Gómez (nicolas.x.gomez@gmail.com) Only Juan Luis Sánchez has continued collaboration on this project and he has owned the right to participate in two of the manuscript that are currently being writing. The remaining students have participated in only one field-work weekend. None of them have received any payments for their effort.
Rafael Giraud is a master-degree student at the UPR-Medical Science Campus and Dr. Josué Pérez, researcher at the Centro Comprensivo de Cancer, are analyzing tissue from A cervicornis reared in farms, to determine how seasonal changes in temperature and solar radiation affect the coral’s microbiome.
Marielys Torres-Diaz Ph.D. candidate at the UPR-Rio Piedras (mtd_24@yahoo.com) and Drs. Liz Diaz, professor at the Chemical Department at the UPR-Rio Piedras (Liz.diaz2@upr.edu) and Brenda Betancourt-Canizales, professor at the Statistics Department University of Florida (bbetancourtc@ gmail.com). MTD and LD have been collaborating in the biochemical analyses (FPs and PO) and BB have been collaborating on the statistical analysis of coral growth and branching. Their collaboration has been significant, thereby they have owned the right to participate in two of the manuscript that are currently being writing. None of them have received any payments for their effort.
List of presentations
UNDERGRADUATE BIOLOGY EDUCATION RESEARCH GORDON RESEARCH CONFERENCE (GRC). EASTON, MA
1. JS Ramírez-Lugo, I Vélez-Gonzalez, Sánchez, JL Nieves-Mártir, G Carmona-Casillas, PJ Méndez-Rojas, JE Silva-Luna, Y Arana-Sepulveda, C Toledo-Hernández & CP RuizDiaz. 2017. Poster Presentation: CREARE: An Interdisciplinary Study of the Response of Corals to Environmental Changes as a Model for the Development of Laboratory Courses Based on Authentic Research Experiences (Oral presentation)
ASLO 2019 AQUATIC MEETING, SAN JUAN, PR
1. JS Ramírez-Lugo, C Toledo-Hernández & CP Ruiz-Diaz. 2019. Course-based undergraduate research experience to investigate the responses of the coral Acropora cervicornis to environmental stress. (Oral presentation)
2. M Torres-Diaz, C Toledo-Hernández & CP Ruiz-Diaz. 2019. Inmune response of Acropora cervicornis to solar radiation and sea Surface temperaturas. (Poster presentation)
3. JL Sánchez-González, C Toledo-Hernández & CP Ruiz-Diaz. 2019. Could rises in water temperature predicted by climate change decrease coral reef complexity by reducing branching capacity of Acropora cervicornis? (Oral presentation)
JUNIOR TECHNICAL MEETING, MAY 4, 2019, MAYAGUEZ PR
1. M Rodríguez-Velez, L Santiago-Pagán, JS Ramírez-Lugo, C Toledo-Hernández & CP Ruiz-Diaz Inmune response of Acropora cervicornis to solar radiation and sea Surface temperaturas. (Oral presentation)
List references peer reviewed publications In Review and published
1. CP Ruiz-Díaz, Brenda Betancourt Canizales, C Toledo-Hernández (In Review). Impact of variable temperature and solar radiation regimes on the growth rate of Acropora cervicornis Conservation Science and Practice Journal
2. JS Ramírez-Lugo, I Vélez-Gonzáles, C Toledo-Hernández, CP Ruiz-Diaz (In review). An interdisciplinary course-based undergraduate research experience (CURE) to investigate the response of the critically endangered coral Acropora cervicornis to environmental stress. Journal of Microbiology and Biology Education
3. Nieves-González A, CP Ruiz-Díaz, C Toledo-Hernández, JS Ramirez-Lugo (2019). A Mathematical Model of the Interactions Between Acropora cervicornis and Its Environments. Ecological Modelling 406:7-22
4. C Toledo-Hernández, CP Ruiz-Díaz, E Hernández-Delgado, S Suelimán. (2018). Devastation of 15-year old community-based coral farming and reef restoration sites in Puerto Rico by major Hurricanes Irma and María. Caribbean Naturalist. 53:1-6
ADDITIONAL MANUSCRIPTS THAT ARE CURRENTLY BEING WRITTEN.
1. M Torres-Díaz, A Rodríguez-Vélez, C Toledo-Hernández, CP Ruiz-Díaz, L SantiagoPagán, JS Ramírez-Lugo (In preparation) Inmune response of Acropora cervicornis to solar radiation and sea Surface temperaturas.
2. C Toledo-Hernández, CP Ruiz-Díaz, JL Sánchez-González. (In preparation) Could rises in water temperature predicted by climate change decrease coral reef complexity by reducing branching capacity of Acropora cervicornis?
3. Nieves-González A, CP Ruiz-Díaz, C Toledo-Hernández, JL Sánchez-González (In preparation). A Mathematical Model of the Branching Growth of Acropora cervicornis in the Caribbean: Assesing the impact of climate change
4. Giraud R, J Pérez, C Toledo-Hernández, CP Ruiz-Diaz (in preparation) Acropora cervicornis microbiome under a climate change.
Outreach
With the support of the current award and additional funding from UPR-RP we have drawn upon the ongoing research project aimed at understanding how the stony coral A. cervicornis responds to seasonal environmental fluctuations to develop an interdisciplinary course-based undergraduate research experience (CURE). Through a semester-long course, entitled CREARE, three cohorts of 6–8 students each have successfully performed biochemical experiments in the laboratory, analyzed environmental data using the R software environment and disseminated the results of their research through a written report and/or oral presentations at a local research symposium. The results of their work in the CURE have been instrumental to the progress of the research project, and lend credence to the idea of creating CUREs to integrate cutting edge research into the classroom.
The impact of CREARE on student learning gains and attitudes towards science has been measured and students report gains in learning, improved attitudes towards science and a higher likelihood of pursuing a career in STEM. Collectively, students who participated in CREARE self-report a perceived gain in learning even though, on average, they do not perform any differently on a 14-item exam about the underlying concepts related to the research project when taking the exam before or after the CURE. Hence, students own perceptions about learning should not be used as the primary measure of effectiveness of CUREs. The discrepancy between the observed and perceived gains that result from participation in research experiences suggests that the mechanisms by which URE impact students goes beyond content acquisition and technical mastery. Using a grounded theory approach to identify emergent themes from focus group interviews of CREARE participants revealed that, when asked to evaluate the experience,
codes associated to mediators of self-efficacy appear with the highest frequency among all variables associated to scientific integration. A detailed analysis of the variables that mediate these processes is currently underway and its results should yield the publication of a research manuscript in biology education. Lastly, one undergraduate student which participated in CREARE is currently pursuing a Master-degree at UPR-RP. Its research topic will include the effects of climate changes in coral reef associated organisms.