Total RNA (5 mg) was extracted from tadpoles and toadlets using Trizol reagent (Invitrogen) according to the manufacturer's instructions and reverse transcribed using the cDNA First Strand Synthesis Kit (Invitrogen). Real time PCR was used to detect larval and adult globin mRNA at selected stages of development. Oligonucleotide sets were larval globin; sense 59-GCTGAAGA-GAAAGCCGC -39, and antisense 59-ATGGCGGTGACATTG-GAC -39 (151 bp), or adult globin; sense 59-CAGATCAC-GAGCTCAAGAG -39, and antisense 59-ATGGCATCAG-CAGAGCCA -39 (151 bp). Results were standardised with cane toad actin (GenBank Accession no. EL595572) by amplifying a 117 bp fragment using sense 59-ATGACACAGATAATGTTT-GAGAC -39 and antisense 59-ATCACCAGAGTCCATCA-CAAT -39 primers. Reactions consisted of QuantiTect SYBR Green RT-PCR Master Mix (Qiagen), 0.5 mM oligonucleotides and 200 ng of first strand cDNA template, and run on a RotorGene 2000 (Corbett). The thermal profile was as follows: 50uC for 2 min, 95uC for 2 min, 40 cycles of 95uC for 30 sec; 58uC for 30 sec; and 72uC for 30 sec. PCR target products were used as reaction standards at concentrations ranging from 1610 2 to 1610 8 molecules per mL. Analysis of real time PCR reactions and melting point dissociation curve reactions were performed using the programme Rotorgene version 6.0 (Corbett).