| Coccotorus beijingensis,belonging to the genus Coccotorus of subfamily Anthonominae of the family Curculionidae,specifically parasitizes Celtis bungeana,forming galls in the process.As a species with a unique gall-inducing characteristic,it serves as an ideal subject for studying the interaction between Coleoptera insects and their host plants,as well as the mechanisms of gall formation.Currently,there is limited research on the localization and recognition mechanisms of C.beijingensis towards C.bungeana,its gall-forming mechanisms,and the physiological and metabolic impacts of gall formation on the host plant.Therefore,this study aims to explore the gallforming mechanism of C.beijingensis and its effects on the physiology and metabolism of its host plant,C.bungeana.Using a timeline from when the beetle bores/oviposits into the buds to the formation of galls,we will employ techniques such as RNA-seq sequencing(RNA-Seq),High-performance liquid chromatography-mass spectrometry(LC-MS/MS),and Headspace-gas chromatography-mass spectrometry(HS-GC-MS)for a combined investigation.The key findings of this study are outlined below.Ⅰ.This study examined the physiological and metabolic impacts of the boring/ovipositing behavior of C.beijingensis on the buds of C.bungeana.Following the boring/ovipositing process,the content of isopentenyl adenosine(IPA)and trans-zeatin riboside(TZR)in the buds of C.bungeana increased significantly.Additionally,the expression of the cytokinin(CKs)related gene was significantly upregulated in the bored/oviposited buds.A comparison between the bored/oviposited buds and the normal buds revealed 26 differential volatile metabolites,with 9 exhibiting upregulated expression.Among the upregulated volatile metabolites,2-phenylethanol was the only one commonly found in both the bored/oviposited buds and the normal buds.The remaining 8 metabolites were exclusively present in the bored/oviposited buds,indicating their uniqueness to this condition.Ⅱ.This study further explored the physiological and metabolic impacts of the initial gall formation by C.beijingensis on C.bungeana.The contents of indole-3-acetic acid(IAA),zeatin,and TZR were highest in the larvae of C.beijingensis,followed by the initial galls,and were lowest in the normal twigs.Conversely,the content of jasmonic acid(JA)was highest in the normal twigs.It is conceivable that the IAA and CKs in the galls primarily originate from the larvae of C.beijingensis.In comparison to the normal twigs of C.bungeana,the genes associated with IAA metabolism were significantly downregulated in the initial galls formed by C.beijingensis.Similarly,the genes related to cytokinin metabolism were primarily downregulated in the initial galls.Additionally,the JA metabolism-related gene CbungACAA1s was predominantly downregulated,ultimately regulating the biosynthesis of jasmonic acid in the initial galls.The metabolic pathways most enriched in both the initial galls of C.beijingensis and the normal twigs of C.bungeana were amino acid metabolism,biosynthesis of secondary metabolites,cofactor and vitamin metabolism,and lipid metabolism.The categories of differentially expressed metabolites were primarily lipids and lipid-like molecules,followed by phenylpropanoids,polyketides,and organic heterocyclic compounds.The main pathways involved in these differential metabolites were flavonoid biosynthesis,tryptophan metabolism,amino acid biosynthesis,and carbon metabolism.The results support the nutritional hypothesis of gall formation.Ⅲ.Clarified the Expression Changes of Plant Hormone Metabolism-Related Genes during the Development of Galls Formed by C.beijingensisAs the galls formed by C.beijingensis continued to develop,the genes related to IAA metabolism were upregulated;the indole-pyruvic acid pathway was the main route for IAA synthesis in the galls.Similarly,the genes related to cytokinin(CKs)metabolism were upregulated.Conversely,the expression of genes involved in JA metabolism showed a downward trend.However,towards the final stage of gall development,all JA metabolism-related genes were upregulated,possibly linked to defense mechanisms against predators such as rodents and birds.Ⅳ.Exploring the Impact of Gall Formation by C.beijingensis on Plant Hormones and Photosynthetic Characteristics of Leaves of C.bungeanaThe concentrations of zeatin and TZR were highest in the galls,followed by the leaves located above the galls,and were lowest in the normal leaves.This pattern suggests that organic matter produced through photosynthesis is transported from tissues with low CK content(leaves)to tissues with high CK content(galls),further corroborating the nutritional hypothesis.The formation of galls by C.beijingensis resulted in a reduction of various physiological indicators related to photosynthesis in C.bungeana.Gall formation led to a decrease in chlorophyll content,carotenoid content,stomatal conductance(Gs),net photosynthetic rate(Pn),transpiration rate(Tr),water use efficiency(WUE),variable fluorescence to maximum fluorescence Ratio(Fv/Fm),and performance index(PI(ABS))in the adjacent leaves.Conversely,the Intercellular CO2 concentration(Ci)and initial fluorescence(Fo)of the leaves increased.Notably,the maximal fluorescence(Fm)and Fv/Fm values of the leaves positioned above the galls were higher compared to those below the galls.Ⅴ.Identification of Chemosensory Genes Closely Associated with the Olfactory Recognition Mechanism of C.beijingensisA total of 59 chemosensory genes were identified in the antennae,tarsi,mouthparts,and abdominal termini of adult C.beijingensis.These genes included 34 CbeiORs,18 CbeiIRs,3 CbeiOBPs,2 CbeiCSPs,and 2 CbeiSNMPs.Among them,six CbeiORs and two CbeiIRs exhibited differential expression patterns across different tissues in male and female insects.These differentially expressed genes are intimately linked to the olfactory recognition mechanism of C.beijingensis.This study delves into the gall-forming mechanism of C.beijingensis at the transcriptional,metabolic,and plant hormone levels.The findings provide a theoretical basis for studying the intricate interactions between Coleoptera insects and host plants,as well as further insights into the mechanisms underlying obligate gall formation. |
