Fire coral, symbionts, and climate change

Question 1

What is symbiosis?

Answer 1

Interactions between two species living in the same place

Question 2

What are the three types of symbiosis?

Question 3

What is mutualism?

Question 4

What is commensalism?

Question 5

What is parasitism?

Question 6

What is a symbiont?

Question 7

What limits tropical corals to the shallow water?

Answer 7

The need for sunlight.

Question 8

What are Symbiodiniceae?

Question 9

What do symbionts do?

Question 10

What do symbionts gain?

Answer 10

Shelter, nutrients, and carbon

Question 11

What’s the difference between generalist and specialist symbiont taxa?

Answer 11

Generalists serve multiple corals and specialists serve one type of coral.

Question 12

What are potential variations insymbionts?

Answer 12

Thermal tolerance, photosynthetic capabilities, cell densities, growth rater, etc.

Question 13

What is genera?

Question 14

What is taxa?

Question 15

What are fire corals?

Question 16

What are pollups?

Question 17

What differentiates fire corals?

Answer 17

They have an alternate life stage where they are baby jelly fish - the medusa stage. Instead of releasing eggs and sperm when they spawn, they release medusa.

They also have two different polyp types. They have feeding polyps (galac..) and hunting/stinging polyps (daclyozoids.) This is a toxin for humans.

Question 18

What are some characteristics of fire corals?

Answer 18

• Strong competitors
• Increasing in abundance due to quick recovery rates
• Some species are endangered, vulnerabal, or threatened however

Question 19

What is the concern about coral?

Answer 19

• Algae cover will destroy reef structure and grow over coral, removing the coral reef and making an algae meadow which cannot support biodiversity.
• Warming oceans and acidification (climate change)
• Water quality
• Physical damage (overfishing, storms, tourism)

Question 20

What is coral bleaching?

Answer 20

The coral goes white. It is caused by heat stress due to marine heat waves which are a result of warming oceans. When temperatures get too high, the coral will bleach.

Question 21

How does heat cause bleaching?

Answer 21

Oxidative damage. A lot of heat and light produce reactive oxygen spieces that damage the DNA and the rest of the cell. No photosynthesis happens, and the harmful oxygen is spit out by the coral. This can lead to host cell death.

Question 22

How does bleaching vary?

Answer 22

Some species are more susceptible to bleaching than other. Some species are really resistant - especially the big reef builders.

Question 23

What is acclimation?

Question 24

What is symbiont switching?

Answer 24

Expelling old symbionts and taking in new ones from the environment.

Question 25

What is symbiont shuffling?

Answer 25

Symbiont shuffling is the movement and exchange of symbiotic organisms between different host species. It allows hosts to dynamically acquire beneficial symbionts.

The new symbiont strains are acquired through horizontal transfer when the coral comes into contact with free-living symbiont populations or other coral colonies hosting different strains. The symbionts are in the surrounding environment.

Question 26

What is a key characteristic of symbiont associations?

Answer 26

They are very stable. Colonies usually revert to their original symbionic makeup.

Question 27

What is the point of the lab?

Answer 27

To identify symbions, and which are more prevelant in that sample. What species are residing in those fire coral colonies? Based on that, we will determine where that coral was collected - warmer shallower waters, or cooler deeper waters

Question 28

What’s the function of the primer?

Answer 28

To identify the symbiont species. They are specific to the species.

Question 29

What’s the point of PCR?

Answer 29

Getting enough copies to be visible on agarose gel AND quantifying of the DNA target

Question 30

Three steps of PCR

Answer 30

Denaturation, anealling, and elongation/extension

Question 31

What is the dye and when does the dye bind?

Answer 31

SYBR green.
During the extension stage.
More binding means more fluorescence.

Question 32

What happens to fluorescence as the cycles increase?

Answer 32

Fluorescence increases because you have more DNA strands and more dye binding to them so it is brighter.

On the graph, the lines shoots up we have enough DNA to measure fluorescence.

Our line has to pass the fluorescent threshold to reveal the sample fluorescence. Any fluorescence before this is just background noise.

Question 33

What’s in the master mix?

Answer 33

Polymerase, dNTPs, fluorescent dye, and buffer with the cofactors polymerase needs to function

Question 34

What does the PCR machine do?

Answer 34

The cycles and recording the fluorescence levels in real time.
This allows you to check how much fluorescence you have after each cycle.
Fluorescence plateaus at a point.

Question 35

What is the point of the lab?

Answer 35

Figuring out the order of prevelance of the three different symbiont species in our sample to tell where in the ocean it came from - hot and high waters or cool and deep waters.

Question 36

Why do we need a different primer pair for each?

Answer 36

We could use a primer that can bind to any of the symbionts but that would not tell us how they are different; which ones are present in which fire corals and how much of it is present (quantification). That is what we want to know because that is what will tell us where it came from. And the point of that is…

Question 37

What is the ct value?

Answer 37

The point at which fluorescence is above background levels.

Comparatively lower ct means the sample had more DNA to start with. Which shows that that sample’s DNA was higher in the coral.

The lowest ct value = the highest amount of DNA = the most presence symbiot

Question 38

What is the exponential phase?

Answer 38

When the amount of PCR doubles each cycle

Question 39

What is the plateau phase?

Question 40

How do you determine the abundance of a symbiot?

Answer 40

Look for the species with the lowest ct value. This is the one with the highest abundance.

Ct is the number of fluorescence required to exceed the background level - now it’s valid data.

Question 41

What is relative abundance?

Answer 41

It is an equation that you plug your figures into x = 2 ^ (higher Ct value - lower Ct value.

It gives you relative abundance as a ratio. You need at least two species present.

For every one of the lower abundance symbionts, there are x of the higher abundance symbionts.

Question 42

What is the melt curve?

Answer 42

Different species will have DNA that denatures at different temperatures due to the difference in base sequence or length of DNA strands (shorter fragments will denature quicker). So, we raise the temperature slowly and assess how many different types of DNA there are biased on the different melting temperatures you derive. We record the temperatures at which our DNA comes apart.

Question 43

How do we know that we have the product that we want?

Answer 43

We can see fluoresence from:
- Primers binding together (primer dimers)
- SYBR binding with random DNA (contaminating DNA)

We need to make sure we are seeing fluorescence from the DNA we care about, not primer dimers or contaminating DNA.

Question 44

What do we use as our control?

Answer 44

We run a PCR test with water, not sample. This will give us a baseline level of fluorescence that comes from primer dimers and contaminating DNA (background fluorescence).

Question 45

How do we read a melt curve?

Answer 45

The peak of the graph tells you the temperature at which the sample denatures.

The line that peaks at a lower temperature is our primer dimers and contaminating DNA (no template/sample peak - our background stuff).

The line that peaks at a higher temperature is the sample’s melting point or the temp. at which it denatures.

If your graph only had one peak, there is no sample DNA in the mixture. If we are suspicious that one species DNA may not be present, we will see one peak - the two lines will denature at the same temp. This means, there is no sample in there because it matches our no template control. We are only seeing primer dimers.

Remember - we want to see two different temperature peaks. One temperature peak with even two lines means there is no sample DNA. It’s just our primer dimers amplifying.

Use-case:

Which samples contain the species DNA?

Question 46

What is irradiance?

Answer 46

Irradiance is the amount of photosynthetic sunlight reaching the coral’s algal symbionts. Corals adapt to different irradiance through symbiont shuffling.

More depth in the ocean means less irradiance which can stress the coral. So, the coral swaps our its symbionts for a species that thrives better in deeper waters with less irradiance - symbiont shuffling.