Ribas L et al. (2009), Expression profiling the temperature-dependent ...

XB-ART-40804

PLoS One 2009 Dec 22;412:e8408. doi: 10.1371/journal.pone.0008408.

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Expression profiling the temperature-dependent amphibian response to infection by Batrachochytrium dendrobatidis.

Ribas L , Li MS , Doddington BJ , Robert J , Seidel JA , Kroll JS , Zimmerman LB , Grassly NC , Garner TW , Fisher MC .

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Amphibians are experiencing a panzootic of unprecedented proportions caused by the emergence of Batrachochytrium dendrobatidis (Bd). However, all species are not equally at risk of infection, and risk is further modified by environmental variables, specifically temperature. In order to understand how, and when, hosts mount a response to Bd we analysed infection dynamics and patterns of gene expression in the model amphibian species Silurana (Xenopus) tropicalis. Mathematical modelling of infection dynamics demonstrate the existence of a temperature-dependent protective response that is largely independent of the intrinsic growth-rate of Bd. Using temporal expression-profiling by microarrays and qRT-PCR, we characterise this response in the main amphibian lymphoid tissue, the spleen. We demonstrate that clearance of Bd at the host-optimal temperature is not clearly associated with an adaptive immune response, but rather is correlated with the induction of components of host innate immunity including the expression of genes that are associated with the production of the antimicrobial skin peptide preprocareulein (PPCP) as well as inflammatory responses. We find that adaptive immunity appears to be lacking at host-optimal temperatures. This suggests that either Bd does not stimulate, or suppresses, adaptive immunity, or that trade-offs exist between innate and adaptive limbs of the amphibian immune system. At cold temperatures, S. tropicalis loses the ability to mount a PPCP-based innate response, and instead manifests a more pronounced inflammatory reaction that is characterised by the production of proteases and higher pathogen burdens. This study demonstrates the temperature-dependency of the amphibian response to infection by Bd and indicates the influence that changing climates may exert on the ectothermic host response to pathogens.

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Species referenced: Xenopus tropicalis
Genes referenced: cct7 grb10 prss1

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	Figure 1. Experimental infection and model outputs.A. Observed prevalence data for iCT animals (blue circles) and modelled prevalence data (blue line, RSS (residual sum of squares) = 0.112), and observed prevalence data for iWT animals (red circles) and modelled prevalence data (solid red line, RSS = 1.99). Dotted orange line shows modelled iWT prevalence data where an identical host response to iCT animals has been assumed. g means the gap between modelled prevalence in iWT with and without differing host response due to temperature. Modelling prevalence without taking into account a differing host response does not fit the observed data well (RSS = 1611). B. Third order polynomial fitted to measurements of temperature against daily exponential growth rate of Bd in culture from [14] (y = -0.00008639x3 + 0.002612x2 – 0.001124x – 0.0004419). Blue circle indicates Bd growth rate at the temperature of iCT animals, and red circle indicates Bd growth rate at the temperature of iWT animals. C. The intensity of infection measured by qPCR as zoospore genomic equivalents.
	Figure 2. Microarray results in spleen of S. tropicalis Bd infected.A. Expression of the top 50 genes obtained from microarrays with known or putative immune-function as shown by two-way gene clustering and clustering analysis (GeneSpring Software). The colour scale shows the fold changes above normalised controls. B. Principal Component Analysis (PCA) showing that S. tropicalis frogs held at cold temperatures (iCT) have the most diverse transcriptional profile during the first stage (7 days) of infection. Axis: X = PCA Component 1 (24.99% variance); Y = PCA Component 2 (18.12% variance); Z = PCA Component 3 (14.95% variance).
	Figure 3. Microarray validation by quantitative RT-PCR for each of the 52 experimental frogs using spleen RNA.For qRT-PCR analysis, samples were analysed in triplicates for each individual (control and infected). A. Microarray validation by qRT-PCR amplification for a subset of 10 genes with known or suspected roles in immunity. Black bars = microarray data (pooled samples); White bars = qRT-PCR validation data (individual samples). Data is expressed by log2 Relative Quantification versus the control group.. B. Further transcriptomic analysis by qRT-PCR of the expression of PPCP and trypsinogen 2 genes individually tested for all frogs (n = 52). Data is expressed by log2 Relative Quantification versus the control group (iWT7/cWT7, iWT42/cWT42, iCT7/cCT7 and iCT42/cCT42) ± Standard Error. * Indicates significance by Student t-test (p<0.05) compared to the control group at each time point.

References [+] :

Ali, Antimicrobial peptides isolated from skin secretions of the diploid frog, Xenopus tropicalis (Pipidae). 2001, Pubmed, Xenbase