Decreased Cuticular Hydrocarbon Biosynthesis And Abnormal Cuticular Melanin Spots Formation Are Induced By CYP4G79 Knockdown In Harmonia Axyridis

The insect cuticle is a multifunctional shield providing shape stabilization and threat protection.It has a complex composition among cuticular components,including chitin,cuticular proteins,and lipids(e.g.,hydrocarbons).Melanin spots produced by the catecholamine pathway can also be observed of cuticle of some insect species.However,the studies on the relationship between hydrocarbon and other cuticular components of insect cuticle are limited.The CYP4 G subfamily is a member of the cytochrome P450 family and has been proved to be involved in the synthesis of insect hydrocarbons.Harmonia axyridis is a kind of completely metamorphosis insect,which belong to Coleoptera Coccinellidae.It is highly adaptable to the environment and is widely distributed in most parts of China.Melanin spots on the cuticle of H.axyridis’ s adults and pupa are very regular,and will change with the changes of the environment,forming a variety of patterns of spots.It plays an important role in regulating body surface temperature and avoiding natural enemies.CYP4G79 is the only CYP4 G gene found in H.axyridis,and its function in the insect needs further study.Based on the above problems,we carried out a series of research experiments: Firstly,we analyzed the spatiotemporal expression of CYP4G79 gene.And then we detected the hydrocarbon composition changes in puae cuticle by GC-MS after the target gene was interfered with,to explore the effect of RNAi on the cuticle composition.After that,we screened the high and low concentrations of ds RNA and carried out gene interference experiments on the fourth instar larvae of H.axyridis,comparing the interference efficiency of the two concentrations of ds RNA and the resulting in phenotypic differences;Meanwhile,real-time fluorescence quantitative PCR was used to detect the expression of candidate genes in the cuticle melanin synthesis pathway in ladybird under low intensity RNAi,and to explore the correlation between cuticular hydrocarbon synthesis and cuticle melanization-related genes.On this basis,we explored other functions of CYP4G79 by combining with the role of cuticular hydrocarbons in insect water retention.We investigated the effects of humidity on the pupal phenotype and the transcription level of CYP4G79;In addition,the expression level of CYP4G79 was regulated by RNAi,and the water content and spiracles size of the puae were detected before and after RNAi treatment to verify the effect of hydrocarbon synthesis and metabolism regulated by CYP4G79 on the water-retention function.We obtained the following research results through the above experiments:Through the analysis of spatiotemporal expression of CYP4G79,we found that the expression levels of CYP4G79 in the fourth instar larvae were the highest among all of the larval stage and the pupal stage,and the expression levels of CYP4G79 in the cuticle and fat body were relatively high too.And then we detected the changes of hydrocarbon composition in the pupa cuticle under gene interference,found that the content of some hydrocarbons of H.axyridis decreased significantly.This shows that CYP4G79 does play a role in the biosynthesis of hydrocarbons of H.axyridis.In different levels of RNA interference experiments,we found that the CYP4G79 expression of the new pupa was significantly down-regulated under high-dose RNA interference,but only a small number of surviving pupa showed serious phenotypic changes;The survival rate of pupa under low-dose ds RNA treatment was significantly higher.In addition,under low-intensity RNAi,the expression levels of the cuticle melanization-related genes lac2 and ebony were significantly up-regulated,and the melanin spots on the dorsal of the H.axyridis pupa were significantly increased;The melanin level of the cuticle at the spiracles of the abdomen segments increased.These results indicate that the synthesis of epidermal hydrocarbons and melanin is related to each other in structure and transcription level.The results of SEM also indirectly confirmed this inference.Under high-intensity RNAi,the melanin spot edges of the pupal cuticle became blurred,and the symmetry of the melanin spot was obviously destroyed.The histological sections of the cuticle under ds-CYP4G79 treatment showed thinner layers,the micro structure of endocuticle layers were also in bad organization.Melanin pigmentation in the exocuticle was obviously close to the border of exocuticle and endocuticle layers under ds-CYP4G79 treatment at the spot-nonspot transverse zone.The results of immunofluorescence staining experiments showed that TH staining was observed immune-positive in the epidermal cells of both treatments,but the tissue fluorescence was detected in lower intensity in CYP4G79-RNAi treatment than that of control.In addition,the distribution of TH protein was relatively irregular in tissues of CYP4G79-RNAi,and TH proteins aggregated into clusters.These experiments prove that abnormal pigments may be physiological changes caused by changes in the microstructure of the cuticle.Under low humidity treatments,the transcription level of CYP4G79 was significantly up-regulated.Under ds-CYP4G79,the water content in the pupa decreased slightly,the spiracles at each side of the abdomen segments were enlarged.Scanning electron microscopy results showed that cuticle shrinkage and collapse were observed at the wingsand abdomen segments under CYP4G79-RNAi treatments.In addition,under higher humidity,the melanin spots on the abdomen of the pupa are larger slightly.These experiments show that the gene does play a vital role in maintaining the water balance of the H.axyridis.It also confirms that the melanin synthesis pathway responds to the stimulus of humidity and generates corresponding feedback.In summary,we can conclude that CYP4G79 is instrumental to the biosynthesis of CHCs in H.axyridis,and there is a certain relationship between cuticular hydrocarbon and melanin.The phenomenon of spot enlargement under high humidity condition or RNAi suggests that this interaction might be physiological based on their upstream regulations and exhibits certain physical functions,and it reflects the environmental adaptations of H.axyridis.Severe phenotypes suggest the importance and necessity of CHCs for insect cuticular formation.This study furthers our understanding of insect physiological adaptation to environment,and cuticle formation and functions.