| Insect cuticular hydrocarbons(CHCs)are long chained and non-polar lipids deposited on insect epicuticle.They play critical roles in maintaining water balance and chemical communication.Insect CHC profile usually displays a high species-specificity and show intraspecific variations due to various internal and environmental factors.Considerably less is known about the sources and extents of the CHC variation in the hemipterous insects,especially in aphids which display multiple intraspecific phenotypic morphs.As a result of biosynthesis and transportation to insect’s surface,CHCs are also under a complex regulation of multiple genes.The cytochrome P450 gene CYP4G1,one of the two CYP4 G subfamily members in Drosophila,are critical for HC biosynthesis;however,functional roles of CYP4 Gs in other insects including the piercing-sucking aphids remain unclear.In the present study,we selected the pea aphid,Acyrthosiphon pisum as a model,and developed a solid-phase microextraction(SPME)method with a high efficiency for aphid CHC sampling.Based on the SPME method,we explored the effects of several factors on CHC profile to understand the sources and extents of CHC variation.Also,we presented functional characterization of the key molecular target,CYP4G51,which is involved in HC biosynthesis in A.pisum.Finally,we tentatively attempted to explore eukaryotic expression and activity detection of the CYP4 G decarbonylase.The major results of this study are described as follows: 1.SPME-based investigation of CHC intraspecific variation in A.pisum.In order to investigate the intraspecific variation of CHC using the SPME method in A.pisum,we compared the CHC profiles achieved from five different SPME fibers with various polarities,and found that 7 μm polydimethylsiloxane(PDMS)demonstrated the most efficient adsorption of CHCs among the tested fibers.SPME sampling showed good reproducibility with repeated collections of CHCs from a single aphid.Validation of SPME was performed by comparing CHC profiles with those from conventional hexane extractions.The two methods showed no qualitative differences in CHCs,although SPME appeared to extract relatively fewer short-chained CHCs.While CHC profiles of a given population differed among the aphid developmental stages,wing dimorphism types,and host plants,wingless adult aphids showed very low variance in relative proportions of individual CHC components.Reproducibility of CHC profiles was explored further to classify wingless adult morph of A.pisum from five different geographic regions which showed no variation in mitochondrial COI sequences.Our results demonstrate that CHC profile can serve as a useful biochemical character independent of conventional morphology and molecular insect identification.2.Functional study of the key gene CYP4G51 involved in HC biosynthesis in A.pisum.To uncover the roles of CYP4 G member in regulating HC biosynthesis in A.pisum,we presented the molecular characterization and a functional study of the only CYP4 G member,CYP4G51,in A.pisum genome.CYP4G51 transcript was detectable across the whole life cycle of A.pisum,and was prominently expressed in the aphid head and abdominal integument.Up-regulation of CYP4G51 under desiccation stress was more significant in the third instar nymphs compared with the adults.Also,up-regulation of CYP4G51 was observed when the aphids fed on an artificial diet compared with those fed on broad bean(Vicia faba)plant,and was positively correlated with a high level of CHCs.RNAi knockdown of CYP4G51 significantly reduced its expression and caused reductions in both internal and external HCs.A deficiency in CHCs resulted in aphids being more susceptible to desiccation,with increased mortality under desiccation stress.The current results confirm that CYP4G51 modulates HC biosynthesis to protect aphids from desiccation.Moreover,our data also indicate that saturated and straight-chain HCs(n-alkanes)play a major role in cuticular waterproofing in A.pisum and CYP4G51 could be a promising RNAi target for environmentally responsible management of aphids.3.Eukaryotic expression and activity assay of the CYP4G51 gene.Moreover,we performed a tentative attempt to explore eukaryotic expression and activity assay of CYP4G51,and strived to clarify its chemical nature of a decarbonylase.Using the Escherichia coli-based expression system,we successfully expressed and purified the prokaryotic CPR protein which serves as an obligatory redox partner of CYP4G51.High potency antibody was obtained by immunizing rabbits with the purified CPR protein.Further,we constructed the donor plasmid of CYP4G51-CPR-pFastBac and followed eukaryotic expression with the Sf9/baculovirus expression system.Western blot analysis using CPR antibody as the primary antibody revealed that the fusion protein was successfully expressed.However,this protein showed no typical 450 nm absorption of P450 from our CO-difference spectrum analysis,indicating that it may not be folding correctly and thus shows no biological activity.In summary,the establishment and optimization of SPME method from this research might provide studies on insect behavior and chemical ecology with a more suitable CHC detection method.The exploration of several factors and extents of CHC intraspecific variation will provide further guidelines for CHC-based delimitation of intraspecific subtypes,and also improve our understanding of CHC phenotypic plasticity and environmental adaptation of insect.The functional characterization of CYP4G51 in modulating HC biosynthesis might make it a potential molecular target of CHC inhibition for environmentally responsible management of aphid pests. |
