Studies On The Toxicity And Insecticidal Mechanism Of The Melaleuca Alternifolia Essential Oil Against Stored-product Insect Pests

Tribolium confusum Jac.du Val. and Sitophilus zeamais are the main insects that cause huge losses to stored grain. The use of chemical fumigants is restricted by their toxicity and the harm to the environment. It is of great significance to develop insecticides of high effectiveness and safety in practice. In this research, we studied Melaleuca alternifolia essential oil's insecticidal activities against stored-product insects,T. confusum and S. zeamais, defined the main active insecticidal components of M.alternifolia essential oil's, explored the biochemical and molecular mechanisms of the insecticidal activities of M. alternifolia essential oil. We further conducted a series of simulation tests in wheat storehouse to preliminarily evaluate the safety of M. alternifolia essential oil. The research provides a technical support for the research and development of botanical insecticides, green prevention and control of stored-product insects, and ensuring quality and safety of agricultural products. The main outcomes are as follow:1. Insecticidal activities of M. alternifolia essential oil against stored-product insectsStored-product insects T. confusum and S. zeamais were selected, to evaluatethe repellent, contact, and fumigation activities of M. alternifolia essential oil against insects under different concentrations and time gradients, and explore the insecticidal performance by different applications. The results showed that M. alternifolia had obviously repellant effects on T. confusum and S. zeamais. The largest dose of1.41mg/cm²2 of essential oil caused the mean repellecy rate of 77.60%and 75.68% in T.confusum and S. zeamais, with IV of repellecy level. M.alternifolia essential oil produced good contact activity against T. confusum and S. zeamais. LD₅₀ were 4.70mg/cm²2 and4.80mg/cm²2 respectively at 24 h. The fumigation activity of M. alternifolia essential oil was outstanding and worth further development. LC50 on S. zeamais adults were8.42mg/L, 7.70mg/L and 6.78mg/L respectively and were 7.45mg/L, 7.09mg/L and 6.37mg/L on T. confusum adults at 24 h, 48 h and 72 h after treatment. M.alternifolia's fumigation toxicity on T. confusum was higher than that on S. zeamais.2. Active fumigant components identified from the essential oil of M. alternifoliaVolatile constituents of M. alternifolia essential oil were quantitatively analyzed with GC-MS. 10 constituents were identified: ?-pinene, sabinene, ?-terpinene, p-cymene,limonene, 1,8-cineole, ?-terpinene, terpinolene, terpinen-4-ol and ?-terpineol, which were mainly terpenoids, and terpinen-4-ol was the highest, up to 40.09%. M.alternifoliaessential oil was of terpinen-4-ol chemotype.The relatively 5 abundant components were selected for further activity research.The results showed that they all possessed good fumigant virulence shown in the following order: terpinen-4-ol > ?-terpineol > 1,8-cineole > ?-terpinene > ?-terpinene.The fumigation activity of terpinen-4-ol was the highest, and the LC50 on S. zeamais and T. confusum were 3.22mg/L and 3.83mg/L respectively, having the potential of further development as fumigants against stored-product insects.3. Insecticidal mechanism of M. alternifolia essential oil against T. confusum and S.zeamais in vivoT. confusum and S. zeamais were treated with triangular flask fumigation to determine the activities of ACh E, GST and Car E. The results showed that the activities of the three enzymes were inhibited at 24 h after treatment with lethal dose?7.45mg/L?8.42mg/L?, then regressed with processing time increased, but were still lowered than those of the control group, and stress response of S. zeamais' s Car E activity appeared under M. alternifolia essential oil's pressure at 12 h after fumigation treatment. Stress response of T. confusum's GST and S. zeamais' s Car E appeared at 24 h after treatment with low dose?5.39mg/L?, S. zeamais' s ACh E activity was almost the same as that of the control group, showing no effect on ACh E activity, but inhibitory effect increased with dose increasing. Stress response of S. zeamais' s Car E appeared after fumigation with7.48mg/L, lower than the control group, showing inhibitory effect.4. RNA-Seq analysis of stored-product insects transcriptome in response to oil fumigationRNA-Seq analysis was carried out to explore the gene expression profiles of stored-product insects in response to essential oil treatment using Illumina sequencing technology. Approximately 8.96 Gb and 8.93 Gb of sequence data for T. confusum and S.zeamais samples were obtained, respectively. A total of 45,114,010 and 44,228,536 clean reads were obtained from sequence data of non-oil and oil-fumigated T. confusum samples, respectively, as well as 44, 697, 706 and 44, 884, 212 clean reads were obtained from zeamais samples. According to stringent quality assessment and data filtering,28,885 and 33,483 unigenes were assembled de novo using the Trinity software with default parameters, a mean length of 944 bp and 1,280 bp, respectively. After assemble, a total of 23,571?81.60%? and 20,811?62.15%? unigenes had significant matches with sequences in the seven databases, including the Non-redundant?Nr?, Nucleotide?Nt?,Cluster of Orthologous Groups?COG?, Kyoto Encyclopedia of Genes and Genomes?KEGG? pathway, Swiss-Prot, Interpro, and Gene Ontology?GO? databases. A total of2,208 and 3,562 differentially expressed unigenes were identified, respectively, including999 up-regulated and 1,209 down-regulated genes after a comparative analysis between oil-fumigated and control T. confusum samples, as well as 2,836 up-regulated and 726down-regulated for S. zeamais samples. To annotate these differentially expressed genes?DEGs?, both GO and KEGG functional analyses were performed. The DEGs with GO annotations were classified into the category of cellular components, molecular functions,and biological processes, respectively. To obtain more information to predict the function of DEGs, the DEGs were mapped in the KEGG database. The pathway functional enrichment was analyzed using hypergeometric test with FDR ? 0.01. Overall, a total of 18,074 and 15,074 Unigene were assigned to 42 different KEGG terms, respectively.According to the threshold of P values and Q values < 0.05. KEGG pathway analysis showed that 23 and 15 pathways were significantly enriched from T. confusum and S.zeamais samples. However, an abundance of DEGs were mapped into the metabolism pathway that were associated with respiration and metabolism of xenobiotics, including cytochrome P450 s, Car Es, GSTs, suggesting that abnormal respiration and metabolic disorders occurred in adult S. zeamais following fumigation with M. alternifolia essential oil.To verify the reliability of RNA-Seq data, fifteen DEGs involved in energy metabolism and detoxification were selected for further q RT-PCR analysis. The house-keeping gene, glyceraldehyde 3-phosphate dehydrogenase?GAPDH? was used as a reference gene. The gene expression?mean ± SD? quantified as a relative fold change was carried out using the 2-??CT method. The result showed that similar trends of up/down-regulation of selected DEGs between q RT-PCR and transcriptome data were observed, indicating that RNA-Seq data was reliable.5. Preliminary evaluation of the safety of M. alternifolia essential oilM.alternifolia essential oil's safety was evaluated preliminarily through a series of acute toxicity tests. The median lethal dose(LD₅₀) of M. alternifolia essential oil was1710 mg/kg b.wt. in female rats and 1470 mg/kg b.wt. in male rats, graded as low toxicity. The LD₅₀ on female and male rats were 3160 and 4300 mg/kg b.wt. respectively,belonging to low toxicity. In the acute inhalation toxicity test, LC50 of M. alternifolia essential oil?2 h? exceeded 5000 mg/m³ in both female and male rats, micro toxicity.Acute skin irritation index of M. alternifolia essential oil on rabbits was 2.5 and returned to normal level on the 7th day, which belonged to moderate irritation. Rabbits acute eyesirritation index was 7.5 before washing, the mean index was 1.0?below 5 4 days later?and returned to normal on the 7th day, graded as mild to moderate irritation. The index was 4.0 washing at 4 s, mean index was 0.0 and returned to normal in 48 h, non-irritative.The index was 4.0 washing at 30 s, mean index was 2.5?lower than 5 48 hours later? and returned to normal at the 72 h, mild irritation. Guinea pigs' skin allergy rate was 6.6%,containing a certain amount of sensitizer. Based on the results mentioned above, M.alternifolia essential oil was safe, with low acute toxicity, and genetic toxicity tests could be carried out in the next stage.6. Simulated wheat storehouse test of M. alternifolia essential oil against stored-product insectsSimulated wheat storehouse test was carried out to determine the insecticidal effect of M. alternifolia essential oil against T. confusum and S. zeamais with different concentration and time gradients. The results showed that M. alternifolia essential oil had good fumigation activities on S. zeamais and T. confusum on the upper layer but less effect on the insects on the lower layer under the condition of 28??20d darkness. The mortality of T. confusum were 68.34%?40.00% for the upper layer and the lower layer respectively, LC50 of fumigation was 373.45mg/L and 748.66mg/L. The mortality rate of S. zeamais on the upper and lower layers were 90.00% and 51.67% respectively after treatment with 598.50mg/L, and the LC50 of fumigation was 290.35mg/L and566.80mg/L respectively. The same fumigation effect could be achieved only when M.alternifolia essential oil was 199.5 times the amount of Aluminium phosphide. The simulated wheat storehouse was fumigated in air tight with 598.50mg/L dose under 28 o C darkness for 1, 7, 14 and 20 days and the fumigation activity was related positively with time. Fumigant virulence was 52.34% and 72.67% respectively for upper layer S.zeamais and T. confusum and 31.67% and 42.34% for lower layer S. zeamais and T.confusum 14 days later, with no obvious difference from the fumigant virulence 20 days later. The results indicted that the wheat had absorbability for M. alternifolia essential oil.The factor, food types and granary stack height, would be taken into account for reasonable administer in the actual application.