| Insects present three ways of carotenoids supporting mechanisms,including ingestion from their diet(for most insects),from endosymbiont(such as whiteflies)and de novo synthesis(such as aphids and mosquitoes).Some suck-feeding insects have obtained carotenoid biosynthesis related genes from the fungus through horizontal gene transfer and maintain de novo carotenoid biosynthesis.In the pea aphid(Acyrthosiphon pisum),a duplicated pattern of carotenoid biosynthesis genes show three Carotenoid Synthase/Cyclase genes(Csc)(CscA/CscB/CscC)and four Carotenoid Desaturase genes(Cde)(CdeA/CdeB/CdeC/CdeD).However,the activity of these duplicated genes and the physiological function of carotenoid biosynthesis remain unclear.To answer this scientific question,in vitro functional identification of these genes was performed using heterologous complementation detection through Escherichia coli system;With the RT-qPCR and HPLC analysis,the expression of these genes and carotenoid content in vivo were investigated;parental silencing of a carotenoid desaturase CdeB showed that carotenoid biosynthesis effected on the red body color and population fitness in the pea aphid;finally,the relationship between the carotenoid light absorption and the resistance to starvation was analyzed through carotenoid desaturase specific inhibitor and the mixture of carotenoid biosynthesis related genes dsRNA.1.Carotenoid biosynthesis related genes present synthase and desaturase activity.In vitro,we analyzed these carotenoid biosynthesis related genes functions in the E.coli expression vector through heterologous complementary assays.Co-expression of pACCRT-E and three synthase/cyclase genes showed the synthase activity of all three synthase/cyclase genes,while the function of cyclase still needs further investigation;Co-expression of the plasmid pACCRT-EB and four desaturase genes showed that the various dehydrogenation functions in the four desaturase genes: CdeA catalyzed the enzymatic conversion of phytoene to neurosporene(third-step product),together with the production of phytofluene and ζ-carotene;CdeC catalyzed the enzymatic conversion of phytoene to ζ-carotene(second-step product),together with the production of phytofluene;CdeD catalyzed the enzymatic conversion of phytoene to lycopene(fourth-step product),together with the production of phytofluene,ζ-carotene,and neurosporene.2.Carotenoid biosynthesis related genes may be mainly expressed and functional in the aphids’ integumentIn vivo,the expressions of the pea aphid carotenoid biosynthesis related genes in different developmental stages,tissues and body color types were performed by RT-qPCR.Most of carotenoid biosynthesis related genes were highly expressed in the adult stages in comparison with the other stages.In different tissues,all carotenoid biosynthesis related genes were highly expressed in integument and embryo implied the relation with body-color formation.In different body colors,CscB was highly expressed in green morph,while CdeB was highly expressed in red morph,suggested that the potential role of these two genes in carotenoid biosynthesis in the red and green morphs.The component and content of carotenoids were also different in red and green morphs.Green morphs contained three carotenoids: α-carotene,β-carotene,and γ-carotene,while three additional carotenoids(cis-torulene,all trans-torulene,and 3,4-didehydrolycopene)were presented in the red morphs.Besides,the total content of carotenoids was higher in green morph than in red morph.Silencing the carotenoid upstream gene GGPPS by RNAi decreased the expression of carotenoid biosynthesis related genes and the carotenoid content in the pea aphid.Taken together,the results indicated that these carotenoid related genes may mainly regulate the pea aphid carotenoid biosynthesis in the integument,and affected by the carotenoid upstream gene GGPPS.3.CdeB influence the body color formation and the population fitness in the red morph of the pea aphid.Parental silencing of dsCdeB continuously from first instar nymphs to adults in red morph of the pea aphid was achieved to explore the effect of ds CdeB on the body color formation and the population fitness.The results showed that the aphid body color of the adults was shallow in the current(F0)and future generations(F1-F3).The content of red pigments(cis-torulene,all trans-torulene,and 3,4-didehydrolycopene)was also reduced.In F0 generation,the aphid developmental period was prolonged and the total offspring production was significantly decreased.In future generations,the biomass and population size were significantly decreased and the age distribution was also changed.These results indicated that carotenoid desaturase gene CdeB regulated the aphid red carotenoid biosynthesis and population fitness in the pea aphid.4.Carotenoid biosynthesis related genes respond to different light spectra and contribute to stress tolerance in the pea aphid.The expression of carotenoid biosynthesis related genes and carotenoid content were increased upon stress(e.g.starvation),imply the association of the carotenoid biosynthesis to starvation stress in pea aphid.Aphids showed a prolonged survival time under the light spectral of carotenoid absorption in comparison with that of non-light spectral of carotenoid absorption.Furthermore,the carotenoid specific inhibitor treatment and the dsRNA mixtures of carotenoid biosynthesis related genes reduced the survival time upon starvation treatment in the pea aphid.The results indicated that carotenoid biosynthesis related genes can respond to different light spectra and enhanced the tolerance to stress in the pea aphid.In conclusion,these carotenoid biosynthesis related genes regulated carotenoid biosynthesis and influenced aphid body color formation;CdeB involved in the fitness of aphid population;meanwhile,the sophisticated carotenoid biosynthesis pathway can respond different light spectra and enhanced the tolerance to stress in the pea aphid.Taken together,the results showed that during long-term evolution,these horizontally transferred genes have established to play important roles in aphid adaptation to various environments. |
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