Functional Analyses Of Key Genes Involved In Cuticle Lipid Barrier Formation In Locusta Migratoria

Lipids in extracellular matrices contribute to the barrier function and stability of epithelial tissues.In insects,skin waterproofness depends on the outermost layer of the extracellular cuticle termed envelope that contains the protein-lipid complex termed cuticulin.To contribute to our understanding of the molecular processes involved in the formation of a lipid-based barrier in insects,three key genes in the migratory locust Locusta migratoria were chosen for detailed characterization:1.Bioinformatic and functional analyses of the key enzyme of the lipid biosynthesis pathway Acetyl-Co A carboxylase(ACC)The L.migratoria genome and transcriptome sequence databases were queried by a BLAST search to identify the corresponding ACC gene in the locust using human and Drosophila sequences.A single L.migratoria ACC gene was identified.As in orthologous proteins,the locust ACC is composed of three domains,including the domains required for the activation of a carboxyl-donor group and for the transfer of this carboxyl group to form malonyl-Co A from acetyl-Co A.RT-q PCR analysis showed that Lm ACC is highly expressed during three days after molting,and decreases thereafter.Tissue-expression pattern showed that Lm ACC is highly expressed in the fat body and the integument.Detection by immunochemistry indicated that Lm ACC is located in the fat body and oenocytes that are tightly associated with the integument.Upon suppression of Lm ACC expression by RNAi,we found that some nymphs died during molting,and some of them died gradually after molting to the next stage.Staining with green-labeled Bodipy showed that the lipid droplets are distributed in the fat body in control nymphs.In ds Lm ACC nymphs,there was less green signal in these tissues.At the ultrastructural level,by transmission electron microscopy,we found that the lumen of the pore canals that are needed for lipid transport from the cell to the cuticle surface,did not harbor less material in ds Lm ACC-injected nymphs than in control nymphs.Quantification of lipids,however,is not possible by this method.By GC-MS analysis,by contrast,we showed that the amounts of cuticular hydrocarbons were decreased by nearly 38.8 %.In conclusion,these results suggest that de novo synthesis of lipids has a minor impact on cuticular lipids,in turn suggesting that nutritional lipids play an important role in this respect.2.Identification and functional analysis of of cytochrome P450 CYP4 G subfamily genes in locustsWe have investigated the function of the cytochrome P450 genes of the CYP4 Gs subfamily,which groups insect-specific genes.We identified two genes in the locust transcriptome database.These genes were named CYP4G62 and CYP4G102.They had different developmental expression patterns.Lm CYP4G62 peaked in the middle of the fifth instar nymphal stage,while Lm CYP4G102 expression was unchanged throughout this stage.Tissue-distribution of these genes was identical,as both are expressed in the integument and the fat body.Fluorescence in situ hybridization showed that both of Lm CYP4G62 and Lm CYP4G102 are localized in oenocytes.Based on the specific-tissue expression pattern,we argued that the respective enzymes may be involved in lipid synthesis.Using RNAi,we identified that Lm CYP4G102 was critical for locust survival.Suppression of Lm CYP4G102 expression in nymphs caused lethality soon after molting.These animals lost weight rapidly,probably by massive water loss.In addition,the cuticle of Lm CYP4G102-knockdown locusts was fragile and easier deformable than the cuticle of control animals.High humidity rescued the lethal phonotype supporting the interpretation that Lm CYP4G102 is implicated in preventing desiccation.Compared to control nymphs,the pore canal shape of ds Lm CYP4G102 nymphs appeared to be normal.This indicates that reduced Lm CYP4G102 function does not have a major impact on lipid transport within the cuticle.RNAi against Lm CYP4G102,however,provoked a decrease in the content of cuticular alkanes at the cuticle surface,which as an important fraction of cuticular hydrocarbons,while reductuion of Lm CYP4G62 did not affect the content of CHC.Taken together,LmCYP4G102 is an essential enzyme required for the construction of lipid-based waterproof barrier and cuticle elasticity.3.Characterization and functional analysis of ATP-binding cassette(ABC)transporters of the H subfamily.ABC transporters constitute a large superfamily of proteins that mediate transport of a diverse number of substrates including nutrients,lipids and xenobiotics across membranes serving a variety of developmental and physiological functions.Here,we identified three genes in total coding for ABCH transporters in locusts,and named them Lm ABCH-9A,Lm ABCH-9B and Lm ABCH-9C.They are half transporters including a nucleotide binding domain(NBD)and a transmembrane domain(TMD).The highly conserved NBD contains three characteristic motifs,including a Walker A box,a Walker B box,and an ABC signature C,which links the two Walker boxes.All three Lm ABCHs were expressed continuously during nymphal development in all tissues including the integument.Expression of Lm ABCH-9B and Lm ABCH-9C were highest just after molting,while Lm ABCH-9A expression is unchanged during all developmental stages tested.Upon suppression of Lm ABCH-9A and Lm ABCH-9B,respective nymphs were normal during or after molting,whereas reduction of Lm ABCH-9C transcript levels by RNAi in nymphs provoked death during or soon after molting to the next stage.These nymphs lost weight within minutes after molting.Moreover,high humidity rescued the lethality of molted ds Lm ABCH-9C-injected nymphs.Bodipy staining showed that the amounts of inner-cuticular lipids were reduced in nymphs with suppressed Lm ABCH-9C expression.These data together indicate that Lm ABCH-9C is needed for lipid-dependent desiccation resistance.In addition,we find that cuticle inward impermeability is compromised in nymphs with reduced Lm ABCH-9C function.Both conclusions are consistent with the model that cuticular lipids are necessary to both prevent desiccation and penetration of xenobiotics in insects.Compared to control nymphs,there is a significant less of the content in the pore canal lumen.We assume that Lm ABCH-9C is needed to load lipids into the lumen of pore canals.These lipids are distributed within the cuticle and at its surface to construct a lipid-based inward and outward barrier.GC-MS analyses of the surface CHCs showed that the amounts of cuticular hydrocarbons were decreased by nearly 63.3 %.In this thesis,I identified three key genes that are important for lipid synthesis and transport to the cuticle,including Lm ACC,Lm CYP4G102 and Lm ABCH-9C.The characterization of their function contributes to our understanding of locust cuticle biology.Moreover,as they are essential,they may serve as excellent targets for RNAi-based insect pest control.