| Frankliniella occidentalis,western flower thrips is an important pest in agricultural and horticulture.It was a typically pest of rasping-sucking mouthpart.It often causes serious damage and results in significant economic losses.Tetranychus urticae,two-spotted spider mites,is similar with F.occidentalis,and also is regarded as an important pest on Phaseolus vulgaris.Both of two pest often occure and damage host plants at the same time,and resul in great economic loss.This paper explored the systemic resistance of kidney bean plants fed by T.urticae and F.occidentalis respectively.At the same time,the systemic resistance of the kidney bean was induced by the feeding of T,urticae and F.occidentalis,and the transcriptome RNA sequencing（RNA-Seq）was carried out to analyze the differences of the first generation and the seventh generation of F.occidentalis adapted to the induced resistance,to reveal the molecular mechanism of F.occidentalis adaptation to the induced resistance of kidney beans.In order to further study the interactions between F.occidentalis and kidney bean.The result would lay a foundation for the competition between F.occidentalis and T.urticae.The main results were as follows:1、Effects of defense enzymes in kidney bean damaged by F.occidentals and T.urticaeCentral leaves of each plant were either fed by F.occidentalis,T.urticae or mechanical damage for 6 h,24 h,48 h,72 h,96 h,respectively.And the activity changes of antioxidant enzymes and defense enzymes in the upper,central and lower leaves were determined.1.1、Changes of antioxidant enzyme activities of POD,CAT,and SODThe activities of peroxidase（POD）、catalase（CAT）and superoxide dismutase（SOD）in damaged central leaves changed obviously after F.occidentalis or T.urticae infestation and mechanical damage.Compared with controls,POD activity in damaged central leaves increased firstly and then decreased to the level of control after different treatments.The activites of POD responded rapidly in the central leaves after infestation of T.urticae,and reached the maximum at 24 h.While the response of POD activities in the central leaves were later and reached the point of peak until 48 h after mechanical damage and F.occidentalis infestation.The response of CAT activities in the central leaves were various after mechanical damage and both the two insect attack.For CAT activity,mechanical damage obviously induced higher activites after 24 hours,however,both two insect infestation caused rose significantly at6 h in the central leaves,and then changed with time extention in different modes.In the central leaves,the SOD activity increased significantly only 96 h after mechanical damage,while T.urticae infestation induced obviously higher activites at 24 h.However,the SOD activity was significantly inhibited at certain time in the leaves challenged by F.occidentalis.The activities of POD in undamaged upper leaves were inhibited or activated in different treatment groups,but the POD activities in lower leaves were induced to different levels.In both upper and lower undamaged leaves,the CAT activities were activated obviously at certain points of time.Mechanical damage and T.urticae infestation resulted in prominent increase of SOD activity in undamaged upper and lower leaves,but F.occidentalis led to unstable changes,the SOD activities were activated or inhibited at different time.1.2、Changes of defense enzyme activities of PPO and PALThe results showed that the activities of polyphenol oxidase（PPO）in central leave rose significantly at 6 h after mechanical damage,and T.urticae or F.occidentalis infestation,however,the changing trends vary with different induction.But the phenylalnine ammonialyase（PAL）activity was activated significantly in central leave until 48 h after mechanical damage.While the activities of PAL in central leave fed by T.urticae and F.occidentalis increased obviously at 6 h and24 h,and reach the maximum,which were 1.8 and 2.8 times of control,respectively.The activities of PPO rose significantly at 6 h at the treatments of all 2 insect pests feeding,while under mechanical damage,it didn’t increase significantly until 48 h in upper leaves.Obviously,the activated time of PAL by mechanical damage was later than pests feeding,whether in the upper or in lower leaves.After T.urticae feeding,PAL activity was activated faster than that of others treatments,andthe activity reached the maximum in the upper and lower leave at 6 h,which were 2.8,and 5.1 times of the control,respectively.The above results indicated that herbivores feeding could induce the systemic defenses of kidney bean,and the effects of temporal and spatial defenses were related to the types of pests.2 Transcriptome analysis of F.occidentalisThe RNA-seq was performed on the F.occidentalis populations of bean pods,healthy bean plants and induced bean by T.urticae infection.A total of 102,696 Unigenes were obtained,which56.55%were genetically annotated（580,77）.42,155,7,495 and 42,740 genes were annotated in KOG,GO and KEGG databases,respectively.There were 10,139 genes in the signal transduction mechanism,and the genes for carbohydrate transport and metabolism were 3,069,and secondary metabolites biosynthesis,transport and catabolism were 1,049.There are 3,402 unigen genes belong to the defense mechanism category.Most of the genes were enriched in 26 and 20 terms of the biological processes and cell components,which were 15,108 and 13,399,respectively.Metabolic pathway analysis revealed that a large number of genes were discovered in the class of infectious diseases,metabolic and immune system pathways,which involved in the growth and development process of the adaptation process of thrips.2.1、Analysis of differential gene expression in F.occidentalis after feeding on different treated plants of kidney beanThe first generation and the seventh generation of F.occidentalis were reared on the plant of healthy beans or kidney bean infected by T.urticae respectively,and the transcriptome differences and expressed genes（DEGs）in different generations at the same age were analyzed.The results showed that 485 up-regulated genes were obtained in the 2nd instar larvae of the first generation of F.occidentalis.And the up-regulated genes and down-regulated genes in the females of F.occidentalis were 78 and 440,respectively.After seventh generation,only 531 differential genes were detected in groups of 2nd instar larvae and female adults of thrips,in 2nd instar larvae of F.occidentalis,145 and 110 genes belong to up-regulated and down-regulated,and 69 and 207 genes belong to up-regulated and down-regulated genes in female adults,respectively.These DEGs were mainly enriched in cellular components and biological processes.After different treatments,the DEGs were enriched in the GO database.The number of up-regulated genes in each term of the 2nd instar larvae were higher than that in down-regulated genes,and some genes of the terms only upregulated genes were annotated.However,the annotation of the female adults performed opposite results,and most of the DEGs were down-regulated in differential expression.Morever,most of the DEGs were involved in metabolic processes,cellular processes,cell and cell parts,catalytic activity,binding and other physiological processes of F.occidentalis.2.2、Transcriptome analysis of F.occidentalis after feeding on the same treated group of kidney beansF.occidentalis feeing on the same group hosts of healthy bean or T.urticae induced bean for the first and seventh generation,were analyzed by RNA-seq.The results showed that the up-regulated and down-regulated genes of the 2nd instar larvae were 1101 and 925,while the females were only 46 and 371,respectively,after feeding on healthy bean plants.After feeding the bean plants damaged by T.urticae,the number of differential genes in the 2nd instar larvae of F.occidentalis increased significantly.There were 8534 and 1698 genes belong to the up-regulated genes and down-regulated genes in the 2nd instar larvae,while only detected 319 and 1377differential genes in female adults,respectively.After feeding healthy beans or kidney bean induced by T.urticae for different generations,the populations of F.occidentalis were obtained.Comparing the different genes to the GO database in the different generations of F.occidentalis.The results of the annotation indicated that most of the DEGs of the 2nd instar larvae and the female adults were involved in physiological processes such as metabolic processes,cellular processes,cellular and cellular components,catalytic activity,and binding of F.occidentalis.2.3、Analysis of differential gene pathway in F.occidentalisThe DEGs what F.occidentalis feed on the same treated beans and breed different generations or fed with differently treated beans and propagated the same generation were detected,which were compared with the KEGG database for Pathyway enrichment and analysis.The results showed that most of the DEGs were enriched the modules of the Metabolism and the Organismal Systems of insects,in which the number of genes were mainly involved the Carbohydrate metabolism,Fat metabolism,Immune system,and Digestion.The number of DEGs in different generations of the F.occidentalis feeding the T.urticae treated bans were significantly higher than that in same generation of the F.occidentalis after feeding heathy bans.These results indicated that the responses of F.occidentalis were activated in the process of adaptation to induced bean plant by T.urticae infection whicn included the disease infestation,metabolism,immune system and digestive system.3、Screening of differentially genes of F.occidentalisAccording to the results of annotation in the GO and KEGG database,the digestive enzymes,detoxifying enzymes and immune system were screened in the process of adapting to the healthy beans and that is attacked by T.urticae.The digestive enzymes,included lipase,serine protease,carboxypeptidase,trypsin and aminopeptidase,which were obtained 240,99,88,85 and 78 genes respectively.The detoxification enzymes included cytochrome P450s oxidases,esterases（carboxylesterases）,superoxide dismutases,and glutathione S-transferases,the numbers of DEGs were 291,131（27）,9 and 5,respectively.In the immune system of F.occidentalis,99,28,22,15and 7 DEGs belonged to serine protease,serine protease inhibitor,peptidoglycan recognition protein,scavenger receptor and C-type lectin,respectively.The above results indicated that reactions of F.occidentalis were actvivted during the process of adaptation to induced bean by T.urticae,and the related genes including the disease infestation,metabolism,immune system and digestive system were activated.Among them,lipase,P450s and serine proteases genes were the main responsive genes in this process. |
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