Abstract: 【Aim】 This study aims to compare the efficiency of different RNAi methods for silencing the heat shock protein genes (GdHsp60 and GdHsp70) of Galeruca daurica, so as to select a method for efficiently reducing the expression level of target gene expression for the functional study to clarify the function of these two genes in the cold hardiness of G. daurica larvae. 【Methods】 GdHsp60 and GdHsp70 in the 1st and 2nd instar larvae of G. daurica were silenced by RNAi via feeding and microinjection, respectively, and their silencing efficiencies were detected by qPCR. After RNAi of GdHsp60 and GdHsp70 by microinjection for 24 h, the super-cooling point and freezing point of the 2nd instar larvae of G. daurica were determined with thermocouple method, and the median lethal temperature (Ltemp₅₀) for the 2nd instar larvae exposed to different low temperatures (-6--14℃) for 2 h and the median lethal time (Ltime₅₀) for exposed to -5℃ for different periods were determined by bioassay. 【Results】 GdHsp60 and GdHsp70 could be silenced by RNAi via both feeding and microinjection, but microinjection-based RNAi had higher silencing efficiency. After microinjection of dsGdHsp60 and dsGdHsp70 into the 2nd instar larvae of G. daurica for 24 h, the expression levels of GdHsp60 and GdHsp70 decreased to the lowest point, being decreased by 84.15% and 92.38%, respectively, as compared to that in the control (microinjected with dsGFP).  In the 2nd instar larvae of G. daurica, the super-cooling point, freezing point, and Ltemp₅₀ and Ltime₅₀ values after microinjection of dsGdHsp60 for 24 h were -10.56±0.42℃, -7.66±0.56℃, -8.33℃ and 49.25 h, respectively, while those after microinjection of dsGdHsp70 for 24 h were -9.08±0.23℃, -6.09±0.28℃, -8.20℃, and 52.21 h, respectively, and those in the control were 14.71±0.11℃, 13.94±0.09℃, -10.63℃ and 87.13 h, respectively. Compared with the control (microinjected with dsGFP), the super-cooling point, freezing point and Ltemp50 in the 2nd instar larvae of G. daurica microinjected with dsGdHsp60 and dsGdHsp70 for 24 h significantly increased, while the Ltime₅₀ value significantly shortened. 【Conclusion】 Microinjection method can be used to efficiently silence the Hsp-related genes in G. daurica. Silencing GdHsp60 and GdHsp70 can significantly decrease the cold hardiness of G. daurica larvae.

Key words: Galeruca daurica, heat shock protein; GdHsp60; GdHsp70, RNAi, cold hardiness