2.4. Expression of CrufCSP1 Gene

The primers that were designed based on the cDNA sequences were used for RT-qPCR (Table 1). The β-actin gene was chosen as a reference gene for the expression levels of CrufCSP1 in different life stages and tissues. The RT-qPCR reactions were conducted using TB Green^® Premix Ex TaqTM II (TaKaRa, Dalian, China) and the LightCycler480 real-time PCR system (Roche Diagnostics, Switzerland). The reaction conditions consisted of an initial denaturation step at 95 °C for 30 s, followed by 40 cycles of denaturation at 95 °C for 10 s, annealing at 55 °C for 30 s, and extension at 72 °C for 40 s. For each sample, three biological replicates and technical replicates were performed. The 2^−ΔΔCT method was used to determine the mRNA expression levels of CrufCSP1.

The data were first checked for normality of distribution and homogeneity of variance. The relative expression levels of CrufCSP1 between different developmental stages and tissues of both female and male specimens were analyzed by one-way analysis of variance (ANOVA), followed by Tukey’s multiple comparison test when means had significant differences at p < 0.05. The expressions of CrufCSP1 between male and female were analyzed by Student’s t-test. Data were analyzed by using statistical software package SPSS 22.0 and shown as mean ± standard error.