Expression analysis of three caspase genes with death-fold domains in amphioxus. (A) The two amphioxus initiator caspases and the caspase family of seven other organisms were used to construct a phylogenetic tree based on their caspase domains using MEGA version 3.1 (neighbor joining tree). Numbers at nodes indicate bootstrap values. hs: Homo sapiens; mm: Mus musculus; gg: Gallus gallus; xt: Xenopus tropicalis; dr: Danio rerio; sp: Strongylocentrotus purpuratus; bf: Branchiostoma floridae; bbt: Branchiostoma belcheri tsingtauense. (B) Quantitative RT-PCR was performed to determine tissue distribution of three caspases in adult amphioxus. Data are expressed as a ratio of the mRNA abundance as calculated with the 2-ΔΔCt method from two parallel experiments performed in triplicate. (C) and (D) Quantitative RT-PCR analysis of three amphioxus caspases at 2, 4, 8, 12, 24, 36, 48, and 72 h after challenge with S. aureus (Gram-positive) and V. vulnificus (Gram-negative). Results are presented as the fold induction in the abundance of mRNA relative to that of samples exposed to PBS and were determined by the 2-ΔΔCt method from two parallel experiments performed in triplicate. The endogenous control for normalization was mRNA for cytoplasmic β-actin. Values were considered to be significant when P < 0.05.