The Coral Holobiont

Question 1

What is the name of the family of dinoflagellates that are associated with coral symbiosis?

Answer 1

Symbiodiniaceae

Question 2

How taxonomically specific did optical techniques allow us to get previously? What new analysis allowed improved understanding?

Answer 2

Only to the species level (thought one single species of Symbiodiniaceae existed).

Genetic Information Analysis allowed multiple species to be identified.

Question 3

What is the name for DNA used to identify evolutionary history via genetic modifications?

Answer 3

Molecular clocks.

Question 4

Name three types of molecular clocks within a cell.

Answer 4

1. Mitochondrial DNA
2. Chloroplast DNA
3. Nuclear DNA

Question 5

Which was the first genetic analysis used to form 4/5 clear diverse groups within Symbiodinium? Which biological clock did this use and how did it work?

Answer 5

Nuclear Ribosomal DNA was used.
Small ribosomal units (18s) were used initially to classify ”Symbiodinium” from different corals -> this was done using Restriction fragment length polymorphism (RFLP) analysis

Question 6

How were six different clades of Symbiodinium identified?

Answer 6

ITS analysis.
This used Internal Transcribed Spaces -> parts that are transcribed but do not encode the ribosomal complex, allowing a deeper understanding of diversity as more significant changes occur in these regions with time.
This was carried out on nuclear ribosomal DNA

Question 7

Summarise the two nuclear DNA analyses used on Symbiodinium.

Answer 7

• Small subunit RNA using RFLP
• ITS analysis

Question 8

Which Clade of Symbiodinium was found to be the most resilient to coral bleaching in 1998?

Answer 8

Clade D

Question 9

What is the mitochondrial DNA sequence commonly used to classify Symbiodinium?

Answer 9

Cytochrome b (a component of the respiratory chain)

Question 10

What are the chloroplast DNA sequences commonly used to classify Symbiodinium?

Answer 10

cp23S rDNA = chloroplast large subunit (23S) - rDNA sequences
psbA = PsbA central reaction centre of polypeptides of PSII

Question 11

What is the new name for a clade? How are these linked?

Answer 11

A genus (plural = genera).
The first name of the genus represents the letter of the former clade

Question 12

What is the new name for sub-clades

Answer 12

Species

Question 13

How did the family name change?

Answer 13

From Symbiodinium to Symbiodiniaceae

Question 14

Where are the symbionts found in relation to the coral?

Answer 14

Within the endoderm.

Question 15

What is the coral holobiont composed of?

Answer 15

Coral animal
Photosynthetic symbiont
Microbes (fungi, protists, viruses, archaea, algae, bacteria)

Question 16

Name three functions of beneficial bacteria in the coral holobiont.

Answer 16

• Nutrient cycling and provision of nutrients
• Protection against pathogens
• Stress tolerance

Question 16

What is the difference between a microbiota and the microbiome?

Answer 16

Microbiota: community of living organisms (microeukaryotes, bacteria, algae, archaea…)

Microbiome: microbiota + surrounding environmental conditions, genetic elements, metabolites and signalling molecules

Essentially: the microbiome also incorporates chemical and physical components as well as the biology

Question 17

Where in the coral structure are the microbes found?

Answer 17

Across all compartments (mucous, epidermis, mesoglea and gastrodermis).
Some are even associated with Symbiodiniaceae cells.

Question 18

What is the coral probiotics technique?

Answer 18

This is a new technique looking at coral resilience to stress based on bacterial communities – inoculate different bacterial communities.

Essentially: Manipulate the coral microbiome to reduce the negative effects of temperature stress and pathogens.

Question 19

What are the limitations of coral probiotics when attempting to increase coral stress resilience?

Answer 19

Lack of long-term studies

Scalability and feasibility

Ethical considerations of introducing a bacterial community to a marine environment

Question 20

What method can be used to assign bacterial taxonomy and estimate the relative abundance of these bacteria and archea?

Answer 20

The 16S rRNA metabarcoding method.

Question 21

What are the steps of the 16S rRNA metabarcoding method?

Answer 21

1. Sample collection
2. DNA extraction
3. Library prep and amplification (specific primer pair for the region required)
4. High-throughput sequencing (produces ASVs)
5. Taxonomic assignment and bioinformation analysis (e.g., Silva database)

Question 22

Why use the barcode used in the 16S rRNA metabarcoding method?

Answer 22

The 16S rRNA gene is used because:
- It contains highly conserved regions (used for ribosomal function and bacterial fitness) that are present across prokaryotes
- Contains hypervariable regions (higher rates of mutation and sequence divergence means it gives insights into evolution)

Question 23

Which regions of the 16S rRNA gene are used for the 16S rRNA metabarcoding method?

Answer 23

Typically, the V3 and V4 regions -> these are both hypervariable regions found between conserved regions.

Question 24

What is an ASV and at what stage of the 16S rRNA metabarcoding method is it produced?

Answer 24

An ASV (Amplicon Sequence Variant) is a unique DNA sequence from a marker gene. It can be obtained from high-throughput sequencing. Each ASV corresponds to a unique bacterial strain or species (depending on the resolution of the gene region sequenced). A higher abundance of an ASV means a higher abundance of the bacteria (or symbiont) associated with that ASV. If multiple ASVs belong to the same genus (e.g., Cladocopium or Vibrio), their combined abundance reflects that genus’s total abundance in the sample.

Question 25

Which marker genes are used for bacteria, coral symbionts, and eukaryotes?

Answer 25

16S rRNA for bacteria/archaea ITS2 for coral symbionts 18S rRNA for eukaryotes

Question 26

Which software can be used to correct errors/quality controls of sequencing output in the 16S rRNA metabarcoding method?

Answer 26

DADA2

Question 27

List the pros of the 16S rRNA metabarcoding method.

Answer 27

16S rRNA metabarcoding gives a broad overview of the bacterial taxa present within our samples Allows characterization of community composition and structure, diversity, and can give insights into potential functions of microbes Construct evolutionary relationships – often don’t get to species level

Question 28

List the cons of the 16S rRNA metabarcoding method.

Answer 28

Cannot differentiate closely related species Absolute abundance is unknown – compositional data Amplification and extraction biases – can underrepresent certain bacterial groups Variations in 16S rRNA gene copy number (1-15) Lack of functional information (purely taxonomical)

Question 29

Does seawater nutrient availability influence coral-associated microbial communities?

Answer 29

Yes - significant differences were found in terms of microbial community diversity between different nutrient conditions. Increasing nutrient imbalances caused decreasing network connectivity (community interactions) -> this is related to reductions in the physiology of the host coral.