Monochamus alternatus adults used in this study were obtained from a laboratory colony. The colony was initially established from 4th instar larvae of M. alternatus in Nanchang city, Jiangxi province, China (28°50′45.6″N, 115°32′55.6″E) in September 2020. The larvae were individually reared on the artificial diet in a plastic cup (4 cm inner diameter × 5 cm height) until emergence . The newly emerged adults fed on the fresh twigs of masson pines (30 cm length) for supplement nutrition lasting approximately 15 days in a plastic box (11.5 cm length × 11.5 cm width × 5 cm height). The sex of the adult is determined by antennal length. Ten pairs of sexually mature adults (sex ratio 1: 1) as a group were maintained in a net cage (35 cm length × 35 cm width × 40 cm height), and were allowed to mate randomly and oviposit on the trunks of Masson pines (10 cm diameter, 30 cm length). Eggs were collected periodically, and kept in Petri dishes (5 cm inner diameter × 1 cm height) with a moist cotton pad for incubation. All insects were kept in an environmental incubator (MTI-201B, Tokyo Rikakikai, Japan) at 25 ± 0.5 °C with a 70 ± 5 % relative humidity and a photoperiod of 14: 10 h (L: D).
Sample preparation for quantification and localization of MaltHSP70-2 under heat stress conditions
To determine the temporal dynamics of MaltHSP70-2 gene expression under heat stress conditions, sexually mature males were kept in the environmental incubator at 40 ± 0.5 °C for 0, 1, 3, 6, and 12 hours, and then were recovered at room temperature for 12, 24, 36, and 48 hours. Vigorous males from each time point were selected and frozen in liquid nitrogen for subsequent tests. Five adults per replicate and three replicates per treatment were used in this experiment. The same sample preparation was performed for female adults.
To determine the spatial dynamics of MaltHSP70-2 gene expression under heat stress conditions, sexually mature males and females kept in the environmental incubator at 42.5 ± 0.5 °C for 3 hours were sampled as the heat stock treatment, and the individuals kept at room temperature were used as a negative control. Five vigorous males and females per replicate and three replicates from each treatment were dissected to obtain various tissues (including antenna, head, leg, gut, wing, malpighian, tubule, and testis or ovary), and frozen in liquid nitrogen for subsequent tests.
For Western blot and immunofluorescence staining of MaltHSP70-2, the scheme of sample preparation was the same as that for determining the spatial dynamics of MaltHSP70-2 gene expression. Only the whole body and reproductive tissues of female and male adults were sampled for Western blot, and the testis of male adults was sampled for immunofluorescence staining.
Determination of gene expression levels of MaltHSP70-2 under heat stress conditions
Coding sequence of MaltHSP70-2 (GeneBank ID: 895064) has been identified in our previous work . The samples for determining the spatiotemporal dynamics of MaltHSP70-2 gene expression under heat stress conditions were crushed into powder in liquid nitrogen for RNA extraction. According to the manufacturer’s protocol, the total RNA of each sample was isolated using an RNA extraction Kit (Tiangen, China). RNA quantity was tested using a Nanodrop 2000 (Thermo Scientific, Waltham, MA, United States), and RNA integrity was monitored on a 1 % agarose gel. Then, the first-strand cDNA was synthesized using the HiScript III RT SuperMix cDNA Synthesis Kit (Vazyme, China) following the manufacturer’s protocol. 2 μL cDNA with a concentration of 200 ng / μL was used as a template for Real-time quantitative PCR (RT-qPCR). According to the manufacturer’s protocol, RT-qPCR experiments were performed using SYBR Premix Ex Taq II (TaKaRa, Japan) on an Applied Biosystem 7500 Real-Time PCR System (Thermo Fisher, Massachusetts, United States) with 20 μL reaction system. PCR conditions were as follows: 5 min at 95 °C, followed by 40 cycles of 10 s at 95 °C and 40 s at 60 °C, and then a melting curve analysis for continuous fluorescence monitoring. Specific primers were designed using Primer Premier version 5.0 (Supplementary Table S1) and synthesized from Sangon, Shanghai, China. Gene expression levels of MaltHSP70-2 were determined by the 2−△△Ct method with RPL10 as the housekeeping genes .
Preparation of specific antibodies against MaltHSP70-2 protein
Our previous study described the preparation of recombinant MaltHSP70-2 protein in vitro . 200 μg of MaltHSP70-2 protein emulsified with complete Freund's adjuvant (CFA) was subcutaneously injected into two New Zealand White Rabbits for the first two times of immunization (an injection is given every 15 days). Then, 100 μg of MaltHSP70-2 protein emulsified with incomplete Freund's adjuvant (IFA) was injected as described above for the next three times of immunization (an injection is given every 15 days). 10 mL of the rabbits’ blood was collected on days 38, 43, 53, 58, and 69 after the first immunization, respectively. Qualitative analyses of these blood samples (cut-off value > 1 after 1: 4000 dilution) were performed using Enzyme-Linked Immunosorbent Assay (ELISA). Approximately 50 mL of positive blood samples was collected for Protein A purification. According to ELISA experiments, the potency of the purified antibody was over 1: 128, 000, and its concentration was over 1 mg/mL. The purified antibody was stored at -80 °C for Western blot analysis and immunofluorescence staining.
Western blot analysis of MaltHSP70-2 under heat stress conditions
The total protein of samples for Western blot analysis was extracted using Tissue & Cell Protein Extraction Kit (Epizyme, China). Protein concentration was measured using bicinchoninic acid (BCA) protein assay kit (Beyotime, China) and diluted to 4 μg / μL with aseptic water. 20 μL of each protein sample was used to conduct 7.5% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and then transformed to polyvinylidene fluoride (PVDF) membrane at 80 V for 2 hours. The PVDF membrane was blocked with 5 % skimmed milk in Tris Buffered Saline with Tween-20 (TBST) for 1 hour at 25 °C, then washed in TBST three times. The membrane was incubated with MaltHSP70-2 antibody (diluted with TBST at a ratio of 1: 200 ) at 4 °C overnight. Subsequently, the membrane was washed in TBST three times again, and incubated with the HRP-conjugated goat anti-rabbit IgG antibody (Beyotime, China) (diluted with TBST at a ratio of 1: 1000) at 25 °C for 1 hour. Finally, the membrane was colored using Clarity Western ECL substrate (Bio-rad, America), and imaged on an Odyssey-Fc imaging system (Gene Company Limited, China).
Immunofluorescence staining of MaltHSP70-2 in testis under heat stress conditions
Testis samples for immunofluorescence staining were fixed in 4 % paraformaldehyde for 24 hours, then were dehydrated with graded alcohol series and embedded in paraffin. Cross-section of the testis was obtained using RM2245 slicer (LEICA, Germany). To observe the basic morphology of cross-section of the testis, hematocylin-eosin staining was performed according to the methods described in Rosenzweig . The section was deparaffinized, rehydrated, and washed in phosphate-buffered solution (PBS) three times for immunofluorescence staining. Subsequently, the section was blocked with two drops of 3% H2O2 methanol solution for 10 min at room temperature, then washed in phosphate-buffered solution (PBS) for three times, and blocked with 100 μL of 5 % Bovine Serum Albumin (BSA) for 30 min at room temperature. After finishing the block, the section was incubated with MaltHSP70-2 antibody (diluted with PBS at a ratio of 1: 200 ) at 37 °C for 2 hours. Then the section was washed in PBS three times and incubated with TRITC-conjugated anti-rabbit IgG antibody (Beyotime, China) (diluted with TBST at a ratio of 1: 1000) at 37 °C for 1 hour. Finally, the section was stained using Diamidino-2-phenylindole (DAPI) and imaged on a confocal scanning fluorescence microscope DM2500 (LEICA, Germany). The excitation wavelength for MaltHSP70-2 was 549 nm, and for DAPI was 450 nm.
RNA interference of MaltHSP70-2 and bioassays
To synthesize double-stranded RNA (dsRNA), a 447 bp fragment of MaltHSP70-2 (see Appendix 1, the region has no similar sequences with other HSP genes) was amplified using the primer containing T7 RNA polymerase promoter at both ends (see Table S1) with T7 RiboMAX™ Express RNAi System (Promega, USA). The dsRNA of the green fluorescent protein (GFP) gene was used as a negative control. The quantity of dsRNA was measured using a Nanodrop 2000 (Thermo Scientific, Waltham, MA, United States), and the size of dsRNA was monitored on a 1 % agarose gel.
To determine the efficiency of RNA interference (RNAi), two doses of dsMaltHSP70-2 (4 μg and 8 μg) were injected into the intersegmental membrane of each adult, and the control injection was conducted with dsGFP (8 μg). At 1, 3, 5, and 7 days post-injection, the whole bodies of eight vigorous adults (sex ratio 1: 1) from each time point were collected for RNA extraction, cDNA synthesis, and RT-qPCR as described above. According to the results of RNAi efficiency, 8 μg of dsMaltHSP70-2 was the optimal dose and 3 days post-injection was the optimal effective time (Fig. S2). Subsequently, the relative expression levels of other HSP genes were measured using RT-qPCR as described above after silencing MaltHSP70-2 (silencing condition: 8 μg dose, 3 days post-injection).
For bioassays, twenty-four males and twenty-four females were respectively exposed to 42.5 °C after silencing of MaltHSP70-2 (silencing condition: 8 μg dose, 3 days post-injection). The survival time of each adult was recorded. The adults were defined as death when there was no muscle response to stimulation with a fine brush. The same amount of adults without injection of dsRNA (group name: control) and with an injection of equal quantity of dsGFP (group name: dsGFP) were used as controls.
mRNA levels of MaltHSP70-2 at different stages of heat shock treatment were compared using one-way ANOVA followed by Tukey’s HSD test (P < 0.05). Statistically significant differences in other quantitative data were analyzed using one-way ANOVA followed by an independent sample t-test (P < 0.05). All analyses were conducted in SPSS version 20.0 software (IBM SPSS Statistics, Chicago, IL, United States), and plotted with OriginPro version 9.0 software (OriginLab Inc., Northampton, United Kingdom).