Presentation at the annual AIMS@JCU seminar day in 2015.
The mutualistic relationship between reef-building corals and intracellular photosynthetic dinoflagellates of the genus Symbiodinium provides the foundation of the coral reef ecosystem. Disruption of this relationship leads to coral bleaching which is occurring on a large scale and poses a real threat to the survival of coral reefs. In spite of the great ecological significance of this partnership to coral reefs, little is currently known about the molecular mechanisms involved in the establishment and maintenance of the symbiosis. A number of studies have investigated host gene expression during the establishment of coral-algal symbiosis, but these have failed to detect host symbiosis-related signals. To better understand the early events occurring during the establishment of symbiosis, infection experiments were conducted during spawning in Sesoko Island, Okinawa, Japan in June 2013 using Acropora digitifera larvae and a competent strain of symbionts, Symbiodinium sp. clade B. Next generation sequencing (Illumina RNAseq) was used for the first time to follow coral transcriptome-wide gene expression after exposure to competent Symbiodinium at 4-, 12-, 48- h post infection. These experiments allowed the detection of transient transcriptomic signals in the coral during the initial infection period. The data imply that the establishment of symbiosis involves cross talk between the partners; an active response is required on the part of the host in order to recognize appropriate partners but at the same time the symbionts appear to suppress some host responses, including immunity
Despite the ecological significance of coral-Symbiodinium symbiosis, very little is known about the molecular mechanisms involved in the establishment of the relationship. Gene expression studies based on microarrays have led to the assumption that host transcriptome is largely unresponsive during the establishment of symbiosis with competent Symbiodinium. In the present study, Illumina RNA-Seq was used to investigate transcriptome changes in Acropora digitifera larvae during exposure to competent Symbiodinium (a clade B strain). A transient period of differential expression involving a small number of genes (1073 transcripts; <3% of the transcriptome) was detected very early in the infection process i.e. 4h after infection. This phenomenon has not previously been detected as a consequence of both the lower sensitivity of the microarray approaches used and the sampling times used. The transcriptome data imply that translation and oxidative metabolism are supressed in the coral host during the infection process, and altered expression of some apoptosis-related genes was also observed. The results also suggest the involvement of both partners in the establishment of the symbiosis; there is an active response on the part of the host in recognizing the symbiotic partner, but an apparent suppression of host immune responses may be initiated by the symbiont. The results are also consistent with the hypothesis that the symbiosome is a phagosome that has undergone early arrest, raising the possibility of a common mechanism of symbiont infection in corals and symbiotic sea anemones.
The document describes an analysis of RNA-seq data from 21 breast cancer samples representing 3 subtypes (TNBC, ER+, HER2+) profiled in 3 different mouse PDX models. The goals were to identify transcriptional differences between cancer subtypes and mouse models in order to select biomarker candidate genes. An overview of the RNA-seq analysis pipeline is provided, including mapping, quantification, normalization, and downstream analyses like PCA, factor regression, and tumor-stroma association studies.
RNA-seq: A High-resolution View of the TranscriptomeSean Davis
The molecular microscopes that we use to examine human biology have advanced significantly with the advent of next generation sequencing. RNA-seq is one application of this technology that leads to a very high-resolution view of the transcriptome. With these new technologies come increased data analysis and data handling burdens as well as the promise of new discovery. These slides present a high-level overview of the RNA-seq technology with a focus on the analysis approaches, quality control challenges, and experimental design.
Whole Transcriptome Profiling of Cancer Tumors in Mouse PDX ModelsTom Koch
This document describes an analysis of RNA sequencing data from patient-derived breast cancer xenograft models. The analysis identified gene expression differences between triple negative and estrogen receptor positive breast cancer subtypes. Factor regression analysis was also used to identify genes influenced by tumor type and mouse strain. Some long non-coding RNAs and genes like CXorf61 were found to be potential biomarkers for triple negative breast cancer.
Transcriptomics is the study of RNA in cells and tissues. The transcriptome refers to the complete set of transcripts in a cell under a specific condition. Understanding the transcriptome reveals the functional elements of the genome and molecular constituents of cells. Techniques for studying the transcriptome include microarray analysis and RNA sequencing. Microarrays measure gene expression levels using fluorescently-labeled cDNA hybridized to probes on an array. RNA sequencing determines expression levels by sequencing individual cDNAs produced from target RNA. Transcriptomics provides insights into development, disease, and varying gene expression under different environmental conditions.
whole genome analysis
history
needs
steps involved
human genome data
NGS
pyrosequencing
illumina
SOLiD
Ion torrent
PacBio
applications
problems
benefits
Deciphering the nature of the coral–Chromera association. Mohamed et al-2018-...Amin Mohamed
Since the discovery of Chromera velia as a novel coral-associated microalga, this organism has attracted interest because of its unique evolutionary position between the photosynthetic dinoflagellates and the parasitic apicomplexans. The nature of the relationship between Chromera and its coral host is controversial. Is it a mutualism, from which both participants benefit, a parasitic relationship, or a chance association? To better understand the interaction, larvae of the common Indo-Pacific reef-building coral Acropora digitifera were experimentally infected with Chromera, and the impact on the host transcriptome was assessed at 4, 12, and 48 h post-infection using Illumina RNA-Seq technology. The transcriptomic response of the coral to Chromera was complex and implies that host immunity is strongly suppressed, and both phagosome maturation and the apoptotic machinery is modified. These responses differ markedly from those described for infection with a competent strain of the coral mutualist Symbiodinium, instead resembling those of vertebrate hosts to parasites and/or pathogens such as Mycobacterium tuberculosis. Consistent with ecological studies suggesting that the association may be accidental, the transcriptional response of A. digitifera larvae leads us to conclude that Chromera could be a coral parasite, commensal, or accidental bystander, but certainly not a beneficial mutualist.
Novel insights into the establishment of symbiosis. Amin Mohamed-2016-thesis JCUAmin Mohamed
Reef-building corals are considered as meta-organisms where the coral animal lives in symbiosis with a wide array of microorganisms. While mutualistic association between corals and Symbiodinium is crucial for the functioning and success of the coral reef ecosystems, surprisingly little is currently known about its molecular basis and this is especially true of the events leading to establishment of the relationship. A morphologically similar alga to Symbiodinium was discovered in Australian corals and has been identified as Chromera. The discovery of Chromera is very significant as it holds a unique position in evolution, between the photosynthetic dinoflagellates and the parasitic apicomplexans. The nature of the association between Chromera and corals is currently unclear. In this thesis, I used high throughput next generation sequencing technology (Illumina RNA-Seq) to explore the molecular mechanisms underlying establishment of coral-algal symbiosis between coral larvae and a competent strain of Symbiodinium. I examined also the nature of the poorly understood relationship between corals and the newly described photosynthetic apicomplexan alga Chromera using RNA-Seq. Finally, I present a functional genomic resource (transcriptome) for a Chromera strain isolated from a Great Barrier Reef coral, and use a comparative transcriptomic approach to examine sharing of functions and pathways among Chromera, Symbiodinium kawagutii and Plasmodium falciparum.
Despite the ecological significance of coral-Symbiodinium symbiosis, very little is known about the molecular mechanisms involved in the establishment of the relationship. Gene expression studies based on microarrays have led to the assumption that host transcriptome is largely unresponsive during the establishment of symbiosis with competent Symbiodinium. In the present study, Illumina RNA-Seq was used to investigate transcriptome changes in Acropora digitifera larvae during exposure to competent Symbiodinium (a clade B strain). A transient period of differential expression involving a small number of genes (1073 transcripts; <3% of the transcriptome) was detected very early in the infection process i.e. 4h after infection. This phenomenon has not previously been detected as a consequence of both the lower sensitivity of the microarray approaches used and the sampling times used. The transcriptome data imply that translation and oxidative metabolism are supressed in the coral host during the infection process, and altered expression of some apoptosis-related genes was also observed. The results also suggest the involvement of both partners in the establishment of the symbiosis; there is an active response on the part of the host in recognizing the symbiotic partner, but an apparent suppression of host immune responses may be initiated by the symbiont. The results are also consistent with the hypothesis that the symbiosome is a phagosome that has undergone early arrest, raising the possibility of a common mechanism of symbiont infection in corals and symbiotic sea anemones.
The document describes an analysis of RNA-seq data from 21 breast cancer samples representing 3 subtypes (TNBC, ER+, HER2+) profiled in 3 different mouse PDX models. The goals were to identify transcriptional differences between cancer subtypes and mouse models in order to select biomarker candidate genes. An overview of the RNA-seq analysis pipeline is provided, including mapping, quantification, normalization, and downstream analyses like PCA, factor regression, and tumor-stroma association studies.
RNA-seq: A High-resolution View of the TranscriptomeSean Davis
The molecular microscopes that we use to examine human biology have advanced significantly with the advent of next generation sequencing. RNA-seq is one application of this technology that leads to a very high-resolution view of the transcriptome. With these new technologies come increased data analysis and data handling burdens as well as the promise of new discovery. These slides present a high-level overview of the RNA-seq technology with a focus on the analysis approaches, quality control challenges, and experimental design.
Whole Transcriptome Profiling of Cancer Tumors in Mouse PDX ModelsTom Koch
This document describes an analysis of RNA sequencing data from patient-derived breast cancer xenograft models. The analysis identified gene expression differences between triple negative and estrogen receptor positive breast cancer subtypes. Factor regression analysis was also used to identify genes influenced by tumor type and mouse strain. Some long non-coding RNAs and genes like CXorf61 were found to be potential biomarkers for triple negative breast cancer.
Transcriptomics is the study of RNA in cells and tissues. The transcriptome refers to the complete set of transcripts in a cell under a specific condition. Understanding the transcriptome reveals the functional elements of the genome and molecular constituents of cells. Techniques for studying the transcriptome include microarray analysis and RNA sequencing. Microarrays measure gene expression levels using fluorescently-labeled cDNA hybridized to probes on an array. RNA sequencing determines expression levels by sequencing individual cDNAs produced from target RNA. Transcriptomics provides insights into development, disease, and varying gene expression under different environmental conditions.
whole genome analysis
history
needs
steps involved
human genome data
NGS
pyrosequencing
illumina
SOLiD
Ion torrent
PacBio
applications
problems
benefits
Deciphering the nature of the coral–Chromera association. Mohamed et al-2018-...Amin Mohamed
Since the discovery of Chromera velia as a novel coral-associated microalga, this organism has attracted interest because of its unique evolutionary position between the photosynthetic dinoflagellates and the parasitic apicomplexans. The nature of the relationship between Chromera and its coral host is controversial. Is it a mutualism, from which both participants benefit, a parasitic relationship, or a chance association? To better understand the interaction, larvae of the common Indo-Pacific reef-building coral Acropora digitifera were experimentally infected with Chromera, and the impact on the host transcriptome was assessed at 4, 12, and 48 h post-infection using Illumina RNA-Seq technology. The transcriptomic response of the coral to Chromera was complex and implies that host immunity is strongly suppressed, and both phagosome maturation and the apoptotic machinery is modified. These responses differ markedly from those described for infection with a competent strain of the coral mutualist Symbiodinium, instead resembling those of vertebrate hosts to parasites and/or pathogens such as Mycobacterium tuberculosis. Consistent with ecological studies suggesting that the association may be accidental, the transcriptional response of A. digitifera larvae leads us to conclude that Chromera could be a coral parasite, commensal, or accidental bystander, but certainly not a beneficial mutualist.
Novel insights into the establishment of symbiosis. Amin Mohamed-2016-thesis JCUAmin Mohamed
Reef-building corals are considered as meta-organisms where the coral animal lives in symbiosis with a wide array of microorganisms. While mutualistic association between corals and Symbiodinium is crucial for the functioning and success of the coral reef ecosystems, surprisingly little is currently known about its molecular basis and this is especially true of the events leading to establishment of the relationship. A morphologically similar alga to Symbiodinium was discovered in Australian corals and has been identified as Chromera. The discovery of Chromera is very significant as it holds a unique position in evolution, between the photosynthetic dinoflagellates and the parasitic apicomplexans. The nature of the association between Chromera and corals is currently unclear. In this thesis, I used high throughput next generation sequencing technology (Illumina RNA-Seq) to explore the molecular mechanisms underlying establishment of coral-algal symbiosis between coral larvae and a competent strain of Symbiodinium. I examined also the nature of the poorly understood relationship between corals and the newly described photosynthetic apicomplexan alga Chromera using RNA-Seq. Finally, I present a functional genomic resource (transcriptome) for a Chromera strain isolated from a Great Barrier Reef coral, and use a comparative transcriptomic approach to examine sharing of functions and pathways among Chromera, Symbiodinium kawagutii and Plasmodium falciparum.
Evaluation of Pool-Seq as a cost-effective alternative to GWASAmin Mohamed
Whole-genome sequencing (WGS) of pools of individuals (Pool-Seq) provides a cost-effective method for genome-wide association studies (GWAS), and offers an alternative to sequencing of individuals that remains cost prohibitive. Pool-Seq is being increasingly used in population genomic studies in both model and non-model organisms. In this paper, the ability of Pool-Seq to recover known GWAS signals was evaluated. Existing GWAS data for 2,112 animals with 729K SNPs were obtained and pooled to simulate data obtained from a pooled WGS approach. Traditional GWAS results was compared with the absolute allele frequency difference (dAF) metric suitable for use with Pool-Seq data. Specifically, we tested the ability of dAF scans to recover known GWAS signals for two different traits with large and moderate gene effects. Pools of different sizes (50, 100 and 200 individuals per pool) were also compared. The results showed the ability of the absolute allele frequency difference (dAF) approach to recover known GWAS peaks obtained by traditional SNP association and recommended the use of a pool size of 100 individuals for DNA pooling.
Molecular basis of sex maturation in Atlantic salmon: Integrated genomics app...Amin Mohamed
This document summarizes a study that used genomics approaches to investigate the molecular basis of sex maturation in Atlantic salmon. The researchers manipulated photoperiod to initiate maturation in female salmon during a specific time window. They collected tissue samples from the brain, pituitary, ovaries and other tissues before, during and after the maturation process to generate transcriptomic and epigenomic data. The goal was to gain mechanistic insights into the triggers for sexual maturation onset in Atlantic salmon, which has implications for the salmon aquaculture industry dealing with issues like early maturation.
Amin Mohamed- PhD Candidate in Coral Reef GenomicsAmin Mohamed
Amin Mohamed is a PhD candidate at James Cook University studying chromerids and apicomplexans associated with coral reefs under a Egyptian Government PhD Scholarship and AIMS@JCU Top-Up Scholarship. He is supervised by professors and doctors at JCU and AIMS. Amin completed his BSc in Zoology in Egypt in 2006 and his MSc provided baseline data on coral health in the Egyptian Red Sea. His PhD project aims to understand the role of chromerids and apicomplexans in coral health using next-generation sequencing to analyze gene expression in corals exposed to Chromera velia. He conducted infection experiments in Japan and Australia and is now analyzing the large sequencing datasets using bio
Since the discovery of Chromera velia, a novel microalga in 2008 during a study of Australian coral symbionts, great interest in this new algal species has developed. C. velia is an important organism being considered to sit phylogenetically between the photosynthetic dinoflagellate algae and the parasitic Apicomplexa. C. velia has been detected and isolated from different scleractinian corals in Australia including corals of the Great Barrier Reef. However little is known about the ecology and genetics of this microorganism. The study aims to use next generation sequencing technology (Illumina RNAseq) to generate a reference transcriptome assembly of C. velia isolated from Montipora digitata at the GBR. Differential gene expression in altered experimental treatments is investigating the diversity and dynamics of C. velia transcripts (mRNAs). Cultures were subjected to heat shock, heat stress, cold shock, cold stress, and darkness with control cultures grown under normal conditions, cultures grown under mixotrophic condition and cultures containing motile/immotile stages. Analysis of the transcriptome of C. velia will provide genomic insight into this novel chromerid alga through identifying genes involved in various physiological and metabolic processes as investigated under different experimental conditions.
Coral diseases, coral bleaching and other health issues affecting Red Sea cor...Amin Mohamed
Research paper presented at the 12th International Coral Reef Symposium (ICRS) Cairns, Australia 9-13 July 2012 in the Climate change and bleaching mini-symposium. The paper has presented the first detailed quantitative data on the distribution and prevalence of coral diseases and other compromised health signs including coral bleaching that affect multiple corals in multiple sites along the Egyptian Red Sea coast.
Status of coral reef health in the northern red sea egypt icrs 2012 paperAmin Mohamed
This study aimed to provide baseline knowledge of coral diseases prevalence and coral health at nine reef sites in the northern Egyptian Red Sea: I (El-Ain Al-Sukhna), II (Ras Za’farana), III (Hurghada), IV (Ras Ghozlani), V (Old Quay), VI (Yolanda reef), VII (Ras Umm Sid, Sharm El-Sheikh), VIII (WoodHouse reef, Sharm El-Sheikh), and IX (canyon reef, Dahab). Sites IV, V and VI are located in Ras Mohamed, marine protected area. Field surveys of coral diseases and signs of compromised health in the studied reefs revealed a mean prevalence o f 1.03 %. Macroscopic observations were diagnosed as: black band disease (0.44%), white syndrome (0.13%), pink line syndrome (0.04%), ulcerative white spots (0.02%), skeletal eroding band (0.001 %), coral bleachi ng (2.2%), Drupella cornus snail predation (0.1%), fish predation (0.2%), pigmentation response (0.13%), sediment damage (0.08%), algae overgrowth (0.06%) and sponge overgrowth (0.04%). Prevalence of coral diseases and bleaching were higher in non-MP A sites than in MPA sites. The highest prevalence of coral diseases was recorded on the coral Favia stelligera, followed by Porites lutea, and Goniastrea edwardsi. Enhanced local anthropogenic stresses and increasing sea surface temperature due to global warming are the suggested potential factors responsible for the initiation and the persistence of some coral diseases in the studied reefs.
Evaluation of Pool-Seq as a cost-effective alternative to GWASAmin Mohamed
Whole-genome sequencing (WGS) of pools of individuals (Pool-Seq) provides a cost-effective method for genome-wide association studies (GWAS), and offers an alternative to sequencing of individuals that remains cost prohibitive. Pool-Seq is being increasingly used in population genomic studies in both model and non-model organisms. In this paper, the ability of Pool-Seq to recover known GWAS signals was evaluated. Existing GWAS data for 2,112 animals with 729K SNPs were obtained and pooled to simulate data obtained from a pooled WGS approach. Traditional GWAS results was compared with the absolute allele frequency difference (dAF) metric suitable for use with Pool-Seq data. Specifically, we tested the ability of dAF scans to recover known GWAS signals for two different traits with large and moderate gene effects. Pools of different sizes (50, 100 and 200 individuals per pool) were also compared. The results showed the ability of the absolute allele frequency difference (dAF) approach to recover known GWAS peaks obtained by traditional SNP association and recommended the use of a pool size of 100 individuals for DNA pooling.
Molecular basis of sex maturation in Atlantic salmon: Integrated genomics app...Amin Mohamed
This document summarizes a study that used genomics approaches to investigate the molecular basis of sex maturation in Atlantic salmon. The researchers manipulated photoperiod to initiate maturation in female salmon during a specific time window. They collected tissue samples from the brain, pituitary, ovaries and other tissues before, during and after the maturation process to generate transcriptomic and epigenomic data. The goal was to gain mechanistic insights into the triggers for sexual maturation onset in Atlantic salmon, which has implications for the salmon aquaculture industry dealing with issues like early maturation.
Amin Mohamed- PhD Candidate in Coral Reef GenomicsAmin Mohamed
Amin Mohamed is a PhD candidate at James Cook University studying chromerids and apicomplexans associated with coral reefs under a Egyptian Government PhD Scholarship and AIMS@JCU Top-Up Scholarship. He is supervised by professors and doctors at JCU and AIMS. Amin completed his BSc in Zoology in Egypt in 2006 and his MSc provided baseline data on coral health in the Egyptian Red Sea. His PhD project aims to understand the role of chromerids and apicomplexans in coral health using next-generation sequencing to analyze gene expression in corals exposed to Chromera velia. He conducted infection experiments in Japan and Australia and is now analyzing the large sequencing datasets using bio
Since the discovery of Chromera velia, a novel microalga in 2008 during a study of Australian coral symbionts, great interest in this new algal species has developed. C. velia is an important organism being considered to sit phylogenetically between the photosynthetic dinoflagellate algae and the parasitic Apicomplexa. C. velia has been detected and isolated from different scleractinian corals in Australia including corals of the Great Barrier Reef. However little is known about the ecology and genetics of this microorganism. The study aims to use next generation sequencing technology (Illumina RNAseq) to generate a reference transcriptome assembly of C. velia isolated from Montipora digitata at the GBR. Differential gene expression in altered experimental treatments is investigating the diversity and dynamics of C. velia transcripts (mRNAs). Cultures were subjected to heat shock, heat stress, cold shock, cold stress, and darkness with control cultures grown under normal conditions, cultures grown under mixotrophic condition and cultures containing motile/immotile stages. Analysis of the transcriptome of C. velia will provide genomic insight into this novel chromerid alga through identifying genes involved in various physiological and metabolic processes as investigated under different experimental conditions.
Coral diseases, coral bleaching and other health issues affecting Red Sea cor...Amin Mohamed
Research paper presented at the 12th International Coral Reef Symposium (ICRS) Cairns, Australia 9-13 July 2012 in the Climate change and bleaching mini-symposium. The paper has presented the first detailed quantitative data on the distribution and prevalence of coral diseases and other compromised health signs including coral bleaching that affect multiple corals in multiple sites along the Egyptian Red Sea coast.
Status of coral reef health in the northern red sea egypt icrs 2012 paperAmin Mohamed
This study aimed to provide baseline knowledge of coral diseases prevalence and coral health at nine reef sites in the northern Egyptian Red Sea: I (El-Ain Al-Sukhna), II (Ras Za’farana), III (Hurghada), IV (Ras Ghozlani), V (Old Quay), VI (Yolanda reef), VII (Ras Umm Sid, Sharm El-Sheikh), VIII (WoodHouse reef, Sharm El-Sheikh), and IX (canyon reef, Dahab). Sites IV, V and VI are located in Ras Mohamed, marine protected area. Field surveys of coral diseases and signs of compromised health in the studied reefs revealed a mean prevalence o f 1.03 %. Macroscopic observations were diagnosed as: black band disease (0.44%), white syndrome (0.13%), pink line syndrome (0.04%), ulcerative white spots (0.02%), skeletal eroding band (0.001 %), coral bleachi ng (2.2%), Drupella cornus snail predation (0.1%), fish predation (0.2%), pigmentation response (0.13%), sediment damage (0.08%), algae overgrowth (0.06%) and sponge overgrowth (0.04%). Prevalence of coral diseases and bleaching were higher in non-MP A sites than in MPA sites. The highest prevalence of coral diseases was recorded on the coral Favia stelligera, followed by Porites lutea, and Goniastrea edwardsi. Enhanced local anthropogenic stresses and increasing sea surface temperature due to global warming are the suggested potential factors responsible for the initiation and the persistence of some coral diseases in the studied reefs.
Status of coral reef health in the northern red sea egypt icrs 2012 paper
Aims@jcu 2015 amin
1. Host Transcriptome Analysis
During Onset and Establishment of Coral-Algal Symbiosis
Amin R Mohamed
ARC CoE Coral Reef Studies, James Cook University
AIMS@JCU, Australian Institute of Marine Science
AIMS@JCU Student Seminar Day, 2015
18th September 2015
2. Rosenberg et al., 2007
The coral holobiontBackground
Corals exist in a
multipartite symbiosis with
• Endosymbiotic algae
• Resident microbiota
Ø Bacteria
Ø Archaea
Ø viruses
5. April 5, 2016
5
Background
Host responses during Establishment of Coral- Algal Symbiosis
Current understanding
5- Disadvantages of the used method Microarrays !!!
• Few genes (1000s)
• Might be dominated by housekeeping genes
1- Competent Symbiodinium might sneak in…and host doesn’t notice
2- Competent Symbiodinium might suppress host responses
3- Response to symbionts is spatially restricted and hard to
detect
4- Previous attempts
• Voolstra et al., 2009 ~ 30 min. and 6 days post infection
• Schnitzler and Weis 2010 ~ 48 h post infection
6. Coral reference Genome and TranscriptomeBackground
Indo-pacific corals
Acropora millepora
Prof David Miller’s lab - JCU
Acropora digitifera
Prof Nori Satoh’s lab - OIST
7. • Using Next generation sequencing (NGS) (illumina RNAseq) to
explore the cross talk between host and symbiont during onset of
symbiosis
• Better understanding the early events occurring during
establishment of coral-algal symbiosis
• A. digitifera larvae infection with a competent symbiont,
Symbiodinium sp. clade B
• Quantitatively measure gene expression (using the whole
transcriptome) in larvae infected with Symbiodinium compared
to to control “aposymbiotic” ones
Aims and Objectives
Aims
Objectives
8. The view in front of the marine station, Sesoko Island, Okinawa
Methodology
Acropora assemblages
and spawning
Infection experiment
Okinawa coral spawning - June/July 2013
10. Infection experiment designMethodology
Sym B No algae
No algae
Sym B
Sym B No algae
Symbiotic
Aposymbiotic
Ø 6-d old larvae were used ~ with oral pore
Ø 700 larvae in 700 ml FSW
Ø 2 groups; Symbiotic Vs Aposymbiotic
Ø 3 replicate per group
Ø Algal cultures (competent Symbiodinium)
were added with a final conc. of 3x105 cells/ ml
after being washed 3 times in FSW
Symbiotic
Symbiodinium sp
clade B cells
Aposymbiotic
= control
Sampling time points
T1= 4h
T2= 12h
T3= 48h
11. Next-gen sequencing (illumina RNAseq)
~ 150 larvae were collected washed in FSW snap frozen in liquid
nitrogen and stored at -80°C
RNA Integrity Number RIN= 9
Methodology
mRNA enrichment
16 cDNA library construction
&
Illumina (HiSeq 2500) high-throughput
sequencing at OIST
Total RNA isolation
RNA Quality
RNA Integrity check using Agilent Bioanalyzer
14. Mapping reads against the reference A. digitifera transcriptome
Results
Illumina Reads Mapping
A screenshot of Acroproa digitifera transcripts reads alignments of both negative
control and Symbiodinium clade B infection reads at 4 h post infection;
Using the Integrated Genomics Viewer (IGV)
15. Coral Host Transcriptome Profiling at 4, 12, and 48 h post infection
Gene expression profiles between the Symbiodinium infected and control larvae
The red dots represent the 1073 significant differentially expressed genes (DEGs) detected
only at the 4h time point at adjusted P < 0.05
Differential Gene Expression Analysis EdgeRResults
Significant DEGs
Non-significant DEGs
1073 DEGs No change No change
16. DEGs clustering at 4h post Symbiodinium infection
symbiodiniumB
control
C1
C2
C3
S2
S1
S3 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samples vs. features
diffExpr.P0.05_C1.matrix.log2.centered
−2 0 2
Value
Color KeyResults
Heat
map
showing
the
expression
profiles
of
Symbiodinium
infected
and
control
samples
Rela7ve
expression
levels
are
shown
in
red
(up)
and
green
(down)
Suppression of
some host genes
18. Coral larvae exhibited varied and significant expression for genes
related to:
Genes of InterestResults
HOST-SYMBIONT INITIAL CONTACT
1. Pattern recognition and cell adhesion proteins (n=17)
2. Immune-related genes (n=5)
3. Transcription regulation (n=21)
SYMBIOSIS ESTABLISHMENT
1. Symbiosome formation (n=6)
2. sym32 signaling
SYMBIONT TOLERANCE
1. Cell cycle (n=10)
2. Apoptosis (n=11)
3. ROS response (n=13)
19. Outcome
Host signals during establishment of coral-algal symbiosis
i- Host- symbiont initial contact
• PRRs- Lectin
• Cell adhesion
• GP2 and immunity
• Ribosomes, ER, and mitochondria
ii- Symbiosis Establishment
• Symbiosome formation
• Sym32 regulation
iii-Symbiont tolerance
• Apoptosis
• Cell cycle
• Response to ROS
Up regulated
Down regulated
20. Take Home Messages
Ø The experiments allowed the detection of transient transcriptomic signals in
the coral during the initial infection period
The data imply that:
Ø Establishment of symbiosis involves cross talk between the partners
Ø An active response is required on the part of the host in order to recognize
appropriate partners
Ø The symbionts appear to suppress some host responses, including
immunity
21. Acknowledgement
Funding
• JCUPRS
• AIMS@JCU Top-up Scholarship
• Egyptian PhD Scholarship
• Prof David Miller lab- Coral Genomics Group -ARC CoE
Collaborators
• Prof Nori Satoh Lab, Marine Genomics Unit, OIST, Okinawa
• Prof Mark Ragan Lab, Computational Biology group, IMB, UQ