Visualizing nuclei in Platygyra daedalea ​

by Minnie Sakulsakpinit

A THESIS

submitted to

Oregon State University

Honors College

in partial fulfillment of the requirements for the degree of

Honors Baccalaureate of Science in Biology (Honors Scholar)

Presented November 15, 2018 Commencement December 2018

AN ABSTRACT OF THE THESIS OF

Minnie Sakulsakpinit for the degree of Honors Baccalaureate of Science in Biology presented on ​ ​ November 15, 2018. Title: Visualizing nuclei in Platygyra daedalea. ​ ​ ​

Abstract approved:______Eli Meyer

Platygyra daedalea, a species of brain coral from the Persian Gulf, can withstand higher ​ ​ ​ temperatures than other coral species. This attribute makes the species a potential model organism for thermal tolerance research. Genomic resources such as a linkage map of this species predict that there are 12 to 14 chromosomes within its genome. An outside validation of this number is required, and karyotyping is a way to confirm this number. In this study, I compared preparation methods for visualizing the nuclei and chromosomes of coral larval tissue while utilizing older samples that have been preserved for over one year. These methods include mechanical preparation of the slide, chemical dissociation of larvae tissue, fixation using different techniques, and staining with different dyes. The most effective methods for visualizing the nuclei of coral cells were determined after various trials. The viabilities of coral larvae ​ samples preserved over time under different conditions were also discussed and compared, which can maximize the efficiency and utilization of limited resources in future projects.

Key Words: Platygyra daedalea, corals, thermal tolerance, karyotype, chromosomes, methods ​ ​

Corresponding e-mail address: [email protected]

©Copyright by Minnie Sakulsakpinit November 15, 2018 All Rights Reserved

Visualizing nuclei in Platygyra daedalea ​

by Minnie Sakulsakpinit

A THESIS

submitted to

Oregon State University

Honors College

in partial fulfillment of the requirements for the degree of

Honors Baccalaureate of Science in Biology (Honors Scholar)

Presented November 15, 2018 Commencement December 2018

Honors Baccalaureate of Science in Biology project of Minnie Sakulsakpinit presented on ​ November 15, 2018.

APPROVED:

______Eli Meyer, Mentor, representing Department of Integrative Biology

______Stephen Atkinson, Committee Member, representing Department of Microbiology

______Nathan Kirk, Committee Member, representing Department of Integrative Biology

______Holland Elder, Committee Member, representing Department of Integrative Biology

______Toni Doolen, Dean, Oregon State University Honors College

I understand that my project will become part of the permanent collection of Oregon State University, Honors College. My signature below authorizes release of my project to any reader upon request.

______Minnie Sakulsakpinit, Author

INTRODUCTION

As an extensive and diverse marine ecosystem, coral reefs provide habitat for countless invertebrate and fish species (Hoegh-Guldberg 2003). However, rising ocean temperatures have increased the frequency of coral bleaching events in which corals lose the symbiotic dinoflagellate partners that they depend on for energy. Such mutualistic symbioses, a fundamental feature of coral reefs, explain their structure, biodiversity and existence

(Hoegh-Guldberg 2003). The phenomenon of increased ocean temperatures disrupts these important symbiotic interactions and has caused coral reef degradation in almost all major coral reef systems. Further damage to coral reef communities will lead to a reduction of biodiversity in the oceans (Veron et al. 2009). This creates urgency in understanding the bleaching process as well as reasons why some coral populations and individuals are more resilient than others to the thermal stress that causes bleaching.

Recent studies have found that a species of brain coral from the Persian Gulf, Platygyra ​ daedalea, can withstand temperatures of 35˚C in the summer (Riegl et al. 2011; Howells et al. ​ ​ ​ ​ ​ 2016). This population and species is of great interest to current research for comparison with other populations because it exists almost everywhere within the coral range. The Meyer Lab at

Oregon State University has been developing genomic resources for this species to facilitate mechanistic studies of thermal tolerance, including a linkage map and genome of the coral.

However, outside confirmation of the number of chromosomes is needed to support this data.

The number of chromosomes per cell is consistent in most species, and comparing chromosomes in different species is a useful way to examine evolutionary relationships and differences or similarities between characteristics (Flot et al. 2006). The linkage map developed in the Meyer Lab indicates that there are 12 to 14 chromosomes, and karyotyping is a way to confirm this number. Karyotyping is a procedure used to visualize chromosomes of cells, and the steps often include preparing a culture to maximize the number of metaphase cells, preparing slides ​ containing chromosome spreads, and utilizing methods to stain chromosomes (Bates 2011).

Chromosomes are visible during cell division when cellular DNA condenses into distinguishable chromosomes pairs. In this study, I compared methods for karyotyping the cells of Platygyra ​ daedalea to determine the most effective techniques for visualizing the nuclei of coral larval ​ tissue.

To examine the nuclei of Platygyra daedalea cells, preserved larval tissue collected from ​ ​ this organism must be prepared through methods of dissociation and fixation, and the nuclei of the cells must be appropriately stained to be visualized. The first part of this project compared three different methods of mechanical preparation, including complete dissociation, smearing using the squash method, and slight crushing of coral larvae. Chemical dissociation methods were then compared by analyzing the effects of two different detergents, CTAB and sodium borate, on the separation of single cells. Varied techniques of cell fixation were also tested in distinct locations (on the microscope slide versus in a 2.0-mL microcentrifuge tube) and in contrasting time intervals (overnight for 24 hours versus within the hour prior to staining). In the last step, different types of dyes including Giemsa, orcein, and DAPI were tested for comparison to see which worked the most efficiently to stain nuclei. The Giemsa stain is often employed in ​ histology due to its high-quality staining of the chromatin and nuclear membrane; orcein can be used in various combinations to stain chromosomes, elastic fibers, and connective tissue; and DAPI is a fluorescent stain that specifically binds to DNA for viewing nuclei and chromosomes in whole cells (Otto 1990; Barcia 2007). ​ ​ ​ Additionally, two different groups of preserved Platygyra daedalea larvae were ​ ​ examined and compared for viability. One group of larvae was treated with 0.02% colchicine and preserved by using a 2:1:1 mixture of ethanol, glacial acetic acid, and distilled water (Kenyon

1997; Flot et al. 2006). Colchicine is an agent that disrupts the assembly dynamics of microtubules during cell division, and it can be used to increase chances of viewing aligned chromosomes during the cell division cycle (Bhattacharyya et al. 2008). The other group of larvae was left untreated and preserved in ethanol. The preserved groups in this study were stored for one year at -20˚C in the case of the GBR group (preserved in ethanol) and two years at

4˚C in the case of the Persian Gulf group (treated with colchicine). Comparing the viability of ​ old coral larvae samples in terms of DNA content is valuable because it wastes fewer animals of a species that is at risk in the wild. Determining successful preservation methods can maximize the efficiency and utilization of limited resources in future projects. Conclusively, an experiment ​ using an adult tissue sample of a Platygyra species was performed to validate the functioning of ​ ​ the cell separation and DNA staining techniques that were determined to be the most effective.

The results of this project contribute to the validation of genomic resources and establishment of this species as a model for thermal tolerance research.

METHODS

Specimen collection

Platygyra daedalea larvae were collected in the Persian Gulf and Australia and preserved ​ using two different methods. Persian Gulf larvae was treated with 0.02% colchicine and preserved in a 2:1:1 mixture of ethanol, glacial acetic acid, and distilled water and were collected during the coral spawn in 2015 (Kenyon 1997; Flot et al. 2006). The other group were from the central Great Barrier Reef (GBR) collected during the coral spawn in 2017. These larvae were not treated with colchicine and were preserved in ethanol. Samples were then brought to Oregon

State University in Corvallis, OR, USA and stored in the Meyer Lab at -20˚C for the GBR samples and 4˚C for the Persian Gulf samples.

Comparing mechanical preparation methods

Three different methods of mechanical preparation were compared: complete dissociation of cells in a media solution, smearing of whole larvae using the squash method, and slight crushing of larvae using a pipette tip. To separate cells from preserved P. daedalea larvae using ​ ​ the first method, a modified cell dissociation technique was applied (Rosental et al. 2017).

Approximately 10-20 preserved larvae were placed into a dish with 1 mL of cell media (3.3 mL of 10x Phosphate Buffered Saline (PBS), 1.0 mL of 100mM 4-(2-hydroxyethyl)-1-piperazine- ethanesulfonic acid (HEPES), 1.5 mL of Fetal Bovine Serum (FBS), and 4.65 mL of deionized water). Using a small pipette tip, the solution was back pipetted for 10 minutes or until the coral larvae broke into smaller fragments. The sample was then filtered through 40-µm mesh, using a ​ ​ blunt edge of a scalpel to push the cells through, and collected in a 2.0-mL microcentrifuge tube. After being centrifuged for 5 minutes at 12˚C and 8,000 RCF, a small white pellet formed at the bottom. The pellet was resuspended in 0.1 mL of cell media. When viewed under the light microscope, smaller clumps and scattered single cells were seen.

As alternative methods to cell dissociation, the squash technique and a simple crushing method were tested for comparison. For the squash technique, one larva was deposited onto one end of a microscope slide. A coverslip was placed over the larva and slid over to the other end of the slide, smearing the larva. The slide was left to dry at room temperature. For the simple crushing method, approximately 2-3 larvae were placed onto a second slide. With the end of a

10-µL pipette tip, the larvae were carefully crushed into smaller pieces and left to dry. ​ ​

Comparing chemical dissociation methods

Detergents or enzymes can be used to assist with the chemical component of the cell dissociation step. When trying to separate single cells, mucinases and collagenases may act as dissociating reagents, and detergents can help to solubilize cell membranes (Lichtenberg 2013).

In this study, chemical dissociation methods were compared by analyzing the effects of two different detergents, sodium borate and CTAB, on the separation of larvae to cells. Each detergent was tested in separate trials. Starting with sodium borate, small quantities of a diluted sodium borate buffer solution (1X) was created in 2.0-mL microcentrifuge tubes. Two samples of the buffer solution were tested, one created with deionized water and the other with ethanol, to see which solvent works best to keep the cells intact. To create a 1X buffer solution in a 2.0-mL sample, 0.1 mL of the 20X sodium borate buffer solution was carefully pipetted into two small microcentrifuge tubes. One tube was then filled with deionized water, and the other tube was filled with ethanol up to the 2.0-mL line. The 1X buffer solutions were to be used as detergents to help segregate large clumps of cells into single cells for viewing under the microscope.

To begin a trial with 1X sodium borate buffer, approximately ten P. daedalea larvae were ​ ​ placed in 1.0 mL of cell media in a small dish. The solution was back-pipetted for 20 minutes to mechanically dissociate the tissue cells. 200 µL of each detergent solution was added to the ​ ​ resuspended cells in the tube. After letting the solutions incubate at room temperature for 3 to 5 minutes, the tubes were centrifuged again at 20˚C and the content at the bottom was resuspended in 0.1 mL of regular cell media. A drop consisting of 20 mL of each sample was placed onto a slide for viewing under the microscope.

For the next detergent dissociation method, a 2% CTAB buffer solution was created with ​ deionized water. CTAB, or cetyltrimethylammonium bromide, is a cationic detergent that is soluble in water (Maki et al. 1991). Approximately ten P. daedalea larvae were placed in 1.0 mL ​ ​ ​ of cell media in a small dish, and the solution was back-pipetted for 15 minutes. Coral larvae cells disintegrated into smaller fragments. The solution was filtered through 40-µm mesh, placed ​ ​ in a 2.0-mL microcentrifuge tube, and centrifuged for 5 minutes at 5,000 RCF and 12˚C. A small white pellet appeared at the bottom, and it was resuspended in 0.1 mL of regular cell media. The detergent was then added as 100 µL of 2% CTAB was placed into the solution with a pipette ​ ​ tube. The solution was left to sit for 3 minutes before viewing under the microscope.

Comparing techniques of cell fixation

When preparing the slides, two different methods of cell fixation were tested and compared in distinct locations (on the microscope slide versus in a 2.0-mL microcentrifuge tube) and in contrasting time intervals (overnight for 24 hours versus within the hour prior to staining).

The fixative solution was created with a 3:1 mixture of methanol and glacial acetic acid. To fix cells onto the microscope slide after preparation, a small amount of the fixative solution

(approximately 500 µL) was placed onto a mechanically prepared slide with a pipette tip. The ​ ​ fixed cells are then dried at room temperature, and the slide is ready for staining. In different trials, the slides were either left to incubate at room temperature overnight prior to staining or they were stained as soon as they were dried within the hour. For testing the second method, whole larvae were fixed by soaking in a small microcentrifuge tube with 1.0 mL of the fixative solution overnight and were prepared (via smearing or crushing) for staining the next day. As comparison, unfixed larvae were prepared on a slide, covered with the fixative, and were stained as soon as they were dried within the hour.

Comparing different types of dye

Three different dyes were compared in this project: Giemsa, orcein, and DAPI. With the

Giemsa dye, a 5-mL stock solution was created by combining 0.75 mL of Giemsa and 4.25 mL of deionized water. Approximately 20-45 mL of the stain was placed onto fixed slides with a ​ ​ coverslip, and the cells were observed under a light microscope for 45 minutes in 15-minute intervals. With the orcein dye, a 2% lacto-aceto-orcein staining solution was transferred onto a fixed microscope slide. The slide was left to incubate at room temperature for 5 minutes and then destained with lactic acid before viewing under a light microscope. Lastly, DAPI (4',

6-diamidino-2-phenylindole) is a specific, highly fluorescent stain suited for viewing DNA in whole cells, in nuclei, and in chromosomes (Otto 1990). The slides containing the fixed cells were incubated for 5 minutes before a few drops containing 50-100 µL of DAPI was placed onto ​ ​ the slide. After placing a coverslip over the thin film of dye on each slide, the samples were viewed under the fluorescence microscope in search of DNA.

Viability of different types of preserved larvae for karyotyping

Two different types of preserved larvae were used in this trial. One group is treated with

0.02% colchicine and preserved in a fixative (2:1:1 ethanol, glacial acetic acid, and distilled water), and the other group consisted of untreated larvae preserved in ethanol (Kenyon 1997;

Flot et al. 2006). Both groups were preserved for one to two years prior to the beginning of this project. Viability was determined through level of nuclei content in each set of larvae, which is observable with DAPI under the fluorescence microscope. The larvae preserved in ethanol remained the base group used for running trials to compare different nuclei visualization methods. After the most effective methods were determined, additional trials were performed to test and examine the colchicine-treated samples. The same preparation steps of slight crushing, fresh fixation, and DAPI staining were used to prepare the cells of both groups of larvae for

DNA visualization, and they were compared in terms of nuclei content under the fluorescence microscope.

Adult sample examination

To conclude this project, an examination of adult Platygyra cells was undertaken to ​ ​ investigate the effectiveness of the methods of nuclei visualization and preservation. In this adult sample experiment, a polyp from a live Platygyra species was obtained using forceps and ​ ​ carefully crushed using a razor blade prior to fixation. The cells were stained with DAPI as soon as they were fixed and dried, and nuclei of the adult cells were viewed under the fluorescence microscope. This conclusive trial validates the mechanism of the developed protocols for visualizing the nuclei of preserved larvae and coral cells. It also shows that the Flot preservation method for colchicine treated samples is not meant for long term storage at 4˚C.

RESULTS

Mechanical preparation

Three different methods were used to mechanically prepare cell samples: dissociation

(Figure 1), smearing (Figure 2), and slight crushing (Figure 3). In the first method, the cells ​ ​ ​ ​ ​ ​ were fully dissociated in media before they are fixed to the slides. The cell counts from the dissociation method yield approximately 19,000 cells per mL, and the imaging results are shown below:

Figure 1 The dissociation method successfully separated large clumps of unstained cells into ​ smaller clumps and eventually yielded single cells. The images above, under 400X magnification using brightfield microscopy, show this succession.

The second method used the squash technique and pre-fixed larvae that were preserved in the fixative solution overnight (Figure 2) . The third method also used pre-fixed larvae but with ​ ​ crushing the larvae with a pipette tip as opposed to smearing (Figure 3). The images below show ​ ​ the results of each trial examples after the specified mechanical preparation:

Figure 2 Unstained Platygyra daedalea cells from larvae that has been smeared across the ​ ​ ​ brightfield microscope slide with a coverslip, at 100X (left) and 400X (right) magnification.

Figure 3 Platygyra daedalea cells from larvae that has been carefully crushed with a pipette tip ​ ​ onto a microscope slide, at 100X magnification. These cells were irregularly shaped, which is to be expected from preserved non-living tissue. Figures 2 and 3 were obtained after recognizing ​ ​ ​ ​ that nuclei can still be visibly seen in dispersed tissue even without the complete separation of single cells. Chemical dissociation using detergents

In the cell dissociation method, large cell clumps were still seen after treatment.

Detergents were tested to see if they can help to gently break up cell groups. With sodium borate, the number of cells present in the sample subjected to the 1X water and sodium borate solution were observably higher than for the ethanol solution. This reveals that the detergent solution worked best with water as opposed to ethanol alcohol. Fewer cells were seen in the alcohol solution, and there was also more cell debris present. This indicated that the addition of chemical agents caused a higher number of cells to burst. Although the clumps were smaller than they were in past trials and a few separated cells were dispersed around the area, most of the cells were still grouped together. The cells were also more often seen as clumps. Due to the extensive amount of cell debris and the majority of cells residing within clumps, specific cell quantification could not be determined. While using CTAB as a detergent, there were visibly fewer cells (very ​ few if any cells were present). This indicated that the detergent was too harsh on cellular membranes. Sodium borate is therefore the more favorable detergent if a chemical dissociation step is required, although the addition of any detergent still created an environment too harsh for the preservation of single cells.

Cell fixation

The trials yielded the most favorable results when cells were freshly fixed and stained immediately after drying. More visible nuclei were seen on slides that were fixed right before staining. Although the differences were minimal for preserved cells, there was a big distinction when visualizing the nuclei of adult tissue cells. Additionally, there were no differences in results whether whole larvae were pre-fixed (soaked in the fixative solution overnight) or freshly fixed onto the slides. A similar concentration of visible nuclei were present in those trials when using preserved larvae tissue.

Staining and DNA visualization

Both Giemsa and orcein stained entire cells too darkly, leaving the nuclei inseparable from the rest of the cell. The only method of viewing nuclei was using DAPI and a fluorescence microscope. After slides of cells were fixed and dried at room temperature, all were subjected to the same staining protocol. The imaging results with DAPI under the fluorescence microscope for all three preparation methods are shown side by side below:

Figure 4 Platygyra daedalea cells that have been stained with DAPI after preparation (left to ​ ​ right) via A) dissociation, B) smearing, and C) crushing. Images viewed at 1000X magnification using fluorescent microscopy.

The first method of complete dissociation yielded many cases of disintegrated nuclei, shown by the smaller fluorescent spots indicating fragmented pieces of DNA (Figure 4A). The ​ ​ second method of smearing presents concentrated areas of whole nuclei along the edges of tissue matter (Figure 4B). Finally, the third technique of crushing proportionally shows dispersed ​ ​ tissue and intact nuclei throughout the cross section (Figure 4C). These same results are ​ ​ consistent and replicable with each trial.

Additional trials of this project examined colchicine-treated larvae along with tissue from an adult Platygyra species, as shown in Figures 5 and 6. Images of the colchicine-treated ​ ​ ​ ​ ​ ​ samples indicated no signs of nuclei or DNA material, while the adult tissue sample demonstrates that the protocol successfully yields images of nuclei.

Figure 5 Colchicine-treated Platygyra daedalea cells that have been stained with DAPI after ​ ​ ​ preparation using the crushing method and fixation. No nuclei can be seen. Images viewed at 1000X magnification using fluorescence microscopy.

Figure 6 Adult sample examination of a polyp from a living Platygyra species after preparation, ​ ​ ​ ​ ​ fixation, and staining with DAPI. Images viewed at 1000X magnification using fluorescence microscopy.

DISCUSSION

Due to increasing ocean temperatures, the resiliency of corals to thermal stress is a vital topic in this research field. The Platygyra daedalea is one of the corals in the Arabian/Persian ​ ​ Gulf that can endure high summer temperatures, which makes them ideal subjects for studying the mechanisms of thermal tolerance (Hume et al. 2013). Through its development as a model organism for thermal tolerance research, it is pertinent to know the specific chromosome number of this species. With the derived techniques refined within this project, I was able to pinpoint which preparation steps work the most efficiently to visualize the nuclei of preserved coral cells.

Varieties of protocols for the mechanical preparation of microscope slides, chemical dissociation of larvae tissue, fixation using different techniques, and DNA staining with various dyes were tested and compared. The results from each experiment were consistently replicated in numerous trials throughout the course of this project. All trials began with the first dissociative step or mechanical preparation method each time.

Although the cell dissociation protocol successfully separated single cells, this technique was proven to be too harsh on cellular membranes. As shown in Figure 4A, this method caused ​ ​ the nuclei within cells to break into smaller pieces of DNA. Therefore, a less abrasive technique was required. In the second technique that exercised the method of smearing, the tissue remained mostly intact. However, when viewed with DAPI under the microscope, a high number of nuclei were seen cluttered along the edges of tissue matter. This made it more difficult to see individual and distinct nuclei, thus harder to find dividing cells. The third method, using a small pipette tip to carefully crush coral larvae onto a microscope slide, yielded the most favorable results. This technique allowed for tissue to be dispersed well enough for viewing proportional amounts of nuclei while not shredding DNA matter in the process.

By using the simple crushing method, a chemical component is not necessary to accompany the mechanical step. When fixing cells to the slide, limiting the wait time between staining maximizes results for both preserved and adult tissue cells. Soaking the larvae overnight in a fixative yields no substantial change in results, since the larvae were already preserved in a fixative solution beforehand. The same approximate number of nuclei were visible in each trial comparing this fixative step.

During the experimentation with staining, the dye used in the Giemsa trials stained the cells too darkly, and the cells as well as the surrounding environment remained a dark shade of purple. Therefore, Giemsa does not work for viewing defined nuclei. In the next dye method using orcein, there is a de-staining method for this purpose. Multiple trials were performed in an attempt to stain the cells on a microscope slide using a lacto-aceto-orcein staining solution.

Afterwards, the method of de-staining with a lactic acid and acetic acid solution was applied to remove stain where it was unnecessary, since the imaging step aimed to only view the features of chromosomes. The de-staining method was applied in hopes that it would remove the orcein stain from all other parts of the cells other than the nucleus. However, the entire cells remained red even after the de-staining step, and the nuclei of cells were not visibly seen. Therefore, DAPI was the only dye that successfully differentiated DNA material from other parts of the cell under fluorescence microscopy. The staining step using DAPI remained consistent for all trials and proved to work every time, including for the adult sample.

Conclusively, the colchicine-treated samples yielded no fluorescent indications of nuclei or any DNA, suggesting that the conserved larvae underwent DNA degradation during the preserving process. Colchicine-treated larvae are therefore not viable when preserved for long periods of time. Only the larvae preserved in ethanol proved to successfully conserve DNA material over time. Revisiting the prospect of using colchicine as an agent to increase chances of finding cells in metaphase would be a progressive step with this intention (Axelrad et al. 1958).

In closing, the adult sample examination proved that the mechanical preparation step of crushing, fixing with a methanol-acetic acid solution immediately prior to staining, and using DAPI as fluorescent dye resulted in a successful viewing of cellular DNA in the form of nuclei. These methods can be applied in future projects with aims to gather imaging proof of the chromosome count of coral species in support of thermal tolerance research and other related fields.

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