Introduction
Diapause is a genetic characteristic gradually formed in the long evolutionary process of insects to avoid adverse living environments for continuation and to improve population abundance. Insect diapause is determined by genes (Gupta, Reference Gupta and Schooneveld1991; Kitagawa et al., Reference Kitagawa, Shiomi, Imai, Niimi, Yaginuma and Yamashita2005) controlled by hormones and specific external stimuli, such as temperature, photoperiod and nutrition (Fischman et al., Reference Fischman, Pitts-Singer and Robinson2017; Pitts-Singer, Reference Pitts-Singer2020; Süess et al., Reference Süess, Dircksen, Roberts, Gotthard, Nässel, Wheat, Carlsson and Lehmann2022). Before entering the diapause period, it is necessary to isolate additional metabolic energy by sensing the shift of environmental conditions (Hand et al., Reference Hand, Menze, Borcar, Patil, Covi, Reynolds and Toner2011; Lehmann et al., Reference Lehmann, Pruisscher, Posledovich, Carlsson, Käkelä, Tang, Nylin, Wheat, Wiklund and Gotthard2016). Even if the environment returns to favourable conditions, diapause continues until the procedure is terminated (Taylor, Reference Taylor1987; Koštál, Reference Koštál2006). In diapause preparation, a series of physiological changes occur in insects, including the specific accumulation and transformation of lipids, proteins, carbohydrates and other nutrients, to ensure the survival of diapausing individuals under adverse and energy shortage conditions. Compared with the non-diapause generation or development stage, the expression profile of genes related to development stagnation, metabolic inhibition and stress resistance increases in diapausing insects (Amsalem et al., Reference Amsalem, Galbraith, Cnaani, Teal and Grozinger2015; Popović et al., Reference Popović, Subotić, Nikolić, Radojičić, Blagojević, Grubor-Lajšić and Koštál2015; Wadsworth and Dopman, Reference Wadsworth and Dopman2015). Lipids are the main energy resource to cope with periods of food deprivation, providing nutrients, transforming other nutrients and serving as cryoprotective substances through lipid metabolism for diapausing insects (Hahn and Denlinger, Reference Hahn and Denlinger2007; Izumi et al., Reference Izumi, Sonoda and Tsumuki2007; Arrese and Soulages, Reference Arrese and Soulages2009; Vukašinović et al., Reference Vukašinović, Pond, Grubor-Lajšić, Worland, Kojić, Purać, Popović and Blagojević2018). Transcriptomics have promoted an understanding of the molecular regulation of insect diapause, and several studies have identified hundreds of diapause-related genes, including in the nutrition metabolism pathway (Poupardin et al., Reference Poupardin, Schöttner, Korbelová, Provazník, Dolezel, Pavlinic, Beneš and Kostál2015; Kang et al., Reference Kang, Cotten, Denlinger and Sim2016; Ragland and Keep, Reference Ragland and Keep2017). Metabonomic studies have shown that diapausing insects store more lipids than non-diapausing individuals, and metabolites related to lipid metabolism during diapause are significantly reduced (Li et al., Reference Li, Zhang, Chen, Koštál, Simek, Moos and Denlinger2015; Batz and Armbruster, Reference Batz and Armbruster2018). The preservation of lipids during diapause is manifested by the downregulation of genes related to lipid catabolism (Reynolds et al., Reference Reynolds, Poelchau, Rahman, Armbruster and Denlinger2012). However, the proximal mechanism leading to changes in lipid metabolism during diapause is still unclear.
Epigenetics are phenotypic inheritance mechanisms independent of genomic changes that are regulated by multiple environmental signals and play an important role in regulating diapause-related gene expression (Bonasio et al., Reference Bonasio, Tu and Reinberg2010; Rando, Reference Rando2012). The expression of heterochromatin protein 1 (HP1) in diapausing pupae of Sarcophaga bullat is downregulated by nearly 50% compared with non-diapausing pupae, while HP1 can interact with histone demethylase dKDM4A and participate in the regulation of developmental plasticity and lipid metabolism (Meister et al., Reference Meister, Schott, Bedet, Xiao, Rohner, Bodennec, Hudry, Molin, Solari, Gasser and Palladino2011; Reynolds, Reference Reynolds2017). Analysis of DNA methylation and the transcriptome of silkworm (Bombyx mori) showed that diapause eggs have higher DNA methylation modification in lipid metabolism-related genes than developing eggs after diapause termination (Li et al., Reference Li, Hu, Zhu, You, Cao, Wang, Zhang, Liu, Toufeeq, Huang and Xu2020). Moreover, miRNAs, another important endogenous regulating factor, are variously expressed in diapausing and non-diapausing insects, and their target genes affect the regulation of diapause-related processes, such as the lipid metabolism pathway (Ragland et al., Reference Ragland, Denlinger and Hahn2010; Batz et al., Reference Batz, Goff and Armbruster2017; Reynolds et al., Reference Reynolds, Peyton and Denlinger2017). These results indicate that epigenetic modification is involved in the regulation of lipid metabolism to affect insect diapause, but the role of m6A methylation regulating insect diapause coupled with lipid metabolism is still unknown. In human and other animal studies, m6A methylation has a significant effect on the regulation of lipid metabolism (Gebeyew et al., Reference Gebeyew, Yang, Mi, Cheng, Zhang, Hu, Yan, He, Tang and Tan2022; Wang et al., Reference Wang, Wang, Gu, Su, Gu and Feng2022). The expression level of demethylase FTO is negatively correlated with the m6A level, and FTO knockout affects lipid metabolism, leading to an increase in energy consumption (Boissel et al., Reference Boissel, Reish, Proulx, Kawagoe-Takaki, Sedgwick, Yeo, Meyre, Golzio, Molinari, Kadhom, Etchevers, Saudek, Farooqi, Froguel, Lindahl, O'Rahilly, Munnich and Colleaux2009; Smemo et al., Reference Smemo, Tena, Kim, Gamazon, Sakabe, Gómez-Marín, Aneas, Credidio, Sobreira, Wasserman, Lee, Puviindran, Tam, Shen, Son, Vakili, Sung, Naranjo, Acemel, Manzanares, Nagy, Cox, Hui, Gomez-Skarmeta and Nóbrega2014; Wang et al., Reference Wang, Zhu, Chen and Wang2015; Takemoto et al., Reference Takemoto, Nakano, Fukami and Nakajima2021). Decreasing the m6A abundance of peroxisome proliferator-activated receptor α (PPaRα) in mice leads to reduced cell lipid accumulation and affects the regulation of the circadian rhythm in lipid metabolism (Zhong et al., Reference Zhong, Yu, Frazier, Weng, Li, Cham, Dolan, Zhu, Hubert, Tao, Lin, Martinez-Guryn, Huang, Wang, Liu, He, Chang and Leone2018). These results indicate that m6A methylation modification of RNA is involved in lipid metabolism in animals. However, the regulatory mechanism of lipid metabolism in diapausing insects remains unclear.
To advance the study of the epigenetic regulation of the developmental pathways of Lepidopteran model organisms, this paper sought to study the egg diapause phenotype of the model silkworm (B. mori). Diapause traits of silkworm are induced by environmental signals, in which epigenetics play an important role. Studies have shown that m6A methylation is involved in the expression regulation of diapause-related genes, and m6A abundance in the early embryonic development stage of diapausing eggs is higher than that of non-diapausing eggs (Jiang et al., Reference Jiang, Li, Qian, Xue, Xu, Chen, Zhu, Shunming, Zhao, Qian and Shen2019). Reader protein YTHDF3 of the m6A modification system is involved in diapause regulation of bivoltine silkworm by altering the expression of Cyp307a1 and Cyp18a1 genes in the pathway of ecdysone synthesis (Chen et al., Reference Chen, Fan, Yasen, Zhu, Wang and Shen2022), but it cannot completely change the diapause fate of silkworm eggs, implying that m6A may regulate diapause in multiple ways. The complex and diverse utilization mechanism of nutrients is a typical characteristic of silkworm in regulating diapause in response to environmental signals, but the relationship between epigenetics and the molecular mechanism of nutrient metabolism is unknown. Based on analysis of previous m6A methylation sequencing data, we found a significant difference in the m6A methylation modification of dihydroxyacetone phosphate acyltransferase (DHAPAT) and phosphatidic acid phosphatase (PAP) genes in the 3 day pupal stage between the non-diapausing egg producer (QFLT) group and the diapausing egg producer (QFHT) group; the expression levels of two genes in the QFLT group were higher than the QFHT group. Triacylglycerol, a product of lipid metabolism, releases fatty acids into the tricarboxylic acid cycle through lipolysis to produce energy. Therefore, we verified that m6A mediates DHAPAT and PAP methylation to change the expression of DHAPAT and PAP and then participates in the diapause regulation of silkworm through the lipid metabolism pathway and tricarboxylic acid cycle. This study clarified the regulation of diapause traits of silkworm by epigenetic coupling lipid metabolism and deepened the understanding of insect metamorphosis and diapause mechanisms, and may provide new insights on how to better utilize insects as resources and for pest management.
Materials and methods
Animals
The materials used in this experiment were univoltine silkworm (B. mori) strain AK4, bivoltine strain Qiufeng (QF), multivoltine with diapause strain ‘SH’ and multivoltine of non-diapause strain Nistari. According to the principle that diapause of the bivoltine strain is regulated by environmental factors, mainly temperature and light, diapause-terminated silkworm egg batches (one batch produced by one female moth) of QF were divided into two semi-batch groups, one of which was incubated at 17°C in darkness (QFLT) to produce non-diapause eggs. To produce diapause eggs of QF, the other sample group was incubated at 25°C under a natural day/night cycle (QFHT) 15 days later for hatching on the same day with QFLT. After hatching, the larvae of both groups were raised with fresh mulberry leaves under 25°C with a relative humidity of 80 ± 5% under natural light. Ovary tissue and fat body were taken for the samples from the 1st day to 6th day of pupal stage and then stored at −80°C for later use. Five pupae were taken as one sample, and each sample was replicated in three groups.
Reagents
The pFastBac-dual vector, pGL-A3-luc-sv40 vector and DH10Bac were provided by the Key Laboratory of Sericultural Research Institute, Chinese Academy of Agri-cultural Sciences (CAAS). Primer synthesis and sequencing were performed by Sangon Biotech (Hangzhou, China). MeRIP kit (No: P-9018) is purchased from Epigentek Company (Guangzhou, China). Actinomycin D was purchased from Macklin (Shanghai, China). TC-100 was purchased from Applichem (Germany), and foetal bovine serum (FBS) was purchased from Corning (USA). Dual-Luciferase Reporter Assay System was purchased from Promega. The plasmid extraction kit, RT-PCR kit and SYBR Green PCR kit were obtained from Vazyme Company.
Cell culture
BmN cells originated from B. mori ovary were cultured in TC-100 insect culture medium supplemented with 10% FBS and 1% penicillin streptomycin. Plasmid transfection was performed with Effectene Transfection Reagent (Qiagen) according to the manufacturer's protocol. After 6 h, the medium was replaced with a culture medium containing 10% FBS for 48 h to observe fluorescence or collect cells.
Quantification of mRNA methylation with m6A-IP and RT-qPCR
The kit was used to enrich m6A modified gene according to the instructions, and then RT-qPCR method was used to quantify the methylation changes of the target gene. The mRNA extracted from the sample and the m6A capture antibody combined with beads were incubated in the buffer for 90 min, and the RNA sequences containing both ends of the m6A target region were cleaved in nuclear digestion enhancer and cleavage enzyme mix reagents. Then, the enriched RNA was released, purified and eluted through RNA binding beads, and the samples were stored at −20 °C. The enrichment of m6A in each sample was analysed by RT-qPCR.
Construction of the baculovirus expression system
Recombinant baculoviruses BmBacJS13-egfp and BmBacJS13-egfp-YTHDF3 were constructed earlier in this project (Chen et al., Reference Chen, Fan, Yasen, Zhu, Wang and Shen2022). Primers of DHAPAT and PAP were designed (table 1). The cloned DHAPAT and PAP were inserted between Not I and Xho I and Sal I and Hind III at the downstream of the vector pFastBac Dual-egfp, respectively. The constructed pFastBac Dual-egfp-DHAPAT and pFastBac Dual-egfp-PAP plasmids were transformed into DH10Bac competent cells to construct recombinant baculoviruses BmBacJS13-egfp-DHAPAT and BmBacJS13-egfp-PAP.
Table 1. Primers
dsRNA synthesis
The dsRNA primers of DHAPAT and PAP were designed according to the NCBI database and the online software SnapDragon – dsRNA design (table 1). After polymerase chain reaction with these two pairs of primers, the template of dsRNA was prepared with T7Megascript Kit (NEB) to synthesize double-stranded dsRNA and achieve gene knockdown.
In vivo injection
Two microlitres of dsRNA at a concentration of 2500 ng μl−1 were injected into 2nd day pupae of QFLT (none-diapause egg producer) with a microinjector. The injected pupae were preserved at 25℃ until eclosion. Then, females were mated with males for 6 h, and the separated females were put on an egg card to produce eggs for phenotypic observation, including egg colour, size and other traits related to diapause.
RNA immunoprecipitation (RIP)
Cells infected with BmBacJS13-egfp and BmBacJS13-egfp-YTHDF3 were washed with PBS and then collected and centrifuged. The cells were then suspended in the cell lysate with protease inhibitor and ribonuclease inhibitor and incubated with 30 min on ice. Then, 1.5 μg of EGFP antibody or control IgG was conjugated to protein A/G beads and incubated at 4℃ for 6 h, washing for three times and then overnight incubation in RIP buffer (5 mM EDTA, 150 mM KCl, 25 mM Tris (pH 7.4), 1× protease inhibitor, 0.5 mM DTT, 0.5% NP40, 100 U ml−1 ribonuclease inhibitor) at 4°C. After washing for three times, resuspension at 100 μl PBS and 30 μg of protease K, digested at 37°C for 15 min. RNA was extracted with TRIzol reagent purchased from TaKaRa.
RNA isolation and quantitative RT-PCR analysis
After RNA was extracted with TRIzol reagent, reverse transcription was carried out with reverse transcription kit according to the requirements of manufacturers, and the expression level of related genes was quantitatively analysed by Power SYBR Green Master Mix. The primers are shown in table 1.
mRNA stability assay
The cultured BmN cells were seeded into a six-well plate at density about 60%（cell inoculum density 2 × 105 ml). After culture at 27℃ for 12 h, the collected baculoviruses were infected with BmN cells for YTHDF3 overexpression. Then, the transcription was blocked with 5 μg ml−1 actinomycin D, and the total RNA was collected at different time points, and the stability of target RNA was analysed by RT-qPCR. Since actinomycin D treatment results in transcription stalling, the change in mRNA concentration at a given time (dC/dt) is proportional to the constant of mRNA decay (K) and mRNA concentration (C), leading to the following equation: dC/dt = −KC, thus the mRNA degradation rate K was estimated by: Ln(C/C0) = −Kt, to calculate the mRNA half-life (t1/2). When 50% of mRNA is decayed (C/C0 = 1/2), the equation was: Ln (1/2) = −Kt1/2.
m6A site validation
The peak obtained by sequencing was used to predict the m6A methylation site by the software SRAMP (a sequence-based N6-methyladenosine (m6A) modification site predictor). According to the difference in the transcription of m6A methylation sites by the Bst I enzyme, reverse transcription primers were designed (table 1), and RT-qPCR site verification was performed (Castellanos-Rubio et al., Reference Castellanos-Rubio, Santin, Olazagoitia-Garmendia, Romero-Garmendia, Jauregi-Miguel, Legarda and Bilbao2019).
Dual-luciferase reporter assay
Wild-type (wt) and point-mutant (A-T) primers of DHAPAT and PAP genes were designed respectively (table 1), and PCR products were connected to pMD19-T for sequencing verification. The wild-type and mutation sequence of DHAPAT and PAP genes containing m6A methylation modification site were cloned into Nco I site of pGL-A3-luc plasmid to construct plasmids pGL-A3-DHAPAT-wt/mut-luc-sv40 and pGL-A3-PAP-wt/mut-luc-sv40, respectively. BmN cells were inoculated in triplicate in 24-well plates and infected with BmBacJS13-egfp-YTHDF3 virus. Seventy-two hours later, according to the supplier's instructions, firefly luciferase (Fluc) and Renilla luciferase (Rluc) activity were measured using Dual-Luciferase Reporter Assay System.
Statistical analysis
SPSS 22.0 software was used for the analysis of the significant difference between treatments. All experiments were reproduced at least three times in separate and independent replicates. Statistical comparisons were performed by using t-tests (two tailed) as indicated in the figure legends. The data are presented as the mean ± standard deviation (SD). The * represents P ≤ 0.05 difference, ** represents P ≤ 0.01 significant difference, ***represents P ≤ 0.001 extremely significant difference.
Results
DHAPAT and PAP involves in the regulation of diapause in B. mori
To explore the effects of DHAPAT and PAP on diapause in B. mori, the DHAPAT and PAP genes in 2-day-old pupae of the QFLT group were knocked down. The synthesized dsDHAPAT and dsPAP were transfected separately into BmN cells at a rate of 500, 800, 1200 and 2000 ng per well. At 12, 24, 48 and 72 h post-transfection, the cells were collected for qPCR. dsDHAPAT and dsPAP showed interference effects, and the best efficiency was obtained at 48 h at 800 ng (fig. 1a, b).
Figure 1. DHAPAT and PAP regulate B. mori diapause. (a) Knockdown of DHAPAT by RNAi in cells. (b) Knockdown of PAP by RNAi in cells. (c) Diapause eggs laid by moths from DHAPAT knockdown pupae of none-diapause producer group. (d) None-diapause eggs in pinkish colour laid by moths from PAP knockdown pupae of none-diapause producer group. (e) Eggs of the control are in light yellow.
Moreover, dsDHAPAT and dsPAP were injected separately into 2-day-old pupae of QFLT, and dsEGFP-injected pupae served as a control. After eclosion, the female moths were mated with males to produce eggs. Compared with the dsEGFP control, which laid non-diapausing eggs, all female moths from dsDHAPAT-treated pupae laid diapause eggs, while the female moths from dsPAP-treated pupae still laid non-diapausing eggs and hatched normally, only to turn pink in egg colour 3 days after oviposition (fig. 1c–e). These results indicate that DHAPAT and PAP have a certain regulation function in the lipid metabolism pathway for diapause of bivoltine silkworm strains.
Transcriptional expression profile of DHAPAT and PAP
Total RNAs were extracted from ovaries and fat bodies of B. mori strains AK4, QF (QFHT, QFLT), SH and Nistari at the pupal stage from 1 to 6 days. RT-qPCR showed that DHAPAT expression in the ovarian tissue of different B. mori strains was similar and stable from the 1st to the 5th day of pupae. The expression level of the non-diapausing egg producer (QFLT) and Nistari was still stable on the 6th day, while the expression level of diapausing egg producers AK4 and QFHT and mixture producer (laying non-diapausing eggs mixed with diapausing eggs) of SH obviously varied (fig. 2a). In the fat body, the expression levels of SH and AK4 in pupae varied significantly, and the expression in QFLT and Nistari remained stable (fig. 2b). From 2 to 72 h in the embryonic developmental stage of eggs, DHAPAT expression showed a steady upward trend in all tested strains (fig. 2c).
Figure 2. Expression profile of DHAPAT and PAP in ovary, fat body and early embryonic stage of different voltine B. mori strains. (a), (b) and (c) DHAPAT expression level in the ovary and fat body during pupa and early embryonic stage of eggs. (d), (e) and (f) PAP expression level in the ovary and fat body during pupa and early embryonic stage of eggs. Different strains are represented by colours: AK4(blue), QFHT (orange), QFLT (grey), SH (black) and Nistari (yellow). 1, 2, 3, 4, 5 and 6 d indicate the first to sixth day of pupal stage. 2, 6, 12, 24, 36, 48 and 72 h indicate the 2nd to 72nd hour of eggs after laying.
Quantitative analysis showed that PAP expression in the ovaries of QFLT and SH remained stable, while the others revealed a downward trend (fig. 2d). The fat bodies of the above B. mori strains were relatively stable, but the expression level of the multivoltine B. mori strains was relatively low (fig. 2e). PAP expression in eggs of the early embryonic stage also showed a relatively stable trend in all strains, but it was generally lower in multivoltine strains (Nistari and SH) and higher in the QFHT and QFLT groups (fig. 2f). These results revealed that the expression levels of DHAPAT and PAP differ from the voltinism of B. mori, indicating that DHAPAT and PAP play important roles in diapause regulation and development of embryos in B. mori.
m6A methylation level of DHAPAT and PAP in the QFHT and QFLT groups
Employing m6A immunoprecipitation and RT-qPCR techniques, the m6A methylation levels of the pupal ovaries and eggs of QFHT and QFLT were analysed. m6A-modified DHAPAT increased from 1 to 6 days in the pupal stage in the ovaries of the QFLT group but decreased slowly in QFHT (fig. 3a). In the eggs, m6A-modified DHAPAT in QFLT increased slowly, but the expression level in QFHT showed a significant downward trend from 24 to 72 h (fig. 3b).
Figure 3. Expression profile of m6A modified DHAPAT and PAP in QFHT and QFLT. (a) and (b) m6A modified DHAPAT expression level in the ovary during pupa and eggs of early embryonic stage. (c) and (d) Expression level of m6A modified PAP in the ovary during pupa and eggs of early embryonic stage. Values are mean ± SD of n = 4 independent experiments. ***P < 0.001; **P < 0.01; two-tailed Student's t-test.
Similarly, m6A methylation level analysis of PAP showed that m6A-modified PAP expression presents an upward trend in the QFLT group but a downward trend in QFHT after the 3rd day of pupae (fig. 3cC). In QFHT and QFLT eggs, the expression levels were basically the same within 36 h and began to increase after 48 h at different speeds, leading to a significantly higher expression level in QFLT than in QFHT (fig. 3d).
This analysis showed that there was a significant difference in the m6A methylation levels of DHAPAT and PAP between the QFLT and QFHT groups, and the modification rate of the QFLT group was higher than that of the QFHT group. Our previous sequencing results showed that there was a higher methylation modification in QFLT, which is consistent with this result (Chen et al., Reference Chen, Jiang, Yasen, Fan, Zhu, Wang, Qian and Shen2023).
YTDHF3 recognizes m6A-modified DHAPAT and PAP
To explore the effect of m6A modification on the expression level of DHAPAT and PAP, the constructed vectors BmBacJS13-egfp-DHAPAT and BmBacJS13-egfp-PAP were expressed in BmN cells, and fluorescence was observed under a microscope 4 days later. DHAPAT and PAP were localized in the cytoplasm (fig. 4a). According to the types of reader proteins found in B. mori and related studies, we speculate that YTHDF3 located in the cytoplasm is a potential reader of these two genes. Expression vectors BmBacJS13-egfp and BmBacJS13-egfp-YTHDF3 were transfected into BmN cells and collected 2 days post-transfection for RNA immunoprecipitation with the EGFP and IgG antibodies, and the pulled RNAs were quantitatively analysed using RT-qPCR. The binding amount of DHAPAT and PAP in the overexpressed BmBacJS13-egfp-YTHDF3/EGFP antibody group was significantly higher than that in the control groups BmBacJS13-egfp/EGFP antibody and BmBacJS13-egfp-YTHDF3/IgG antibody (fig. 4b), indicating that YTHDF3 recognized and bound the m6A methylation sites of DHAPAT and PAP.
Figure 4. YTHDF3 recognizes and binds to m6A modified DHAPAT and PAP. (a) Localization of BmBacJS13-egfp, BmBacJS13-egfp-DHAPAT and BmBacJS13-egfp-PAP in BmN cells. (b) Effect of overexpressing YTHDF3 and egfp on m6A modified PAP and DHAPAT by RNA immunoprecipitation (RIP) and RT-qPCR. Values are mean ± SD of n = 4 independent experiments. *P < 0.05; **P < 0.01; two-tailed Student's t-test.
The collected viruses of overexpressing BmBacJS13-egfp-DHAPAT and BmBacJS13-egfp-PAP were injected into 2-day-old pupae to observe the phenotype of egg colour after eclosion and mating. The overexpression of DHAPAT and PAP had no significant effect on the diapause phenotype.
YTHDF3 mediates DHAPAT mRNA translation in the lipid metabolism pathway
To explore how YTHDF3 regulates DHAPAT expression, the stability of DHAPAT mRNA was tested by YTHDF3 overexpression and actinomycin D inhibition in the cells. Compared with the control group, the half-life of DHAPAT in the experimental group was significantly increased, indicating that YTHDF3 affected the stability of DHAPAT mRNA and promoted the stability of the molecule (fig. 5a). To further investigate whether m6A mediates the regulation of DHAPAT by YTHDF3, we used catRAPID software to predict the potential binding sites of YTHDF3 with DHAPAT. Then, the sequencing peak was predicted using SRAMP software, and two methylation sites were obtained. RT-qPCR showed that the m6A modification site of DHAPAT was A (293), which is the binding region of YTHDF3 (fig. 5b, c). To validate this, we designed wild-type and mutant (A-T) primers (table 1) for this site to clone the correct and mutated sequences and construct a dual-luciferase gene reporter plasmid, respectively (fig. 5d). Then, the constructed wt, mutant and blank control plasmids were transfected into BmN cells. The results showed that the luciferase activity of wt DHAPAT was significantly higher than that of the mutant and the blank control, indicating that YTHDF3 promoted the expression of DHAPAT mRNA, while the expression level of the mutant was similar to that of the blank control, indicating that YTHDF3 has no regulatory effect on the mutated DHAPAT (fig. 5e). This demonstrated that the m6A modification site of DHAPAT was correct. The quantitative results showed that the expression level of DHAPAT mRNA in the wt group was significantly higher than in the two control groups under YTHDF3 overexpression. Although DHAPAT could not be modified by m6A or recognized by YTHDF3 and its mRNA level was also at a high level in the mutant group, it was significantly lower than that in the wt group (fig. 5f). This verified that YTHDF3 recognizes and binds the m6A site of DHAPAT mRNA, increasing the stability of DHAPAT and promoting its translation.
Figure 5. YTHDF3 promotes DHAPAT mRNA translation. (a) Overexpressing YTHDF3 increases the DHAPAT mRNA half-life in BmN cells. (b) Graphical representation of m6A site of DHAPAT and PAP. (c) m6A levels of DHAPAT and PAP in BmN cells. (d) Structure of the dual-luciferase reporter assay vector. (e) and (f) Relative firefly luciferase (Fluc) activity (protein level) and DHAPAT mRNA level of DHAPAT-wt/DHAPAT-mut/blank control reporters in BmN cells with overexpressing YTHDF3. Values are mean ± SD of n = 4 independent experiments. Two-tailed Student's t-tests were used (**P < 0.01, *P < 0.05).
YTHDF3 mediates PAP mRNA translation in the lipid metabolism pathway
Similarly, to explore the regulatory effect of YTHDF3 on PAP, the stability of PAP mRNA was tested. PAP mRNA stability was significantly increased under YTHDF3 overexpression (fig. 6a). Using SRAMP software, two methylation modification sites were predicted in PAP mRNA. RT-qPCR verification showed that A (161) was one methylation modification site (fig. 5b). Then, double luciferase reporter plasmids for the m6A wt and A-T mutant were constructed according to the schematic diagram in fig. 5d. The plasmids were transfected into BmN cells for the luciferase activity assay. The luciferase activity of wt PAP was significantly higher than that of mutated PAP and the two control groups (fig. 6b). RT-qPCR showed that mRNA expression of PAP in the wt and mutated groups was significantly higher than that in the two groups (fig. 6c). It has been confirmed that YTHDF3 increases PAP stability and promotes its translation.
Figure 6. YTHDF3 promotes PAP mRNA translation. (a) Overexpressing YTHDF3 increases the PAP mRNA half-life. (b) and (c) Relative firefly luciferase activity (protein level) and PAP mRNA level of PAP-wt/PAP-mut/blank control reporters in BmN cells with overexpressing YTHDF3. Values are mean ± SD of n = 4 independent experiments. Two-tailed Student's t-tests were used (**P < 0.01, *P < 0.05).
Discussion
Diapause is a complex insect biological characteristic, and environment-induced epigenetics play an important role in regulating, accumulating and transforming nutrients during this process(Reynolds and Hand, Reference Reynolds and Hand2009; Reynolds et al., Reference Reynolds, Nachman and Denlinger2019). However, the complex and diverse mechanisms of these metabolites in diapause regulation are still being uncovered. In our study, we validated that m6A modification-related genes in the lipid metabolism pathway regulate diapause traits in the bivoltine strain of B. mori. The m6A methylation rate of DHAPAT and PAP in the ovary and fat body tissue of pupae and eggs in the early embryonic stage after oviposition was higher in the non-diapausing QFLT group than in the diapausing QFHT group. DHAPAT knockdown in the lipid metabolism pathway in QFLT resulted in non-diapause destined eggs becoming diapause destined eggs, while knockdown of PAP, a downstream gene in the lipid metabolism pathway, induced a colour change in non-diapause destined eggs from light yellow to pink 3 days after oviposition, but they hatched normal non-diapausing eggs, indicating an increase in 3-hydroxycanine expression. In addition, the reader YTHDF3 recognized the m6A methylation sites of DHAPAT and PAP, increasing stability and promoting their translation. m6A methylation mediated the change in expression levels of DHAPAT and PAP and affected the diapause traits of bivoltine B. mori through the lipid metabolism pathway and tricarboxylic acid cycle. These results demonstrated that m6A methylation of epigenetic modification plays an important role in regulating the expression level of DHAPAT and PAP in the lipid metabolism pathway in response to diapause-induced environmental signals and has a certain correlation with the control of the basic energy demand for the preparation and maintenance of diapause in bivoltine B. mori.
Epigenetics are systematic regulatory processes that have biological functions and can cooperate in all insect development stages (Stoll et al., Reference Stoll, Wang and Qiu2018). Epigenetic processes, such as histone modification, DNA methylation and non-coding RNA, have been involved in insect diapause regulation (Li et al., Reference Li, Hu, Zhang, Toufeeq, Wang, Zhao, Xu, Xu and Huang2019; George and Palli, Reference George and Palli2020; Duan et al., Reference Duan, Li, Wang and Pang2022). Research on RNA methylation modification is very limited to insect diapause regulation. It has been reported that histone deacetylation participates in the regulation of juvenile hormone and metamorphosis and in the development of insects (George et al., Reference George, Gaddelapati and Palli2019; George and Palli, Reference George and Palli2020). Interference by miR-277-3p in Aedes aegypti activates insulin signalling to enhance the nuclear output of FOXO, leading to the failure of lipid storage and ovarian development (Ling et al., Reference Ling, Kokoza, Zhang, Aksoy and Raikhel2017). Diapausing and non-diapausing Culex pipiens showed significant differences in the expression of several miRNAs related to lipid metabolism in the fat body and ovary, and the change in miRNA abundance was related to the phenotypic change in diapause (Meuti et al., Reference Meuti, Bautista-Jimenez and Reynolds2018). This shows that epigenetic modification interacts with lipid metabolism signals to affect the diapause fate in insects, but the response mechanism of m6A modification coupling lipid metabolism to the diapause-induced environment is still not completely clarified.
We also showed that the m6A methylation levels were significantly different between non-diapause destined and diapause destined bivoltine B. mori. However, the complicated regulation network of the B. mori diapause mechanism with epigenetic involvement is deficient. The function of a specific gene in the diapause phenotype depends on its expression abundance and tissue specificity, as well as its upstream and downstream regulatory pathways (Reynolds et al., Reference Reynolds, Peyton and Denlinger2017; Sahoo et al., Reference Sahoo, Dutta, Dandapat and Samanta2018). In particular, lipids are the main nutrients for diapause insects to cope with energy deprivation, and their accumulation and utilization mechanisms are complex and diverse (Vukašinović et al., Reference Vukašinović, Pond, Worland, Kojić, Purać, Popović Ž and Grubor-Lajšić2015; Batz and Armbruster, Reference Batz and Armbruster2018). Studies have shown that epigenetic modification mainly affects the splicing, transport, stability and translation efficiency of RNA mediated by a series of reader proteins (Dominissini et al., Reference Dominissini, Moshitch-Moshkovitz, Schwartz, Salmon-Divon, Ungar, Osenberg, Cesarkas, Jacob-Hirsch, Amariglio, Kupiec, Sorek and Rechavi2012; Du et al., Reference Du, Zhao, He, Zhang, Xi, Liu, Ma and Wu2016; Slobodin et al., Reference Slobodin, Han, Calderone, Vrielink, Loayza-Puch, Elkon and Agami2017). A typical feature of diapause is that the gene expression level is widely downregulated (Denlinger, Reference Denlinger2002). In this experiment, a higher m6A methylation modification rate of DHAPAT and PAP in the ovary and early embryos of the QFLT group than in the QFHT group may be conducive to recognition and translation mediated by YTHDF3, suggesting that the increased expression abundance of these two genes can promote lipid metabolism in QFLT and provide more energy for embryo development.
In summary, our experimental results showed that the environmental signals, such as temperature and photoperiod, received during the parental embryo period of bivoltine B. mori changed the m6A RNA modification level of some genes, thus affecting YTHDF3 expression (higher expression level in pupae from eggs incubated under a low temperature in the dark and low expression level in pupae from eggs incubated under a normal temperature in a natural light cycle). A higher level of YTHDF3 promotes the expression of m6A-modified DHAPAT and PAP genes (including m6A modification abundance, mRNA stability and translation) in the pupal stage to provide energy for embryo development, resulting in offspring eggs developing being non-diapausing; otherwise, the offspring are diapausing. These results indicate that m6A methylation mediates the regulation of environmental signals in the diapause of bivoltine B. mori. Taken together, the results partly explain the molecular mechanism of bivoltine B. mori diapause changes induced by environmental signals, which provides a reference for studying the relationship between lipid metabolism and diapause and a new target for controlling pests in agriculture and forestry.
Acknowledgements
This work was supported by the National Natural Science Foundation of China (Grant No. 32072791 and No. 32102609) and the Postgraduate Research & Practice Innovation Program of Jiangsu Province (No. KYCX21_3508)
Author contributions
Y.-H. C., T. J. and X.-J. S. led the experiments and designed the analytical strategy; Y.-H. C., A. Y. and B.-Y. F. performed the experiments; Y.-H. C., T. J., J. Z., M.-X. W. and X.-J.-S. analysed the data; Y.-H. C. and X.-J. S. wrote the manuscript. All authors have made a contribution to the final manuscript, and have read and approved the final manuscript.
Conflict of interest
None.
Data availability statement
All data are contained within the article.
