Reference genes are commonly used for normalization of target gene expression during RT-qPCR analysis. However, no housekeeping genes or reference genes have been identified to be stable across different tissue types or under different experimental conditions. To identify the most suitable reference genes for RT-qPCR analysis of target gene expression in the hepatopancreas of crucian carp (Carassius auratus) under various conditions (sex, age, water temperature, and drug treatments), seven reference genes, including beta actin (ACTB), beta-2 microglobulin (B2M), embryonic elongation factor-1 alpha (EEF1A), glyceraldehyde phosphate dehydrogenase (GAPDH), alpha tubulin (TUBA), ribosomal protein l8 (RPL8) and glucose-6-phosphate dehydrogenase (G6PDH), were evaluated in this study. The stability and ranking of gene expression were analyzed using three different statistical programs: GeNorm, Normfinder and Bestkeeper. The expression errors associated with selection of the genes were assessed by the relative quantity of CYP4T. The results indicated that all the seven genes exhibited variability under the experimental conditions of this research, and the combination of ACTB/TUBA/EEF1A or of ACTB/EEF1A was the best candidate that raised the accuracy of quantitative analysis of gene expression. The findings highlighted the importance of validation of housekeeping genes for research on gene expression under different conditions of experiment and species.

Tadpoles of two sympatric anurans, Bufo gargarizans Cantor, 1842 and Rana zhenhaiensis Ye, Fei & Matsui, 1995, were used as model organisms to examine the effects of different levels of tail loss on swimming performance and survival. On average, B. gargarizans tadpoles were shorter and had smaller tails and body mass than R. zhenhaiensis. After 75% tail loss, the survival rate of experimental and control B. gargarizans tadpoles, and of experimental tadpoles of the two species, differed significantly; the number of tadpoles surviving a complete impairment of their swimming ability did not differ between B. gargarizans and R. zhenhaiensis. After 50% tail loss, the swimming performance (swimming speed, maximum distance and number of stops) of the two species was significantly affected. However, the adverse influence of tail loss on the swimming speed of B. gargarizans tadpoles was greater compared to R. zhenhaiensis tadpoles. Our data indicates that a 50% tail loss results in swimming costs for B. gargarizans and R. zhenhaiensis tadpoles, and that 75% tail loss decreases the survival rate of B. gargarizans tadpoles. Therefore, we conclude that tadpoles of different species and with the same degree of tail loss use distinctive strategies to improve individual fitness in the face of predator pressure.