RESUMO
Quantitative reverse transcription-polymerase chain reaction (qRT-PCR) is a widely used tool for measuring gene expression; however, its accuracy relies on normalizing the data to one or more stable reference genes. Eocanthecona furcellata (Wolff) is a polyphagous predatory natural enemy insect that preferentially feeds on more than 40 types of agricultural and forestry pests, such as those belonging to the orders Lepidoptera, Coleoptera, and Hymenoptera. However, to our knowledge, the selection of stable reference genes has not been reported in detail thus far. In this study, nine E. furcellata candidate reference genes (ß-1-TUB, RPL4, RPL32, RPS17, RPS25, SDHA, GAPDH2, EF2, and UBQ) were selected based on transcriptome sequencing results. The expression of these genes in various samples was examined at different developmental stages, in the tissues of male and female adults, and after temperature and starvation treatments. Five algorithms were used, including ΔCt, geNorm, NormFinder, BestKeeper, and RefFinder, to evaluate reference gene expression stability. The results revealed that the most stable reference genes were RPL32 and RPS25 at different developmental stages; RPS17, RPL4, and EF2 for female adult tissue samples; RPS17 and RPL32 for male adult tissue samples; RPS17 and RPL32 for various temperature treatments of nymphs; RPS17 and RPS25 for nymph samples under starvation stress; and RPS17 and RPL32 for all samples. Overall, we obtained a stable expression of reference genes under different conditions in E. furcellata, which provides a basis for future molecular studies on this organism.
RESUMO
The honey locusts (genus Gleditsia) are a genus of high-value trees in Asia. Seed beetle, Megabruchidius dorsalis (Fåhraeus) (Col.: Chrysomelidae: Bruchinae), is a Gleditsia oligophagous pest that causes severe yield reduction. To understand the cold tolerance of M. dorsalis adults, this study investigated its cold tolerance strategy and the influence of low temperatures on its physiology and biochemistry. The low-temperature treatments were divided into three groups: long-term temperature acclimation (Group 1; 15°C, or 20°C, or 25°C, or 28°C [control check, CK] for 10 days), short-term low-temperature exposure (Group 2; 0°C or 4°C for 2 h), and long-term low-temperature induction (Group 3; 0°C or 4°C for 1, 3, or 5 d). The supercooling point (SCP; temperature at which spontaneous nucleation and ice lattice growth begin), freezing point (FP; temperature at which insect fluids freeze), low lethal temperature (LLT; temperature at which all individuals are killed), water, lipid, glycerol, and total sugars contents were measured under different temperature stresses. The results showed that M. dorsalis adults were a freeze-avoidant species. The SCP and LLT at 28°C were -10.62°C and -19.48°C, respectively. The SCP and FP of long-term temperature acclimation (15°C, or 20°C, or 25°C) were significantly lower than that of the control group (28°C). The water content of the long-term low temperature induction (0°C) group was significantly lower than that of the control group. The lipid and glycerol content in the acclimated group at 20°C and 25°C were significantly higher than in the control group. M. dorsalis adults may maintain their biofluids in a supercooled state via cryoprotectant accumulation and cryoprotective dehydration to prevent ice nucleation. This study provides a theoretical basis for future research on overwintering and potential distribution and related prediction of M. dorsalis adults.