Growth and gonadal transcriptome changes in the largest extant amphibian after prolonged heat stress

Rising environmental temperatures may disrupt gonadal differentiation and alter sex ratios in amphibians, posing risks to temperature-sensitive populations. The Chinese giant salamander ( Andrias davidianus ; CGS), the world’s largest extant amphibian, is critically endangered in the wild and is also an important aquaculture species in China. However, how prolonged heat exposure affects gonadal differentiation in CGS remains poorly understood. Here, we combined growth measurements, gonadal histology, fluorescence in situ hybridization, and multi-timepoint transcriptomic profiling to characterize responses under control-temperature (CT) and high-temperature (HT) rearing conditions. Animals reared in the HT tank showed lower growth during later development and delayed ovarian differentiation in genetic females. Integrating histological and transcriptomic evidence, we identified 270 dph as a key developmental stage at which delayed ovarian differentiation coexisted with masculinization-associated molecular changes in HT genetic females. During this stage, ectopic Dmrt1 expression was detected in HT genetic females but was absent or nearly absent in control females. HT genetic females also showed reduced Cyp19a1 expression, increased Hsd17b3 expression at 270 dph, and increased Cyp17a1 expression at 360 dph, indicating stage-dependent steroidogenesis-associated transcriptional remodeling. WGCNA and protein–protein interaction analyses further identified Hsp90aa1 , Hsp90ab1 , and other molecular chaperone genes as candidate hubs within an HT-associated co-expression network. Together, these findings indicate that HT rearing was associated primarily with delayed ovarian differentiation and attenuation of female-biased developmental programs, accompanied by limited, stage-dependent masculinization-associated molecular remodeling. These results provide a framework for understanding temperature-associated gonadal plasticity in CGS and may inform future conservation and aquaculture studies.