1. The Role of Complement Molecules in Cnidarian Immunity
Hello, my name is Ellen Dow. I have been researching the role of complement molecules in cnidarian immunity under my mentor Dr. Virginia Weis. I am interested in connecting marine ecosystems and organisms with molecular biology.
Ellen Dow
Mentor: Dr. Virginia Weis
HHMI 2012

2. Coral Colonial animals Polyp: identical individual units Reef builders
Secrete calcium carbonate skeleton
Biodiversity
Cnidarians
Related to anemones
polyp
Corals are colonial animals that grow through individual polyps (picture), which are identical individual animal units (picture). Some corals are reef building corals, which secrete a calcium carbonate skeleton that slowly builds over time. They create a habitat that allows for great biodiversity, which is important for marine ecosystems. Corals belong to the phylum of cnidaria and are therefore closely related to anemones.

3. Coral reef symbiosis Dinoflagellates Give carbon to the coral host
Photosynthetic
Live within coral cells
Primary productivity
Give carbon to the coral host
Dinoflagellates receive inorganic nutrients and a stable intracellular environment
Symbiosis is important to the infrastructure of coral reefs. Dinoflagellates are photosynthetic and live within coral cells (picture). The symbionts provide a foundation for reef primary productivity. The symbiotic dinoflagellates also provide the host corals with organic carbon in exchange for inorganic nutrients and a stable intracellular environment. (picture)
Dinoflagellates
serc.carleton.edu

4. Coral bleaching Dinoflagellate loss resulting in white appearance
Caused by stressors, including rising ocean temperatures, pollutants, UV stress, salinity, and disease
Leads to coral death
Decline of the reef ecosystem
The loss of symbionts, which results in a white appearance of corals and anemones is called coral bleaching. This process is caused by a variety stressors, which include rising ocean temperatures, pollutants, UV stress, salinity and disease. Bleaching may lead to coral death and a decline of the reef ecosystem. Disease is a main focus of my research on cnidarian immunity, because I want to know how cnidarians respond to bacterial infection.
snf.fra.affrc.go.jp

5. Immunity: Complement System
Innate immune pathway
Three distinct mechanisms of activation
Classical pathway
(only present in vertebrates)
Lectin pathway
Alternative pathway
Series of proteolytic cleavage events
Cleavage of protein C3
Classical
Alternative
Lectin
Factor B
MASPs
MBL
Antibodies
Cleavage of C3
And one innate immune pathway that has potential to be involved is the complement system, which is what my research focuses on. Innate immunity is what organisms are born with, it is not learned, but a built in protection mechanism. There are three distinct mechanisms of activation. The classical pathway, which is only present in vertebrates, uses antibodies. The Lectin pathway using Mannose Binding Lectin and Mannose Binding Lectin Serine Proteases. There is also the alternative pathway which uses Factor B. These three pathways are a series of proteolytic cleavage events and converge at protein C3 as a common event. Further downstream from this event, the membrane attack complex is formed and destructs the microbe, which only occurs in vertebrate immunity. The complement system is activated through identifying what are the invaders to the organism. This can be through chemical signals or sensing what molecules are present on the surface of a bacterium.
Formation of membrane attack complex
Destruction of microbe

6. Invertebrate immunity
Classical
Alternative
Lectin
Factor B
MASPs
MBL
Antibodies
Cleavage of C3
Formation of membrane attack complex
Invertebrate immunity is composed of the lectin and alternative pathways as the present activating pathways. The components of the complement system, known to be in vertebrates, of these pathways include MASPs, Factor B and the well researched C3.
Destruction of microbe

7. How do cnidarians respond to disease?
Complement molecule mRNA expression responds to exposure to bacterial molecules in other invertebrates
Complement system as a pathway of innate immunity
What triggers cnidarian immunity?
Expression level change
How do cnidarians respond to disease? In other invertebrates, complement molecules mRNA expression has been shown to respond to exposure to bacterial molecules. Cnidarians could respond to disease through the complement system innate immune pathway. But what triggers immunity for cnidarians? If there is immune stimulation that does trigger this, the complement molecule mRNA expression may change in regulation.
translatingscience.wordpress.com

8. Aiptasia pallida Model organism that is easy to maintain in lab
Complement genes
One MASP gene
Two Factor B genes (Bf-1 and Bf-2)
Two C3 genes
Corals and Anemones are both cnidarians and closely related. Aiptasia pallida the the anemone used in this study as a model organism because it is easy to keep in the lab and create new anemones. Multiple replicates can be used for experiments through individual animals. Recently, several complement genes have been discovered in aiptasia. There is one MASP gene, two factor B genes, that will be called Bf-1 and Bf-2 and two C3 genes. Aiptasia can also become apo-symbiotic, this is when the anemone ejects it symbionts, just as in bleaching.
seaslugforum.net

9. My Previous Research
Experiment using Lipopolysaccharide (LPS) shows up-regulation in complement molecules *
Previously, I used Lipopolysaccharide, LPS, which is a component in gram-negative cell walls to induce an immune response in Aiptasia pallida. The experiment was shown to up-regulate the complement molecules of Bf-1 and MASP after LPS exposure. There is significant difference between 0 and 72 hours for this time point. Because Bf-2 does not show any significant regulation changes, it may not be involved in the immune response to bacterial infection.
Keep???????
Relative quantities of genes of interest after LPS exposure. Data analyzed using ANOVA with Tukey post-hoc. (*denotes expression significant compared to 0 hours time point)

10. Research Objectives
How does complement molecule mRNA expression in Aiptasia pallida change with immune stimulation from Peptidoglycan (PGN) and live bacteria?
Gene Expression
quantitative PCR (qPCR)
Gene Localization
In situ hybridization
Using this information, I want to know how invertebrate complement molecule expression changes with immune stimulation through PGN and live bacteria. Peptidoglycan is a component in gram-positive bacteria cell walls. As a component of bacteria, it may elicit an immune response. I am examining the gene expression using qPCR and gene localization using in situ hybridization.
Aiptasia pics
AIPTASIA PICS

11. 2. Complement gene mRNA expression localized to phagocytic cells
Hypothesis
The mRNA expression of complement genes up-regulated in immune stimulated A. pallida
2. Complement gene mRNA expression localized to phagocytic cells
I hypothesized that the mRNA expression of complement genes will be up-regulated in Aiptasia pallida that are immune stimulated than in the negative control anemones and that gene expression will be localized to phagocytic cells.

12. 1. Gene expression Separate Relative qPCR to examine anemones
gene mRNA expression
Bf-1, Bf-2 & MASP
PGN
treatment
Relative qPCR to
determine reference genes:
L12, PABP, Ef-1 alpha
Freeze
replicates
In the PGN treatment I used to determine gene expression, first I separated apo-symbiotic anemones into individual wells. Next the PGN treatment was added using two concentrations and the aiptasia frozen at designated time points. From this step, the mRNA was extracted and cDNA synthesized. The cDNA is then used in relative qPCR. I determined the reference genes, which was used as a relative comparison for the target complement molecules between different treatments. Then relative qPCR to show gene expression of Bf-1, Bf-2 and MASP.
State why reference genes are important-normalization etc
Extract mRNA and
synthesize cDNA

13. PGN does not have a significant effect on gene expression
Relative qPCR Results
PGN does not have a significant effect on gene expression
The relative qPCR results show that PGN does not have a significant effect on the change in regulation of gene expression. Between the initial timepoint at 0hr and after 24 hr exposure to both concentrations of PGN, there is not a significant change in gene expression. There is a trend however, that the 10ug/ml concentration shows upregulation, while the 50ug/ml shows a down regulation. The results may be insignificant because there is so much individual variation between replicates and some may have significance. A third treatment was added using 0.08% DMSO for 24hr treatment. This same concentration of DMSO is also in the PGN treatments. It is used as a vehicle to transport PGN across the cell membrane. There was no significant change in this, infact, the average of replicates stays approximately the same throughout each gene. This means that DMSO does not have a direct effect of gene expression and is not impacting the change of expression in the PGN treatments.
Relative quantities between Bf-1, Bf-2 and MASP genes with two different PGN concentrations and DMSO treatments.

14. Gene Expression Analysis
PGN does not effectively induce a significant immune response in A. pallida
Observations in Horseshoe Crabs (Tagawa K, et al., 2012)
Exposed to PGN and LPS without a significant change in complement protein activation
Live bacteria
Use live bacteria instead of individual molecules from bacterial surfaces
I can be summarized that PGN does not have effectively induce a significant immune response in A. pallida. Similar results have been observed in Horshoe Crabs. When exposed to bacterial molecules, like PGN and LPS, the complement proteins did not active. However, the horseshoe crabs did respond to live bacteria.
This has drawn the conclusion to use live bacteria instead of individual molecule on bacterial surfaces.

15. Vibrio coralliilyticus
Coral pathogen
Virulent around 28 ºC
Always present
Harmful at higher temperatures
V. coralliilyticus infecting A. pallida
Cultured on plates
28 ºC
25 ºC
Because of these results, it was decided to use v. coralliitycus, which is a coral pathogen. It becomes virulent around 28 degrees celsius. And although it is always present, as temperatures rise, the bacteria becomes harmful to cnidarians. There has also been previous research on a. pallida using v. coralliitycus for infection.
V. Coralliitycus will be used as an immune stimulant, to measure gene expression and localization in A. pallida. So far, successful colonies have been cultured. Individual cultures incubated at RT for about hours, while the vibrio grows quickly at 28 degrees celsius.
Verbal statement that we decided to use v. coralliitycus
Temps rising, bacteria can infect--virulence
Cultures-issues with right temps and individual colonies
B/w 28 and 25 degree plates

16. 2. Gene localization vs Treat anemones with Vibrio coralliilyticus
Incubate in Detection Buffer
Dunn, et al, 2008
Treat anemones
with Vibrio coralliilyticus
Incubate in Anti-DIG
antibody solution
Hybridize with
anti-sense and sense probes
labeled with DIG
Freeze and section
anemones
For gene localization. The anemones are treated with v. coralliitycus, then frozen at time points in media and sectioned. In situ hybridization makes the target genes visible. First, the sections are fixed to slides. Then the sections are hybridized with anti-sense and sense probes. The anit-sense is complementary to the target genes and will attach, while the sense probe is the same as the target gene and acts as a negative control. These probes are labeled with DIG. The DIG label will attact to anti-dig when the slides are incubated in anti-dig antibody solution. The slides are then incubated in detection buffer, which reacts with the antibody solution to create a blue stain. This highlights where the target genes are located and will be used to compare and contrast with negative control anemones.
IN SITU HYBRIDIZATION IS VERY COMPLEX-THIS IS A SIMPLIFICATION&MANY WASHES ARE USED
PROBES W/DIG THEN ANTI DIG USED IN DETECTION TO STAIN BINDING SITE WHERE THE TARGET GENES ARE LOCATED
Fix sections to slides vs

17. Northern Blot of Bf-2 probe
In situ Data
Northern Blot of Bf-2 probe
control sense anti-sense
Designed probes
Bf-1, Bf-2 and MASP
Sense and anti-sense
100ng/ul
Northern Dot Blot technique
Sectioning
Control probe and target probes
Actin
Sense
Anti-
So far for the in situ data, probes have been designed to use for Bf-1, Bf-2 and MASP. There are sense and antisense probes tested along with a control on a membrane for the concentrations of 100ng/ul using northern blots. (picture sense&antisense w/decreasing concentrations and positive control) Sectioning protocols are being improved to create more accurate and similar sections. These probes have been tested on sections using Actin, as a control to test the hybridization protocol and also with the target gene probes. (picture of actin probes) This will be continued in further research and analysis.

18. Conclusion Further Research Pursue with V. coralliilyticus
PGN does not cause a significant immune response
In situ probes effective
Further Research
Bacterial infections = realistic
Pursue with V. coralliilyticus
Gene expression and gene localization
Methods from this study

19. Acknowledgements Dr. Virginia Weis The Weis Lab Angela Poole
Howard Hughes Medical Institute
CRIPPS Scholarship
The Weis Lab

20. Questions?

21. Thank you
coralscience.org

22. Reference Genes: L12, PABP, Ef-1 alpha
The reference genes for PGN stimulation are L12, PABP and Ef-1 alpha.The process for this is involved as many different genes are targeted to find the most stable ones. This graph of genorm results shows that L12 and PABP are the most stable pair and Ef-1 alpha is the next most stable reference gene. (point to stability on x axis and low deviation on y axis) I also used another program, normfinder, which demonstrated similar results.