WEBVTT 1 00:00:06.860 --> 00:00:12.581 Hello, my name is Meri Bilan and an I'm a PhD student of the University of Salento. 2 00:00:13.230 --> 00:00:17.959 My thesis focused on the physiology and ecology of cold-water corals from Blanes 3 00:00:18.259 --> 00:00:23.520 submarine canyon. In this presentation, we will discuss the methods for measuring 4 00:00:23.820 --> 00:00:28.910 growth in scleractinian corals and respiration for non-symbiotic animals. 5 00:00:30.360 --> 00:00:35.672 Before we go into more detail, here is a reminder that the organisms used in the 6 00:00:35.872 --> 00:00:40.722 experiments are collected from wild populations using remotely operated 7 00:00:41.022 --> 00:00:43.300 vehicles during research cruises. 8 00:00:44.810 --> 00:00:49.952 Due to their life characteristics such as longevity, low fecundity, and vulnerability 9 00:00:50.152 --> 00:00:54.413 to environmental change, it is difficult to maintain them in large numbers for 10 00:00:54.613 --> 00:00:59.594 long periods to establish a controlled population as done with model organisms. 11 00:01:00.924 --> 00:01:05.273 But significant progress is made in the biology and ecology of these species 12 00:01:05.573 --> 00:01:09.460 and in the future we may be able to use them as model organisms. 13 00:01:11.100 --> 00:01:15.430 Here is a video collected during the ABRIC cruise in February 2020. 14 00:01:15.730 --> 00:01:20.461 The lasers are 10cm apart so you can have an idea of the size of the colony. 15 00:01:20.970 --> 00:01:26.360 We are sampling Muriceides lepida, a gorgonian coral with the ROV Liropus. 16 00:01:27.272 --> 00:01:33.394 The ROV needs to settle on a stable surface such as the seabed from where it can collect 17 00:01:33.694 --> 00:01:39.296 the animal in the fastest way possible. Then it uses this arm which we modified 18 00:01:39.596 --> 00:01:42.377 a bit using strainers to facilitate the collection. 19 00:01:43.607 --> 00:01:49.709 After collection, the coral is put in a biobox which is like a drawer on the ROV where it 20 00:01:49.909 --> 00:01:51.900 is stored until the arrival to the deck. 21 00:02:14.770 --> 00:02:20.221 We will be talking about nubbin preparation as it is a part of experimental preparation. 22 00:02:20.932 --> 00:02:25.963 Once we come back from the cruise and select the corals to be used in the experiment, 23 00:02:26.363 --> 00:02:31.405 we can put them on support bases, like you can see here, that can help maintain them 24 00:02:31.705 --> 00:02:34.790 in the same position during the experimental period. 25 00:02:35.360 --> 00:02:39.819 Growth is one of the basic measurements acquired in experiments. For scleractinian 26 00:02:40.019 --> 00:02:46.330 corals, this can be done with buoyant weight since they have extensive aragonite skeletons. 27 00:02:47.590 --> 00:02:54.121 Octocorals and black corals have mainly protein skeletons and tissues, so buoyant 28 00:02:54.421 --> 00:02:59.160 weight is not the best option, as their calcification rates are none for black 29 00:02:59.360 --> 00:03:05.810 corals and very, very small for octocorals. In those cases, photographs can be used 30 00:03:06.110 --> 00:03:07.210 to measure growth. 31 00:03:09.310 --> 00:03:15.531 So after the collection of animals, we leave them for some time to adjust to 32 00:03:15.831 --> 00:03:19.454 the new environment, which is the laboratory. Based on which species 33 00:03:19.754 --> 00:03:25.287 you're working with, you need to prepare the laboratory, meaning stabilised water 34 00:03:25.587 --> 00:03:31.529 temperature, salinity, water flux, choose what food to give them, how and when 35 00:03:31.829 --> 00:03:34.400 to clean the aquarium, and so on. 36 00:03:35.300 --> 00:03:40.270 When setting up an experiment, we choose healthy-looking fragments. A fragment is a 37 00:03:40.570 --> 00:03:47.480 piece of a coral colony. A healthy fragment has extended polyps and no tissue damage. 38 00:03:48.281 --> 00:03:54.200 We then cut them into smaller pieces called nubbins which we will use in the experiment. 39 00:03:54.400 --> 00:04:00.500 When using a zip tie cutter or similar tool, be careful not to harm yourself. 40 00:04:01.420 --> 00:04:06.773 When we have the nubbins ready, we set up a high-precision balance on a stable 41 00:04:06.973 --> 00:04:11.692 surface where we can slide in an aquarium because we don't want to remove the 42 00:04:11.992 --> 00:04:16.170 nubbins from the water, as it can change the their weight. 43 00:04:18.100 --> 00:04:21.380 We want to measure the weight of the corals while in seawater. 44 00:04:22.128 --> 00:04:26.418 So we hook a so-called basket on the bottom side of the balance. 45 00:04:27.449 --> 00:04:32.488 You can check their manufacturers' notes on how to install it, and then we measure 46 00:04:32.788 --> 00:04:39.751 temperature, salinity with a separate probe and weight of the nubbin from the balance. 47 00:04:40.481 --> 00:04:44.533 Temperature and salinity are important factors for density calculations and we 48 00:04:44.833 --> 00:04:47.480 will go into more detail about that later. 49 00:04:48.900 --> 00:04:53.591 When we finished weighing all the nubbins, we can choose to put them on support bases 50 00:04:53.891 --> 00:04:58.669 to keep them upright, but you can choose not to do that. Depends what you want to 51 00:04:58.869 --> 00:05:03.781 do with experiment. In case you choose to put them on bases, firstly you should have 52 00:05:04.081 --> 00:05:10.300 bases available. They can be made of glass, plastic, rock or some other inert material. 53 00:05:11.300 --> 00:05:16.801 Then we use aquaria-suitable glue or epoxy to fix the nubbins on the support. 54 00:05:17.101 --> 00:05:22.294 It is usually good to leave the corals on a support overnight to make sure the glue 55 00:05:22.494 --> 00:05:28.543 or epoxy has settled. Once they have settled you can repeat the same procedure to weigh 56 00:05:28.843 --> 00:05:30.430 the corals on the bases. 57 00:05:31.320 --> 00:05:35.259 Every subsequent measure in the experiment will be done with the base. 58 00:05:35.559 --> 00:05:40.181 So in order to know how much the coral grew, you need to subtract the weight 59 00:05:40.481 --> 00:05:41.580 of the base. 60 00:05:45.650 --> 00:05:50.270 Here's a summary on the nubbin preparation and buoyant weight technique, 61 00:05:50.570 --> 00:05:54.160 and don't forget to add a probe measuring salinity and temperature. 62 00:05:58.200 --> 00:06:03.712 Here is a video from the lab at the Institut de Ciencies del Mar, ICM, in Barcelona. 63 00:06:04.982 --> 00:06:09.583 This is the high precision balance and as you can see it is on a support that enables 64 00:06:09.883 --> 00:06:14.284 the aquaria to slide below. There are many different ways of doing this, so feel free to 65 00:06:14.584 --> 00:06:19.675 experiment. Then we have the coral with the base and the temperature and salinity 66 00:06:19.975 --> 00:06:23.613 probe that we saw before. Here is the basket that is hooked at the bottom 67 00:06:23.913 --> 00:06:30.495 of the slide and here we are putting the coral onto the basket, making sure it 68 00:06:30.795 --> 00:06:35.373 doesn't touch the walls of the aquarium. And now we wait for the balance to 69 00:06:35.574 --> 00:06:40.300 stablise and note down the weight and temperature and salinity. 70 00:06:48.430 --> 00:06:53.739 So what do we do next with that data? Jokiel et al published this paper "Coral 71 00:06:53.939 --> 00:06:58.868 growth buoyant weight technique" in 1978. They described the method of measuring 72 00:06:59.168 --> 00:07:03.410 scleractinian corals in seawater following the Archimedes principle. 73 00:07:04.840 --> 00:07:09.102 To recap, the Archimedes principle states that the weight of an object 74 00:07:09.302 --> 00:07:14.891 in air is equal to the weight of the object in a liquid plus the weight of 75 00:07:15.191 --> 00:07:17.200 the displaced water by the object. 76 00:07:19.660 --> 00:07:25.220 We can do several substitutions based on density, volume, and masse relation. 77 00:07:25.820 --> 00:07:32.731 Therefore the weight of the displaced liquid equals the volume of the liquid multiplied 78 00:07:33.031 --> 00:07:38.591 by its density, and since the volume of the liquid is the same volume of the object, 79 00:07:39.191 --> 00:07:45.882 we can use the same relation - density volume and mass relation - to calculate 80 00:07:46.182 --> 00:07:47.500 the volume of the object. 81 00:07:49.640 --> 00:07:56.160 This brings us to the formula where the weight of the coral in air equals the 82 00:07:56.460 --> 00:08:02.610 weight of the coral in sea water divided by 1 minus the density of seawater 83 00:08:02.910 --> 00:08:07.601 divided by the density of the object, and in this case for scleractinian corals, 84 00:08:07.901 --> 00:08:10.800 it's aragonite, calcium carbonate. 85 00:08:11.360 --> 00:08:15.180 For more details you can read the paper, it's available for download. 86 00:08:18.860 --> 00:08:25.559 Respiration rate is an important indicator of individual stress levels and it is 87 00:08:25.859 --> 00:08:28.867 influenced by environmental variables such as temperature. 88 00:08:29.567 --> 00:08:33.115 Therefore, it is commonly used in experimental biology in order to 89 00:08:33.415 --> 00:08:37.215 determine whether the treatment you're testing has an influence on the basic 90 00:08:37.515 --> 00:08:45.925 metabolism of a coral. It is expressed in micromoles of oxygen per weight per time. 91 00:08:47.240 --> 00:08:53.604 Sometimes it is converted to carbon, using the relation that the carbon respired 92 00:08:53.843 --> 00:08:58.700 equals the oxygen consumed times the respiration quotient. 93 00:09:02.300 --> 00:09:07.460 The respiration quotient is a ratio between carbon dioxide expired 94 00:09:08.340 --> 00:09:14.042 and the oxygen consumed. It is based on the metabolism of a species and how it 95 00:09:14.342 --> 00:09:19.170 uses different forms of energy, like carbohydrates, fats, and proteins. 96 00:09:19.470 --> 00:09:22.540 For corals, it is approximately 0.8. 97 00:09:25.100 --> 00:09:29.300 The respiration incubations start with putting the corals in an incubation vial 98 00:09:29.500 --> 00:09:31.760 that can be hermetically closed. 99 00:09:33.460 --> 00:09:35.580 We measure the oxygen and temperature. 100 00:09:36.310 --> 00:09:40.970 In case you have probes that can measure salinity or pH, you can use them as well. 101 00:09:41.470 --> 00:09:45.320 It is important to make sure that we seal the vials well, 102 00:09:46.280 --> 00:09:51.505 making sure there is no gas exchange during the incubation. Depending on 103 00:09:51.705 --> 00:09:56.730 the vial volume, temperature in species' incubation time may vary. 104 00:09:58.100 --> 00:10:03.680 Also keep in mind that since the corals will be respiring carbon dioxide, 105 00:10:03.880 --> 00:10:06.450 the pH in the vial will decrease. 106 00:10:07.150 --> 00:10:11.332 When the incubation is over, you carefully open the vial and measure 107 00:10:11.532 --> 00:10:15.180 the initial measurements again, temperature and oxygen. 108 00:10:15.730 --> 00:10:21.103 Later we need to standardise the respiration rate by volume, incubation 109 00:10:21.303 --> 00:10:24.660 time, weight of the coral or number of polyps. 110 00:10:28.230 --> 00:10:29.320 So here 111 00:10:29.780 --> 00:10:33.762 we are starting an incubation, a respiration incubation. 112 00:10:34.272 --> 00:10:39.400 My colleague is putting an impermeable foil that doesn't permit gas exchange. 113 00:10:39.950 --> 00:10:44.509 The corals are put in separate vials on a magnetic stirrer and inside of each 114 00:10:44.809 --> 00:10:49.450 there's a small magnet that maintains the water 115 00:10:50.100 --> 00:10:54.760 in movement during the incubation. We're closing it with a rubber, 116 00:10:55.480 --> 00:10:59.640 and they will be maintained during several hours like this. 117 00:11:00.730 --> 00:11:03.370 Now we're opening the incubation 118 00:11:04.520 --> 00:11:09.900 slowly that we don't stir the water too much and we measure oxygen and temperature. 119 00:11:19.130 --> 00:11:24.550 In this presentation we saw how cold- water corals are sampled with the ROV. 120 00:11:24.850 --> 00:11:27.860 Later we talked about nubbin preparation for experiments. 121 00:11:28.360 --> 00:11:33.510 We discussed buoyant weight, one of the main methods for measuring growth 122 00:11:33.710 --> 00:11:38.481 in scleractinian species. And finally we talked about respiration incubations, 123 00:11:38.681 --> 00:11:43.750 how to prepare, what is the basic equip- ment, and how to standardise the results. 124 00:11:44.390 --> 00:11:45.680 Thank you for listening.