Studies On The Synthesis And Function Of Hydrocarbons In Blattella Germanica

Insect cuticular hydrocarbons(CHCs)are long-chain alkanes or alkenes that deposited on the outermost layer of insect integuments.They present remarkable importance in water conservation,drought tolerance,and chemical communication in insects.CHCs show highly intra-species diversity and plasticity in insects,and different biosynthetic steps in HC biogenesis pathway play key roles in the variation of CHCs.However,the molecular basis of CHC variation in insects has not been systematically studied.Sexual dimorphism is one of the most commonly percented intra-species variation of insect CHCs,the key molecular mechanisms behind the formation of sexually dimorphic CHCs as well as their biological significance still need to be throughly studied.CHCs of the German cockroach Blattella germanica contain only saturated alkanes,and their intra-species diversity is mainly reflected in the carbon chain lengths and the patterns of methyl branches.Therefore,B.germanica is an ideal experimental material for studying the molecular basis of CHC variations.This study employed the hemimetabolous B.germanica as a mode insect,relying on the genomic and transcriptomic data,using RNAi screening and gas chromatographic–mass spectrometry(GC–MS)systemicly studied the fatty acid synthase(BgFas)and fatty acid elongase gene families(BgElo),and identified the BgFas and BgElo genes that are involved in HC biosynthesis and variation in B.germanica,and further revealed the biological function of CHCs in insects.The main findings of this study are shown as follows:1.We identified the BgFas genes involved in hydrocarbon synthesis in B.germanicaFatty acid synthase(FAS)is a key enzyme in lipid biogenesis.In this study,seven different fatty acid synthase genes(BgFas1–7)were identified from the genomic data and the third-generation transcriptome data of B.germanica.Bioinformatics analysis showed that different BgFas proteins of B.germanica contained the typical domains of FAS.Tissue expression showed that four BgFas genes were highly expressed in the abdominal integument.RNAi screening combined with GC–MS analysis revealed that repression of BgFas1 significantly reduced the content of both internal and external HCs,especially for the methyl branched HCs.Quantitative PCR analysis showed that the expression level of BgFas1 was up-regulated by desiccation and molting.Finally,desiccation assay demonstrated that BgFas1-RNAi could significantly accelerate the evaporation of water from the cuticle,and reduce the tolerance of B.germanica under an arid environment.These results strongly suggest that BgFas1 is involved in HC biosynthesis.In addition,we also found that the fat body highly expressed BgFas2 plays an important role in the accumulation of free fatty acids and triacylglycerols in B.germanica;and BgFas3 plays important roles in waterproofing the respiratory system of B.germanica.2.BgElo12 and BgElo24 participate in hydrocarbon synthesis in Blattella germanicaFatty acid elongase(ELO)mainly regulates the biosynthesis of very long chain fatty acids(VLCFA)and their derivatives in insects,which play important roles in the normal life activities of insects.In this study,bioinformatics analysis and molecular cloning identified a total of 24 BgElo genes(BgElo1–24)from B.germanica,in which 14 BgElo genes were found to be highly expressed in the abdominal cuticle or fat body,suggesting that there may be some genes involved in HC synthesis within these 14 BgElo genes.Systematic RNAi screening showed that Bg ELO12-RNAi and Bg ELO24-RNAi significantly down-regulated internal and epidermal HCs,and both BgElo12 and BgElo24 had certain selective regulation characteristics.Furthermore,in vitro expression of BgElo12 and BgElo24 using heterologous yeast expression suggests that BgElo12 is able to convert saturated straight-chain C28 FA to saturated straight-chain C30 FA,which functions as a precursor of n-C29 hydrocarbon.However,BgElo24 has a wider range of catalytic activity,which can catalyze the yeast endogenous fatty acids to saturated straight-chain C28 and C30 FAs,the precursors for n-C27 and n-C29 alkane,respectively.Finally,desiccation assay found that only BgElo24 regulated the desiccation tolerance of B.germanica,while BgElo12 showed little effect on the drought tolerance.3.Sex-differentiation genes maintain sexually dimorphic hydrocarbons via regulation of BgElo12 in B.germanicaHydrocarbon dimorphism is commonly presented in insects,but the mechanism of HC dimorphism in insects is unknown.In this study,we found that the formation of sexually dimorphic HCs in B.germanica was synchronized with the process of sexual maturation,and mainly manifested by the gradually increased proportions of C27 CHCs and 9-;11-;13-;15-Me C29,while 3,7-;3,9-;3,11-Dime C29 was gradually decreased;In females,the CHC profiles are consistent,and always show high levels of C29 HCs(especially for the contact sex pheromone precursor 3,7-;3,9-;3,11-Dime C29)and low levels of C27 CHCs,these results suggest that the chain-length is an important factor in sexually dimorphic HC generation.Further,by differential expression analysis of BgElo12 and BgElo24 between males and females,and by comparing their effects on HC profiles of B.germanica,we confirmed that BgElo12 was a key terminal synthetic gene involved in regulating the formation of sexually dimorphic CHCs in B.germanica.In addition,we found that the female HC profiles are advantageous for keeping high levels of contact sex pheromone.Repressing female-enriched BgElo12 masculinized female HC profiles and decreased the contact sex pheromone level,which largely weaken female sexual attractiveness.Moreover,RNAi of sex-determination genes such as Bg Tra or Bg Dsx mosaicked both BgElo12 transcripts and HC profiles in females or males,and dual-luciferase reporter assay demonstrated that the male-specific Bg Dsx-M can directly inhibit BgElo12 transcription by interacting with the unstream regulatory sequence.Our current study confirmed that BgElo12 is the terminal gene in regulating the formation of sexually dimorphic HCs in B.germanica,and also proved evidences that the asymmetric expression of BgElo12 between males and females is regulated by sex differentiation genes.Thus our results revealed a novel mechanism of the generation of sexually dimorphic HCs in B.germanica.In summary,the present study systematically studied the BgFas1,BgElo12 and BgElo24 genes that involved in the biosynthesis of HCs in B.germanica,and clarified their roles in the control of the methyl branch patterns and carbon chain-lengths,which partially explained the molecular basis of HC variations in B.germanica.And we also found that BgElo12 was a key gene for maintaining sexual dimorphic HCs and high levels of contact sex pheromone in B.germanica,and its differential expression between males and females was regulated by sex-determining genes.This study also revealed the roles of BgFas1 and BgElo24 in water retention and desiccation tolerance of B.germanica,which provided new targets for pest management via inhibiting lipid biosynthesis;Moreover,the studies on the regulation of sexual dimorphic HCs and contact sex pheromone generation in B.germanica is expected to provide a new avenue for green pest control technology based on the regulation of sex pheromone biosynthesis.