The interplay among environment, demography, and host-parasite interactions is a challenging frontier. In the ocean, fundamental changes are occurring due to anthropogenic pressures, including increased disease outbreaks on coral reefs. These outbreaks often include multiple parasites, calling into question how coral immunity functions in this complex milieu. Corals provide an interesting model to study ecological immunity during co-infection, being highly sensitive to environmental change, susceptible to many diseases, and defended by the innate immune system. Our work investigates the interplay of factors influencing coral co-infection using metrics of the innate immune response: levels of cellular immunity and the expression of candidate immune genes. We used existing copepod infections and live pathogen inoculation with Aspergillus fungus to test the effect of sequential co-infections in a laboratory experiment. We profile significant increases in the expression of the immune recognition gene Tachylectin 5A in response to both of these naturally occurring parasites of the Caribbean sea fan octocoral, Gorgonia ventalina. Cellular immunity increased significantly by 8.16% in copepod infections compared to controls and single Aspergillus infections. We evaluated immunity in reef populations and again detected activation of cellular immunity, with a 13.6% increase in copepod infections and no detectable increase in fungal infections. Thus, cellular immunity measured in the field and lab were similar, increasing with copepod infections and not Aspergillus. We found random co-occurrence of copepods and fungus across 15 reefs in Puerto Rico, suggesting other factors prevail in structuring parasite infection. Sea fan colony size strongly predicted infection by the copepod parasite. Moreover, the effect of parasitic infection on immunity was small relative to the explanatory power of site differences and coral cover, and roughly equivalent to the effect of reproductive status. We suggest that host size, reproductive status, live coral cover, and site-specific factors have large effects on parasitic infections and host immunity that overwhelm effects of the parasites on each other. Thus, host size and site-specific features emerge as critical drivers in this multi-parasite system. Parsing the effects of immunity and ecological factors in coral co-infection shows how disease depends on more than one host and one parasite.

These data are from a laboratory experiment and a field survey. The laboratory data on amoebocyte density and copepod severity were collected using histology slides of the sea fan G. ventalina. Amoebocyte density was calculated using percent area of amoebocytes (immune cells) in ImageJ. The gene expression data for T5A, MMP, IKB and EF1 were obtained using quantitative PCR. We calculated fold change in gene expression relative to the reference gene, EF1. Real-time PCR miner was used to calculate primer efficiencies and cycle threshold values, which were incorporated into the following equation to calculate relative expression: 1/(1 + Efficiency)^Cq (Zhao and Fernald, 2005).

Notes are included in R code for each set of files (R code and related Excel spreadsheet).