Melatonin Participates In Photoperiodic Response Of Rhopalosiphum Padi By Regulating Reactive Oxygen Species

The bird cherry-oat aphid,Rhopalosiphum padi?L.??Hemiptera:Aphididae?,is one of the most important wheat pests worldwide.There are four different life cycles in R.padi.In the holocyclic life cycle,the cyclical parthenogenesis?CP?lineages would engage in sexual reproduction once per year under inductive conditions?short photoperiod and low temperature?.However,the obligate parthenogenesis?OP?lineages can not produce sexual generation even in inductive conditions.Exogenous melatonin?Mel?injection can affect the aphid-type differentiation of CP lineage under short photoperiod and low temperature,however the molecular mechanisms of melatonin regulating the photoperiod response has not yet been reported.Melatonin is a small molecular substance widely existing in the organisms.The secretion of Mel is rhythmic,which can regulate the response of organisms to photoperiod.Mel is also an antioxidant,which can clear the active oxygen of organisms,improve immunity and maintain cells'functioning.In this study,we explored effects of exogenous melatonin injection as well as light and temperature on melatonin and reactive oxygen species?ROS?of R.padi,effect of melatonin on the the expression of antioxidant enzyme genes,and variation of antioxidant enzyme gene expression pattern under inductive conditions.Our study provide knowledge of the roles of melatonin and reactive oxygen in reproduction mode transformation of R.padi.The main findings are as follows:1. Effects of exogenous Mel injection on Mel and ROS in R.padiContent of Mel and ROS in R.padi were determined on the first and third day after exogenous Mel injection.The results showed that the level of ROS in R.padi was singinficantly lower than that in the control.2. Effects of light and temperature on Mel and ROS in R.padiThe contents of Mel and ROS in R.padi were determined under long photoperiod?16L:8D?and normal temperature?24??at 1h,2h and 4h after the light-dark junction.Within 4hours after the transition from dark phase to light phase?ZT?Zeitgeber time?1,ZT2,ZT4?,the Mel content was gradually decreased in R.padi,while the content of ROS was gradually increased with the length of light rise.Within 4 hours after the transition from light phase to dark phase?i.e.ZT17,ZT18,ZT20?,the Mel content was higher at ZT17 and ZT18,but decreased significantly at ZT20.The ROS content was lower at ZT17 and ZT18,and increased at ZT20,but the difference was not significant.The lowest concentration of Mel?44.44±1.74 ng/g?in the dark period was significantly higher than that in the light period?39.04±0.33 ng/g?.The R.padi were treated with metal bath at 12?and 24?.For 1 hour post-treatment,the Mel content of R.padi under low temperature condition was significantly lower than that of the normal temperature condition,whilst ROS contents was not significantly different.For3 hours post-treatment,the Mel level of R.padi increased to the similar level of that in the normal temperature control,whilst the ROS content decreased significantly.3. Acquisition of antioxidant enzyme genes of R.padiIn this study,transcriptome sequencing and PCR verification techniques were used to clone the open reading frames sequences of 15 antioxidant enzyme genes of R.padi,including 7 peroxidase genes?Rppod-1,Rppod-2,Rppod-3,Rppod-4,Rppod-5,Rppod-6,Rppod-7?,5 superoxide dismutase genes?Rpsod-1,Rpsod-2,Rpsod-3,Rpsod-4,Rpsod-5?,2glutathione peroxidase genes?Rpgpx-1,Rpgpx-2?and 1 catalase gene?Rpcat?.Sequence analysis and phylogenetic tree showed that the respective amino acid sequences of the gens obtained had the highest similarity to the corresponding amino acid sequence of Rhopalosiphum maidis.4. Effects of exogenous Mel injection on the expression level of antioxidant enzymes genes in R.padiThree different concentrations?500?mol/L,1500?mol/L,3000?mol/L?of exogenous Mel were injected into the R.padi,and the fluorescence quantitative PCR was used to determine the expression level of antioxidant enzyme gene in R.padi on the third day post-injection.The results showed that exogenous Mel could raise the expression of Rppod-2,Rppod-4,Rppod-7,Rpsod-2 and Rpgpx-2 genes.Injection of 500?mol/L Mel significantly increased the expression of Rpsod-4 and Rppod-7 genes.Injection of 1500?mol/L Mel significantly inreased the expression of Rppod-4.Injection of 3000?mol/L Mel significantly increased the expression of Rpgpx-2.The expression of Rppod-2 gene was significantly up-regulated when injection of 1500?mol/L and 3000?mol/L Mel.5. Effects of short photoperiod and low temperature on antioxidant enzyme gene expression level in R.padiThe aphid strains reared under long photoperiod and normal temperature?LN?or under long photoperiod and low temperature?LL?were used in the experiments.CP/OP-LN?L:D=16h:8h,24±1??and CP/OP-LL?L:D=16h:8h,12±1??strains were induced at short photoperiod and low temperature,respectively.The results showed that the expression levels of 7 antioxidant genes?Rppod-2?Rppod-5?Rppod-7?Rpsod-4?Rpgpx-1?Rpgpx-2?Rpcat?in the CP strain were significantly higher than that in the OP strain The expression levels of antioxidant enzyme genes in CP strain were significantly higher than those in the OP strain in the G₁ generation when the condition being changed from changed from LN to SL?short photoperiod and low temperature?.When the condition being changed from LL to SL,The expression levels of antioxidant enzyme genes in CP strain were significantly higher than those in the OP strain in the G₀ generation.