Effect And Molecular Target Of Wilforine On Muscle Tissue Of Mythimma Separata Walker

Alkaloids are primary insecticidal compounds isolated from Tripterygium wilfordii Hook.f.,among which wilforine showed the hightest insecticidal activity against Mythimna separata,Plutella xylostella and Pieris rapae,etc.With the breakthrough in the research of biosynthesis of T.wilfordii alkaloids,industrialization production of the insecticidal alkaloids is possible.However,the insecticidal mechanism of wilforine has not been elucidated,which restricted its further development and application.Preliminary observations indicated that M.separata larvae were paralyzed by total alkaloids and wilforine,and muscle tissue was damaged.The target of wilforine maybe located in the muscle tissue of insects,as a new nerve-muscle agent.In order to explore the insecticidal mechanism of wilforine,ultrastructural changes in the muscle cells of M.separata treated with wilforine were observed by transmission electron microscopy,and the effect of wilforine on[Ca²⁺]_i in the muscle cells was tested by laser scanning confocal microscope.Then,RNA-seq and RNAi technology were used to reveal the transcriptomic alterations and the function of target gene,respectively.Finally,the possible binding sites of wilforine were predicted by homologous modeling and molecular docking.Following results were obtained:1.The poisoning symptoms and musculopathy of M.separate treated with wilforine were observated on the basis of bioassay.Following results were obtained:?1?wilforine showed high insecticidal activity against larvae and adults,with the LC₅₀0 values of 31.99 and61.03 mg/L against third-instar and fifth-instar larvae and 53.46 mg/L against adults,respectively;?2?M.separata are paralyzed by wilforine,and muscle tissues was lesion with myolemma and organelles dissociation.2.Cytoxicity assay and[Ca²⁺]_i of M.separate muscle cells treated with wilforine were tested on the basis of muscle cell culture.The results show that:?1?the IC₅₀ values of wilforine against muscle cells was 25.14 and 19.65?g/mL by CCK-8 and MTT method,respectively.?2?The[Ca²⁺]_i was rise by wilforine in 1 nmol/L¹00 nmol/L concentrations,and reduced in 1?mol/¹ mmol/L concentrations.?3?The active site of wilforine maybe located in sarcoplasmic reticulum of insects,with RyR and IP₃R as action targets.The acting site of wilforine maybe differ from caffeine in RyR,and the same as ryanodine.3.Transcriptomic alterations in M.separata in response to wilforine were determined and analyzed with RNA-seq,and the results were verified by qRT-PCR.?1?1400 differently expressed gens including 453 up and 947 down were obtained;?2?Examining the relationship between RNA-seq and qRT-PCR expression levels within-transcript comparisons revealed a close correlation between the expression changes?fold difference?measured by each method?R²=0.889?.?3?Muscle contraction related genes induced by wilforine and chlorantraniliprole were changed significantly.Relative expression levels of RyR decreased gradually in response to wilforine as time and concentration increasing,and increased in response to chlorantraniliprole.4.Full-length sequence of M.separata RyR gene?MsRyR?was cloned by RT-PCR and RACE,and relative expression levels of MsRyR in different developmental stages and tissues was tested by qRT-PCR.The open reading frame?ORF?of MsRyR was obtained,and bioinformatics analysis showed that the ORF of MsRyR gene was 15372 bp,which encoded a protein of 5124 amino acids.The MsRyR was expressed at a high level in fifth-instar larvae and adults among different developmental stages,and at a high level in head and somatic muscle among different tissues.5.The function of RyR gene in the insecticidal acivity of wilforine was verified by RNAi.Two dsRNA?dsMsRyR1 and dsMsRyR2?were synthesized,and the mRNA levels of MsRyR in the treated larvae was significantly reduced to 47%and 52%compared to that in corresponding controls.Though the weight of M.separate larvae after injection of dsMsRyR reduced 40.46%and 43.07%?48 h?,the mortality of M.separate larvae did not rise significantly.Both wilforine and chlorantraniliprole at the dosage of LC₅₀ showed weak insecticidal activity against M.separata pretreated with dsMsRyR2,with a derease motality of33.33%and 30%?48 h?.Thus,RyR may be the action target of wilforine.6.The possible binding sites of wilforine on RyR were predicted by homologous modeling and molecular docking.Wilforine and chlorantraniliprole could combine with MsRyRNTD Pocket 1,MsRyRSPRY1 and MsRIH2.Wilforine showed stronger combing ability on MsRyRNTD Pocket 1 and MsRIH2 than chlorantraniliprole.Five amino acid residues?Arg51,Gly74,Asn75,His106 and Ser108?in MsRyRNTD Pocket 1 and three amino acid residues?Arg2344,Asp2350 and Arg2406?in MsRIH2 may be the binding site of wilforine.In conclusion,the insecticidal mechanism of wilforine is suggested as follows:wilforine enters the body of insects by feeding,then combines with RyR and IP₃R in sarcoplasmic reticulum of muscle cells,inducing calcium ions in calcium store unnormally releasing,futher leading to calcium dyshomeostasis;calcium dyshomeostasis causes the damage of cellular structure,and even death or apoptosis,resulting on muscle tissues pathological change and function loss;poisoned insects reduce movement and are paralyzed.Thus,RyR is an important action target of wilforine,and IP₃R is also one of the action targets.