Table of Contents

• Dataset Description
  • Methods & Sampling
  • Data Processing Description
• Data Files
• Parameters
• Instruments
• Deployments
• Project Information
• Funding

Accession numbers to NCBI's Gene Expression Omnibus (GEO) are provided for the Gorgonia ventalina Illumina sequences collected from reefs off the coast of Puerto Rico.

References:
Burge CA, Mouchka, ME, Harvell, CD & S Roberts. (In review) Immune response of the Caribbean sea fan, Gorgonia ventalina exposed to an Aplanochytrium parasite as revealed by transcriptome sequencing.

Sea fan collection and husbandry
Twelve G. ventalina individuals were collected at Laurel Patch Reef, La Parguera, Puerto Rico in May 2010. Sea fans were cut into two, 6x9 cm pieces, and suspended in situ to heal on the reef for three days. Sea fans were then collected from the reef and moved into static 38 L aquariums at the University of Puerto Rico, Isla Magueyes Laboratories in La Parguera, PR. Fans were distributed equally, in a clonally replicated design, between six aquariums (n=4 sea fans per aquarium with sea fan specimens 1-4, 5-8, 9-12 held in individual aquariums in duplicate). Sea fans were acclimated for two days and water was changed twice daily.

Experimental Inoculations
One piece of each sea fan (n=12) was injected three times with a solution of a 5 day culture of an Aplanochytrium previously isolated from a sea fan. Complementary clonal pieces (n=12) were injected with QPX media only to serve as a control. After 24 hours, an area including each of the three injection points was collected from each sea fan and flash frozen in liquid nitrogen and subsequently stored at -80 C. Sea fan samples were shipped on dry ice overnight to Cornell University and stored at -80 C until sample preparation.

Sample preparation
Each individual sea fan sample was ground in liquid nitrogen using a mortar and pestle, and the resulting powder was placed in a 2.0 mL microcentrifuge tube. Total RNA was extracted using a modified Trizol/Qiagen RNeasy protocol, whereby following the ethanol precipitation step of the Trizol manufacturer's instructions (Invitrogen, The Life Technologies CorporationTM, Grand Island, NY ), the aqueous solution was added to an RNeasy column, and the Qiagen RNeasy manufacturer's instructions were subsequently followed (Qiagen, Valencia, California). DNA was removed from extracted RNA using the Turbo DNA-free treatment according to the manufacturer's instructions (Ambion Inc, The Life Technologies CorporationTM, Grand Island, NY). Removal of DNA was confirmed by using RNA (1 µl) as template in a PCR reaction targeting 18s ribosomal DNA as previously described. RNA concentrations were quantified using the NanoDrop ND-1000 (NanoDrop Technologies, Wilmington, DE).

Sequencing and Analysis
For each treatment, samples were pooled (n=12) using 900 ng of total RNA from each sea fan. RNA quality was assessed using an Agilent BioAnalyzer 2100 at the Cornell University Life Sciences Core Laboratory Center (CLC). Library preparation was done at the Cornell Microarray facility using the mRNA-Seq 8-Sample Prep Kit (Illumina, San Diego, California) followed by sequencing preparation using the standard cluster generation kit and 36 cycle Illumnia sequencing kit (Illumina, San Diego, California) at the Cornell University CLC. Each library was sequenced in its own individual lane where 86 bp reads were captured using an Illumina/Solexa Genome Analyzer at the Cornell University CLC. The data have been deposited in NCBI's Gene Expression Omnibus.

Data Processing:
Initially, all sequences were trimmed based on quality scores of 0.05 and the number of ambiguous nucleotides (>2 on ends). Sequences smaller than 20 bp were also removed. De novo assembly was carried out using CLC Genomics Workbench v4.0 (CLC Bio) with the following parameters: similarity=0.90, length fraction=0.8, insertion cost=3, deletion cost=3, mismatch cost=2 and minimum size = 400.

Consensus sequences (or contigs) were compared to the UniProtKB/Swiss-Prot database. Comparisons were made using the BLASTx algorithm with a maximum of 1E-6 e-value threshold.

RNA-Seq analysis was performed to determine differential gene expression patterns between Aplanochytrium exposed and control libraries. CLC Genomics Workbench v4.0 (CLC Bio) was used to map the reads to the assembled transcriptome and to obtain raw counts of sequencing reads using following parameters: unspecific match limit = 5, maximum number of mismatches = 2, minimum number of reads = 10. Statistical comparisons of count data between the control and Aplanochytrium exposed libraries was carried out using DESeq. Genes were considered differentially expressed in a given library when the adjusted p-value was less than or equal to 0.05.

To identify enriched biological themes and GO terms, the Database for Annotation, Visualization and Integrated Discovery (DAVID) v6.7 was used. Specifically, corresponding Swiss-Prot accession numbers for differentially expressed genes were used as the gene list and corresponding Swiss-Prot accession numbers for the assembled transcriptome was used as the background. Biological Process terms were considered significantly enriched when the p-value was less than 0.05.

This award is funded under the American Recovery and Reinvestment Act of 2009 (Public Law 111-5).

Coral reef ecosystems are highly endangered by recent increases in temperature and by projected increases in ocean acidification. Although temperature has been identified as a driver of some coral disease outbreaks, nothing is known about direct effects of acidification on host immunity and pathogen virulence, or the potential for synergism with temperature. Natural coral populations often suffer from simultaneous infection by multiple pathogens that can also influence host immune responses, but co-infection dynamics have not been investigated in invertebrate systems lacking classical adaptive immunity. Changing climate will very likely influence the outcome of single and co-infection.

This project will investigate the influence of environmental stress on co-infection dynamics of the sea fan coral, Gorgonia ventalina, with a fungal pathogen, Aspergillus sydowii and a protist parasite, SPX. The goal is to identify the mechanisms through which multiple infections, temperature and acidification modify host resistance, leading to changes in within- and among-colony rates of disease spread.

The objectives of this project are to:
(1) Identify incidence and co-infection frequency of Aspergillus sydowii and SPX. Detailed field surveys of the two diseases will test the hypothesis that co-infection is significant, provide valuable information about drivers of aspergillosis, and will help to characterize an emerging new sea fan disease.
(2) Investigate how co-infection influences sea fan susceptibility, resistance, and within host disease dynamics. Through manipulative lab inoculation experiments we will test the hypothesis that single infections increase susceptibility to a second pathogen.
(3) Examine the effects of temperature increase and ocean acidification on pathogen virulence, on underlying host resistance, and on the dynamics of single and co-infections.

The hypotheses that acidification will increase pathogen virulence and host susceptibility will be tested in a temperature and pH controlled experimental system. This system will also allow the potential synergistic effects of temperature and acidification on host immunity and co-infection dynamics to be explored. The primary intellectual merit of the proposed work will be a greater understanding of how changing climate mediates co-infection and immunity in a non-model invertebrate. While fungal pathogens are primarily opportunistic, labyrinthulid protozoans are recognized as primary pathogens in shellfish. Even in shellfish, little is known about co-infections involving labyrinthulids, and these protists are entirely unstudied in corals.

Publications associated with this project:
Burge CA, Douglas N, Conti-Jerpe I, Weil E, Roberts S, Friedman CS & CD Harvell. (May 2012) Friend or foe: the association of Labyrinthulomycetes with the Caribbean sea fan, Gorgonia ventalina. Dis Aquat Org. 101:1-12. doi: 10.3354/dao02487

Burge CA, Mouchka, ME, Harvell, CD & S Roberts. (In review) Immune response of the Caribbean sea fan, Gorgonia ventalina exposed to an Aplanochytrium parasite as revealed by transcriptome sequencing.