Modulation Of Behavior By Histone Acetylation In The Phase Transition Of Locusta Migratoria

The migratory locust, Locusta migratoria, is an intercontinental and major agricultural pests.L.m.manilensis is the most widely distributed and damaging sub-species among which.Migratory locust shows a typical phenotypic plasticity, transitioning between a reclusivesolitary phase and a swarming gregarious phase in response to different population densitypressure conditions. There are significant differences in the body color, behavior and physiologybetween them. The phenotypical changes from the solitary phase to the gregarious phase andlong-distance migration was seen as a key factor in locust outbreaks.In the entire process of locust phase transition, the most easily observed is behavioralchanges. Gregarious is very active; while Solitary exhibits a more quiet state. In order to accurateand quantitative study of the behavior of the locust, our analysis based on binary logisticregression equation, screened out the eleven behavioral characteristic parameters which isclosely related to the two phases transition, and constructed two sets of methods in quantitativedetection of behavioral phase state of the locusts, that is Open Field Test (OFT) and ForcedSwimming Test (FST). By the two behavioral detection methods, we can well distinguish thebehavioral phase state between the solitary and gregarious locusts, which helps further explainthe molecular mechanism of locusts phase transition and is of important and practicalsignificance.Histone acetylation is an important post-translational modification, which is regulated byhistone acetyltransferases (HATs) and histone deacetylases (HDACs). The dynamic level ofhistone acetylation in vivo controls the activity of chromatin and gene expression. Usuallyhyperacetylation activates the expression of related genes; while hypoacetylation inhibitscertain genes activities. This research found that there was a closely link between histoneacetylation and the behavior of locust phase transition.We used three different doses in low, middle, high (100μg,300μg,500μg) of the histonedeacetylase inhibitor Sodium Butyrate (SB, or NaBut), which were injected into solitary,gregarious and the crowding of solitary in the mid-fourth stadium locusts (0h and48h injectionrespectively). At72h after drug injection, we began to evaluate behavioral phase transition byOpen Field Test (OFT). This study found that solely elevated histone acetylation level can not directly change the behavior of either solitary or gregarious locusts. However, high doses ofsodium butyrate injection (500μg group) promoted the crowding processes of solitary locusts(P <0.05). In addition, in order to further identify the role of increased acetylation in promotinggregarization of solitary locusts, we used the Forced Swimming Test (FST) to make furtherevaluation of the behavioral phase changes. We found that the group of injecting500μg sodiumbutyrate exactly promoted the crowding processes in compared with the control group, such asTotal Distance Moved improved significantly (P <0.01), meanwhile Mean Immobile Timedecreased sharply (P <0.05).Firstly, by transcriptome sequencing (RNA-seq) analysis in migratory locust, we foundthat the acetylated transcription factor PCAF showed a significant expression difference ingregarious and solitary locusts (P <0.01); meanwhile histone deacetylase HDAC5also showeda significant difference between two phases (P <0.05). On the other hand, by the real-timefluorescence quantitative PCR experimemts (qRT-PCR), we found that CBP, PCAF, HAT1acetylated factors or enzymes significantly increased in crowding1hour (P <0.05); whiledeacetylated factors or enzymes SIRT1and HDAC5rapidly improved in crowding4hour (P <0.05). This study implys that the crowding process is going with the increase in acetylation level.When the acetylation level reaches a certain degree, deacetylation plays an important role innegative feedback regulation in response to histone acetylation, thereby maintain homeostasis ofthe acetylation level in vivo in migratory locust.Secondly, we used Western blotting experiments to detect the site of H3K9acetylationprotein level at each time point (0h,1h,4h,24h,48h and72h) of crowding process insolitary nymphs. We found that at4hour of gregarization process, histone acetylation levelssignificantly increased in compared with the control group (P <0.05), then descended into astable level in the next time points. Such a series of experiments demonstrate that the acetylationincreased in mRNA level at crowding of1h, while increased in protein level at crowding of4h,but the behavioral changes was observed at crowding of72h. The results imply that acetylatedchanges in molecular level is earlier than behavioral phase shift, which means that beforebehavioral phase transition of the migratory locusts, the acetylation level has changed.Finally, we used RNA interference technology to verificate the function of histoneacetylation in migratory locust phase transitions, which helps further prove the connectionbetween acetylation and the behavioral phase shift of locusts. We specificly silenced two keygenes in acetylated processes, that is PCAF and HDAC5genes for functional verification. Thisstudy found that silencing PCAF gene in fourth-stadium of gregarization nymphs by dsRNAinterference and simultaneously injecting high doses of sodium butyrate, the crowding process slowed down in compared with the control group (P <0.05); while dsRNA interfering withHDAC5gene accelerated the crowding trend.Through this study, we demonstrated that histone acetylation was involved in the regulationof behavioral phase changes of migratory locusts. At the same time, we proved that theacetylation changes in gene transcription and protein levels in the crowding process of solitarynymphs. Through drug injection and RNA interference, we proceeded the functional verificationof histone acetylation in migratory locusts behavioral phase transitions. From the perspective ofepigenetics for the first time, we further explained the molecular mechanisms of locusts phasetransition. This paper helps to prevent large-scale outbreaks of the locusts and provides a newstrategy to migratory drug screening, with the great guiding significance in protecting the cropsfrom the devastating disaster.