Ariana Symbiont Integration Project Physiological Samples Collected in July 2020

Processing Ariana’s Montipora capitata Larval Development Physiological Samples from July 2020

Goal:

Process all of the physiological data (i.e. counts, size, symbiont counts) for the Montipora capitata (vertical spawner) developmental timepoint samples (n=40). Samples were collected at the Hawaii Institute of Marine Biology in July 2020 and are apart of a larger project which focuses on early life history energetics and symbiotic integration in vertical and horizontal transmission corals.

Contents

Count Protocol

Materials

Protocol Steps

  1. I removed the physiological larval samples (H1-40) from the -4 °C fridge behind the titrator.
  2. I carefully used 70% ethanol and the glass pipette to remove all of the contents of each tube into a cell culture dish.
  3. I first counted one of the fertilized egg replicates by hand to get a total of 1,047 fertilized eggs total in sample H1. After discussing with Ariana, we decided to take a subset (roughly 200-300 fertilized eggs) of samples that had large amounts in them. The first 8 samples (H1 to H8) had large amounts so I labeled new tubes (ex: H1_1, H2_1, H3_1….H8_1) and took a subset sample (~200-300).
  4. To count each sample, I repeated step 2 and then counted all the individuals in each tube moving the contents in one cell culture dish to the next whilst keeping track of counts using the hand held tally counter.
  5. If I was ever concerned that individuals were stuck together or too small to count by eye, I placed the cell culture dish under the 4x magnification on the compound light microscope.
  6. In between each count, I cleaned the glass pipette and cell culture dishes with the 70% ethanol solution.

Count data for all samples (n=40) completed on 20210304

tube.ID lifestage date num.sampled count initials notes
H1-1 Egg Fertilized 20210304 subset 356 DMB  
H2-1 Egg Fertilized 20210304 subset 259 DMB  
H3-1 Egg Fertilized 20210304 subset 256 DMB  
H4-1 Egg Fertilized 20210304 subset 269 DMB  
H5-1 Embryo 1 20210304 subset 254 DMB  
H6-1 Embryo 1 20210304 subset 232 DMB  
H7-1 Embryo 1 20210304 subset 306 DMB  
H8-1 Embryo 1 20210304 subset 214 DMB  
H9 Larvae 1 20210304 full.sample 218 DMB some slightly degraded
H10 Larvae 1 20210304 full.sample 294 DMB some slightly degraded
H11 Larvae 1 20210304 full.sample 189 DMB some slightly degraded
H12 Larvae 1 20210304 full.sample 119 DMB some slightly degraded
H13 Larvae 2 20210304 full.sample 105 DMB  
H14 Larvae 2 20210304 full.sample 38 DMB  
H15 Larvae 2 20210304 full.sample 49 DMB  
H16 Larvae 2 20210304 full.sample 40 DMB  
H17 Larvae 3 20210304 full.sample 66 DMB  
H18 Larvae 3 20210304 full.sample 88 DMB  
H19 Larvae 3 20210304 full.sample 76 DMB  
H20 Larvae 3 20210304 full.sample 38 DMB  
H21 Larvae 4 20210304 full.sample 208 DMB  
H22 Larvae 4 20210304 full.sample 118 DMB  
H23 Larvae 4 20210304 full.sample 107 DMB  
H24 Larvae 4 20210304 full.sample 176 DMB  
H25 Larvae 5 20210304 full.sample 55 DMB  
H26 Larvae 5 20210304 full.sample 48 DMB  
H27 Larvae 5 20210304 full.sample 40 DMB  
H28 Larvae 5 20210304 full.sample 47 DMB  
H29 Larvae 6 20210304 full.sample 16 DMB  
H30 Larvae 6 20210304 full.sample 19 DMB  
H31 Larvae 6 20210304 full.sample 21 DMB  
H32 Larvae 6 20210304 full.sample 16 DMB  
H33 Recruit 1 20210304 full.sample 13 DMB  
H34 Recruit 1 20210304 full.sample 19 DMB  
H35 Recruit 1 20210304 full.sample 20 DMB  
H36 Recruit 1 20210304 full.sample 33 DMB  
H37 Recruit 2 20210304 full.sample 10 DMB  
H38 Recruit 2 20210304 full.sample 12 DMB  
H39 Recruit 2 20210304 full.sample 6 DMB  
H40 Recruit 2 20210304 full.sample 7 DMB  

Notes for count data completed on 20210304

  • Noticed that the larvae 1 timepoint replicates were slightly degraded but still had a sufficient amount of larvae that was good

Size Protocol

Materials

Protocol Steps

  1. I used a glass pipette to carefully remove ~20-30 individuals from each of the sample tubes.
  2. I aligned the cell culture dish underneath the dissecting microscope with the light on medium to high and positioned the cell culture dish next to the 0.01 mm stage micrometer so that the total length of the scale could be seen within the photo along with all the individuals.
    • make sure to keep some ethanol/water in the sample to reduce reflections of light and to have a more even photo
    • it is very important to have the scale micrometer within each individual photo as the zoom and focus may change on the microscope in between samples and you will need it to calibrate your photos later on in ImageJ

  1. Once the stage micrometer and individuals were focused and clear in view, I used the ToupView software to snap a photo and save it to the windows computer.
  2. I uploaded the photos to Google Drive and then when I was ready to size the individuals I would open them in ImageJ.
    • Refer to the ImageJ manual with any questions on how to calibrate or how to use the freehand tool
  3. I opened (File > Open) each individual photo into ImageJ and used the text tool to randomly select and label a sub-sample of 10 individuals per photo.
    • make a small text box next to each individual you will size and label from 1-10, press command D after each text box to overlay it on the photo
    • save the labeled photo before calibrating the scale in ImageJ
  4. I selected the Straight Line Selection Tool in ImageJ and zoomed in to the scale bar on the stage micrometer. I then carefully drew a line from each end of the 0.01 mm scale bar. I then set the number of pixels to a known distance of 1.0 and the unit length to mm. I also click on global so that it will be set for as long as I have this image open.

  1. I then use the freehand selections tool to carefully draw a circle around the entire outline of each individual.
    • make sure to go slow and zoom in as far as possible with the individual still in focus when doing this step.
  2. After outlining the individual, I press command M (measure) and a small data box will pop up with the calculated surface area of the individual in mm.
    • make sure to keep an eye on these measurements and if you see them fluctuating or having large numbers, do the set scale step above again
  3. I then save the photo again and transfer the measured values into a csv document.
  4. All labeled images were uploaded here.

Symbiont Count Protocol

I had to first troubleshoot various methods so that we could properly homogenize the eggs to larvae developmental stage samples. I had to ensure that the symbiont cells were not lysing or degrading due to the sonicator. I used 1 mL aliquots of Emma’s adult Mcap (n = 2) and Pacuta (n = 1) tissue homogenate samples and Hollie’s Mcap developmental timepoint samples (n = 6) to test sonicator and homogenizing methods.

Materials

Protocol Steps

Followed the E5 Symbiodiniaceae Cell Density Counting Protocol with a few modifications for sonicator testing.

  1. Since the sonicator had a 220V outlet intended for use in Mo’orea, we had to order a transformer to convert the voltage safely.

Always read the instructions carefully when operating a transformer and make sure there is no liquid around the transformer

  1. Once unpacked, I plugged the transformer into a 110V outlet and read the instructions carefully. I changed the INPUT to the transformer to 110V (this step is extremely important, make sure the voltage you plug the transfromer into matches the INPUT tab located in the back of the transformer).

  1. With the transformer turned to OFF, I plugged in the sonicator into the 220V outlet on the transformer and tested that it turned on with no noise or spark.

  1. Following the sonicator instruction manual, I filled the sonicator 2/3rds of the way which was 2,000 mL.
    • This specific sonicator does not have a way to change the frequency settings, you can only adjust the time it runs for and the temperature it runs at.

  1. Placed a combination tube rack with the 1.5 mL tube size facing up to hold the test tubes while the sonicator runs.

  1. Tested the sonicator by adjusting the time setting in increasing intervals and using subsequent counts to make sure cells were not being lysed or degraded.

Symbiont count data for test samples completed on 20210321

  • I used Emma’s adult tissue homogenate Pacuta and Mcap samples for this first round.
tube.ID time.sonicated date.counted num.squares count1 count2 count3 count4 count5 count6 count7 count8 # individuals volume (uL) avg.per.square cells.uL avg.per.individual std.per.square cv initials notes
TEST_P1432 before sonication 20210321 3 194 232 200 176 195 182         65.5     6.517497816 9.950378345 DMB 1 mL aliquot of adult tissue slurry from Emmas Holobiont Integration samples; sonicator test samples
TEST_M2184 before sonication 20210321 2 119 144 147 126 156 115 128 135     66.875     7.170126518 10.72168451 DMB 1 mL aliquot of adult tissue slurry from Emmas Holobiont Integration samples; sonicator test samples
TEST_P1170 before sonication 20210321 6 109 122 148 136 138 133         21.83333333     2.275473089 10.42201415 DMB 1 mL aliquot of adult tissue slurry from Emmas Holobiont Integration samples; sonicator test samples
TEST_P1432 1 minute 20210321 3 189 194 206 211 210 169         65.5     5.377938473 8.210593089 DMB symbionts look good, consistent counts
TEST_M2184 1 minute 20210321 2 142 112 135 125 131 127 137 138     65.4375     4.761733628 7.276765811 DMB symbionts look good, consistent counts
TEST_P1170 1 minute 20210321 6 120 118 139 128 131 140         21.55555556     1.540803061 7.14805544 DMB symbionts look good, consistent counts
TEST_P1432 2 minutes 20210321 3 229 222 193 213             71.41666667     5.202385493 7.284553782 DMB symbionts look good, consistent counts
TEST_M2184 2 minutes 20210321 2 131 115 123 154             65.375     8.410063416 12.86434175 DMB symbionts look good, consistent counts
TEST_P1170 2 minutes 20210321 6 120 144 147 146             23.20833333     2.14896616 9.259459216 DMB symbionts look good, consistent counts
TEST_P1432 3 minutes 20210321 3 210 230 196 210             70.5     4.662696724 6.613754219 DMB symbionts look good, consistent counts
TEST_M2184 3 minutes 20210321 2 135 126 121 126             63.5     2.915475947 4.591300705 DMB symbionts look good, consistent counts
TEST_P1170 3 minutes 20210321 6 110 121 127 146             21     2.512929528 11.96633109 DMB symbionts look good, consistent counts
TEST_P1432 4 minutes 20210321 3 202 222 212 180             68     5.987641593 8.805355285 DMB symbionts look good, consistent counts
TEST_M2184 4 minutes 20210321 2 128 121 114 103             58.25     5.330728531 9.151465289 DMB symbionts look good, lower counts
TEST_P1170 4 minutes 20210321 6 132 119 148 136             22.29166667     1.992462649 8.938150201 DMB symbionts look good, consistent counts
TEST_P1432 5 minutes 20210321 3 173 209 210 211             66.91666667     6.172669751 9.224413077 DMB saw a few lysed/degraded cells, lower counts
TEST_M2184 5 minutes 20210321 2 112 114 128 121             59.375     3.63719214 6.125797289 DMB saw a few lysed/degraded cells, lower counts
TEST_P1170 5 minutes 20210321 6 122 133 121 132             21.16666667     1.062840359 5.021293037 DMB saw a few lysed/degraded cells, lower counts

Notes for 20210321

  • I started to notice that cells started to lyse and degrade after a total time in sonicator of 15 minutes. The next step was to test Hollie’s 2018 Mcap developmental timeseries samples, making sure to test samples from eggs to larvae.

Symbiont count data for test samples completed on 20210323

  • I used Hollie’s 2018 Mcap developmental timeseries samples from fertilized eggs, gastrula, and larvae stages.
tube.ID time.sonicated date.counted num.squares count1 count2 count3 count4 count5 count6 count7 count8 # individuals volume (uL) avg.per.square cells.uL avg.per.individual std.per.square cv initials notes
TEST_84 10 minutes 20210323 9 24 46 43 30 26 31         3.703703704     1.007788599 27.21029217 DMB were not breaking up in 5 minutes, used plastic pestle to breakdown, sonicated for another 10 minutes and that worked
TEST_55 10 minutes 20210323 8 103 111 109 112 106 110         13.5625     0.423895624 3.125497688 DMB were not breaking up in 5 minutes, used plastic pestle to breakdown, sonicated for another 10 minutes and then had to homogenize
TEST_35 10 minutes 20210323 9 67 44 48 53 60 45         5.87037037     1.01206711 17.24025992 DMB were not breaking up in 5 minutes, used plastic pestle to breakdown, sonicated for another 10 minutes and that worked

Notes for 20210323

  • I noticed that the sonicator seemed to be more just mixing the samples but did not seem strong enough to breakdown the samples on its own. We decided to use a plastic pestle to break apart the initial layer of the individuals and sonicated them again. It seemed to work on two of the samples but not the last one and I did not want to reach the limit where the cells would start to lyse. I instead used the plastic pestle, the homogenizer, and the vortex to get the samples to fully homogenize within the tubes. We decided this was the best step moving forward since the sonicator did not have a setting to adjust the frequency.

Symbiont count data for test samples and Ariana samples completed on 20210325

  • I used Hollie’s 2018 Mcap developmental timeseries samples from fertilized eggs, cleaving eggs, and swimming larvae stages to test the plastic pestle, homogenizer, and vortex sequence of steps again before moving forward. Once we were satisfied with how well the samples were homogenized using this method, I moved forward with counts for Ariana’s samples.

Symbiont count data with Hollie’s samples
tube.ID time.sonicated date.counted num.squares count1 count2 count3 count4 count5 count6 count7 count8 # individuals volume (uL) avg.per.square cells.uL avg.per.individual std.per.square cv initials notes
TEST_38 none 20210325 9 85 94 89 108 87 85     19 500 10.14814815 5074.074074 133528.27 0.980467681 9.661542837 DMB testing pestle, homogenizing, vortexing; fertilized eggs
TEST_56 none 20210325 8 124 121 124 125 112 110     55 500 14.91666667 7458.333333 67803.03 0.827898947 5.550160539 DMB testing pestle, homogenizing, vortexing; cleaving eggs
TEST_348 none 20210325 7 112 105 109 111 104 106     18 300 15.4047619 4621.428571 77023.81 0.473085113 3.071031643 DMB testing pestle, homogenizing, vortexing; swimming larvae
TEST_344 none 20210325 8 104 103 106 100 105 104     38 500 12.95833333 6479.166667 85252.19 0.25819889 1.992531625 DMB testing pestle, homogenizing, vortexing; swimming larvae

Symbiont count data with Ariana’s samples (n=4)

tube.ID lifestage date.counted num.squares count1 count2 count3 count4 count5 count6 count7 count8 avg.per.square std.per.square cv total.volume.ul num.individuals initials notes
H1-1 Egg Fertilized 20210325 2 135 133 157 170 174 170 175 173 80.4375 8.756375229 10.88593657 500 356 DMB  
H2-1 Egg Fertilized 20210325 2 179 167 165 171 171 169     85.16666667 2.422120283 2.843976849 500 259 DMB  
H3-1 Egg Fertilized 20210325 2 160 154 163 171 155 170     81.08333333 3.625143675 4.470886342 500 256 DMB  
H4-1 Egg Fertilized 20210325 2 156 159 174 170 177 171     83.91666667 4.212085786 5.019367371 500 269 DMB  

Notes for 20210325

  • Happy with how the sample counts turned out and how homogenized they were for the symbiont counts. There was some tissue remains left within the samples under the microscope but not enough to disturb the accuracy of the counts.

Symbiont count data for Ariana samples completed on 20210328 (n=6)

tube.ID lifestage date.counted num.squares count1 count2 count3 count4 count5 count6 count7 count8 avg.per.square std.per.square cv total.volume.ul num.individuals initials notes
H5-1 Embryo 1 20210328 2 160 184 193 187 189 190     91.91666667 6.028404985 6.558554834 500 254 DMB  
H6-1 Embryo 1 20210328 2 192 188 181 186 187 182     93 2.024845673 2.177253412 500 232 DMB  
H7-1 Embryo 1 20210328 2 186 180 179 186 183 179     91.08333333 1.655797894 1.817893388 500 306 DMB  
H8-1 Embryo 1 20210328 2 187 179 169 170 156 178     86.58333333 5.342440142 6.170286978 500 214 DMB  
H9 Larvae 1 20210328 2 112 119 120 118 107 125     58.41666667 3.184598352 5.451523571 500 218 DMB  
H10 Larvae 1 20210328 2 137 130 129 122 131 136     65.41666667 2.709551008 4.141988803 500 294 DMB  

Notes for 20210328

  • All the counts went well and the CV’s all checked out.

Symbiont count data for Ariana samples completed on 20210330 (n=10)

tube.ID lifestage date.counted num.squares count1 count2 count3 count4 count5 count6 count7 count8 avg.per.square std.per.square cv total.volume.ul num.individuals initials notes
H11 Larvae 1 20210330 3 112 116 110 110 108 105     36.72222222 1.236781965 3.367938786 500 189 DMB  
H12 Larvae 1 20210330 3 135 143 143 156 151 155     49.05555556 2.735906648 5.577159644 500 119 DMB  
H13 Larvae 2 20210330 3 129 131 128 125 131 130     43 0.76011695 1.767713837 500 105 DMB  
H14 Larvae 2 20210330 5 115 117 115 118 110 111     22.86666667 0.640832792 2.802475765 500 38 DMB  
H15 Larvae 2 20210330 5 122 115 116 125 120 130     24.26666667 1.129011367 4.652519369 500 49 DMB  
H16 Larvae 2 20210330 5 128 130 123 129 112 110     24.4 1.775387282 7.276177384 500 40 DMB  
H17 Larvae 3 20210330 3 100 109 103 106 101 105     34.66666667 1.115546702 3.217923179 500 66 DMB  
H18 Larvae 3 20210330 3 120 113 107 111 112 107     37.22222222 1.600925658 4.300994306 500 88 DMB  
H19 Larvae 3 20210330 3 115 114 106 101 103 102     35.61111111 2.059305888 5.782762244 500 76 DMB  
H20 Larvae 3 20210330 4 105 110 110 120 121 106     28 1.724818831 6.160067254 500 38 DMB  

Notes for 20210330

  • All the counts went well and the CV’s all checked out.

Symbiont count data for Ariana samples completed on 20210401 (n=20)

tube.ID lifestage date.counted num.squares count1 count2 count3 count4 count5 count6 count7 count8 avg.per.square std.per.square cv total.volume.ul num.individuals initials notes
H21 Larvae 4 20210401 1 101 100 102 101 105 102     101.8333333 1.722401424 1.691392561 500 208 DMB  
H22 Larvae 4 20210401 2 120 146 143 135 146 151     70.08333333 5.598362856 7.988151519 500 118 DMB  
H23 Larvae 4 20210401 2 147 148 140 143 134 154     72.16666667 3.473710792 4.813456063 500 107 DMB  
H24 Larvae 4 20210401 2 180 179 181 186 193 180     91.58333333 2.709551008 2.95856343 500 176 DMB  
H25 Larvae 5 20210401 3 107 117 114 109 108 105     36.66666667 1.5202339 4.146092455 500 55 DMB  
H26 Larvae 5 20210401 3 102 104 116 110 100 108     35.55555556 1.962613526 5.519850541 500 48 DMB  
H27 Larvae 5 20210401 4 138 113 110 112 112 124     29.54166667 2.731376332 9.245843719 500 40 DMB  
H28 Larvae 5 20210401 4 121 117 111 122 118 125     29.75 1.21449578 4.082338757 500 47 DMB  
H29 Larvae 6 20210401 6 115 111 110 112 116 118     18.94444444 0.523520844 2.763453135 500 16 DMB  
H30 Larvae 6 20210401 7 114 112 108 113 121 115     16.26190476 0.608891041 3.744278727 500 19 DMB  
H31 Larvae 6 20210401 6 118 121 115 123 120 116     19.80555556 0.510083508 2.575456703 500 21 DMB  
H32 Larvae 6 20210401 6 107 105 111 109 110 109     18.08333333 0.361324723 1.998109068 500 16 DMB  
H33 Recruit 1 20210401 7 101 100 102 104 103 100     14.52380952 0.233284737 1.606222782 500 13 DMB  
H34 Recruit 1 20210401 6 117 121 118 119 121 118     19.83333333 0.278886676 1.406151305 500 18 DMB  
H35 Recruit 1 20210401 6 114 113 116 110 112 112     18.80555556 0.340206909 1.809076619 500 20 DMB  
H36 Recruit 1 20210401 5 118 120 116 117 110 116     23.23333333 0.67428975 2.902251434 500 33 DMB  
H37 Recruit 2 20210401 6 110 115 108 110 108 107     18.27777778 0.479196859 2.621745733 500 16 DMB  
H38 Recruit 2 20210401 9 110 120 111 113 108 115     12.53703704 0.473581921 3.777462881 500 12 DMB  
H39 Recruit 2 20210401 9 89 88 92 75 93 96     9.87037037 0.817755636 8.284953908 500 6 DMB  
H40 Recruit 2 20210401 s 84 85 71 77 86 91     9.148148148 0.794010501 8.679467009 500 7 DMB  

Notes for 20210401

  • All the counts went well and the CV’s all checked out. All symbiont counts completed.

Coral Plugs Protocol

Process all of the physiological data (i.e. size and symbiont counts) for the Montipora capitata (vertical spawner) coral plugs with recruits (n=8).

Notes for 20211018

First - I took photos of each individual plug for surface area measurements.

Materials

Protocol Steps

  1. I aligned the coral plug underneath the dissecting microscope with the light on medium to high and positioned the coral plug next to the 0.01 mm stage micrometer so that the total length of the scale could be seen within the photo along with all the individuals.
    • it is very important to have the scale micrometer within each individual photo as the zoom and focus may change on the microscope in between samples and you will need it to calibrate your photos later on in ImageJ
  2. Once the stage micrometer and coral plugs were focused and clear in view, I used the ToupView software to snap a photo and save it to the windows computer.
  3. I uploaded the photos to Google Drive and then when I was ready to size the individuals I would open them in ImageJ.
    • Refer to the ImageJ manual with any questions on how to calibrate or how to use the freehand tool
  4. I selected the Straight Line Selection Tool in ImageJ and zoomed in to the scale bar on the stage micrometer. I then carefully drew a line from each end of the 0.01 mm scale bar. I then set the number of pixels to a known distance of 1.0 and the unit length to mm. I also click on global so that it will be set for as long as I have this image open.

  1. I then use the freehand selections tool to carefully draw a circle around the entire outline of each recruit on the top of the coral plugs.
    • make sure to go slow and zoom in as far as possible with the individual still in focus when doing this step.
  2. After outlining the individual, I press command M (measure) and a small data box will pop up with the calculated surface area of the individual in mm.
    • make sure to keep an eye on these measurements and if you see them fluctuating or having large numbers, do the set scale step above again
  3. I then save the photo again and transfer the measured values into a csv document.
  4. All labeled images were uploaded here.

Second - I counted the number of endosymbionts within each coral recruit per plug for symbiont density.

Materials

  • Glass pipette
  • OMAX compound light microscope
  • Vortex
  • Haemocytometer
  • Hand held tally counter
  • 200 uL pipette
  • 1.5 mL microcentrifuge tubes
  • Homogenizer
  • 70% ethanol
  • 10% bleach
  • DI water

Protocol Steps

Followed the E5 Symbiodiniaceae Cell Density Counting Protocol with a few modifications for removing recruits from coral plugs.

  1. First, I put 100 uL of Type II DI water into 8 individually labeled microcentrifuge tubes.
  2. I then sterilized a scalpel with 10% bleach, DI water, and 70% ethanol before carefully removing each individual coral recruit from the coral plugs. I lightly applied pressure underneath each coral recruit with the scalpel and slowly moved the scalpel underneath the coral recruit until it was fully removed and could be placed into the labeled 1.5 mL microcentrifuge tube.
  3. I used the small attachment head on the homogenizer and individually homogenized each microcentrifuge tube sample for 30 seconds and sterilized the homogenizer in between each sample with the 10% bleach, DI water, and 70% ethanol solutions.
  4. I then followed the E5 Symbiodiniaceae Cell Density Counting Protocol and recorded the final endosymbiont counts here.

Symbiont count data for coral plug samples completed on 20211018

lifestage date.counted num.squares count1 count2 count3 count4 count5 count6 count7 count8 avg.per.square std.per.square cv total.volume.ul num.individuals initials
Plug Mcap1 20211018 2 142 132 123 137 113 150   66.41666667 6.658953872 10.02602841 100 NA DMBP
Plug Mcap2 20211018 2 136 140 141 133 129 145   68.66666667 2.909753712 4.237505406 100 NA DMBP
Plug Mcap4 20211018 2 150 141 147 156 151 143   74 2.75680975 3.725418582 100 NA DMBP
Plug Mcap5 20211018 2 189 176 200 196 195 201   96.41666667 4.641300105 4.813794405 100 NA DMBP
Plug Mcap6 20211018 2 200 196 189 176 191 201   96.08333333 4.619704175 4.808018222 100 NA DMBP
Plug Mcap8 20211018 2 189 199 187 175 202 187   94.91666667 4.841659495 5.100958204 100 NA DMBP
Plug Mcap9 20211018 2 172 180 198 200 186 191   93.91666667 5.370443805 5.718307512 100 NA DMBP
Plug Mcap10 20211018 2 198 204 191 196 199 186   97.83333333 3.172801076 3.243067539 100 NA DMBP
Written on March 4, 2021