| Autophagy is a highly conserved physiological process of degradation and recycling of cell contents during the growth and development of eukaryotes.Autophagy plays a key role in midgut remodeling during metamorphosis development and overwintering of holometabolids such as silkworm,drosophila and honeybee.Megalurothrips usitatus(Bagnall)is a hyperpaurometamorphosis insect,in which the function of autophagy is unclear.In this study,laser confocal microscopy and transmission electron microscopy were used to observe the morphology and ultrastructure of the alimentary canal of Megalurothrips usitatus at every developmental stage.Then,RT-q PCR,immunofluorescence and immunoelectron microscopy were used to determine the activation of autophagy during the growth and development of Megalurothrips usitatus.Finally,the regulation of autophagy on its growth and development was studied by injecting autophagy inducer and inhibitor.The research results are of great significance for in-depth analysis of the mechanism of autophagy regulating the development of thrips.The main findings obtained in this study are as follows:1.The alimentary canal of Megalurothrips usitatus is composed of foregut,midgut and hindgut.The foregut is divided into pharynx,oesophagus,crop and cardia.The intestinal wall is composed of the outer layer of myoepithelial cells,muscle layer,epithelial cell layer and cuticle from the outside to the inside.The midgut is divided into three parts,namely anterior,middle and posterior parts.The muscle cell layer and the epithelial cell layer are distributed from the outside to the inside,and the side near the midgut lumen is specially formed into the microvilli structures.And myelin-like structures and more autophagosome-like vesicle structures were observed in the midgut cells of 2nd instar nymphs.The hindgut is composed of ileum and rectum,and has developed muscle layer and cuticle at each developmental stage.There are four Malpighian tubules.There are no muscle layers in the Malpighian tubules,but organelles or structures such as mitochondria,rough endoplasmic reticulum,basal labyrinth and zonulae adherens still in epithelial cells.2.Four autophagy-related genes: Atg5,Atg6,Atg8 and Atg12 were cloned by RT-PCR technology.The Gene Bank accession numbers are:OK574307,OM937919,OM721594 and OM721595.RT-q PCR was used to analyze the expression level of Atg5,Atg6,Atg8 and Atg12 gene of Megalurothrips usitatus at different developmental stage.It was found that the transcription level of four genes in the 2nd instar nymphs stage was significantly higher than that in other developmental stages.The results of immunofluorescence labeling showed that the expression level of the autophagy-specific marker protein Atg8 in the alimentary canal of the 2nd instar nymphs was significantly higher than other developmental stages.Transmission electron microscopy showed that many double-membrane autophagosome-like vesicle structures could be observed in the alimentary canal of the 2nd instar nymphs of Megalurothrips usitatus.Immunoelectron microscopy showed that colloidal gold particle labeled Atg8 could locate to the vesicle structure containing organelles and macromolecular substances,indicating that the autophagy-like vesicles in the alimentary canal are autophagosomes,and the autophagy was activated in the 2nd instar nymphal developmental stage of Megalurothrips usitatus.3.Rapamycin,an autophagy inducer,and 3-methyladenine(3-MA),an autophagy inhibitor,were used to successfully induce and inhibit the autophagy in alimentary canal of Megalurothrips usitatus.Life table was constructed to study the influence of autophagy on its growth and development of Megalurothrips usitatus.The results showed that compared with the control group,the 2nd instar nymphal,pupal development duration and female adult lifespan of Rapamycin group were significantly prolonged,the generation period of female adult was significantly prolonged,and the average number of egg laid by single female was significantly increased.When 3-MA was injected to inhibit the autophagy,the average number of egg laid by single female was significantly decreased,and the intrinsic rate of increase,finiterate of increase,net reproductive rate and the gross reproductive rate were significantly decreased.It indicated that autophagy was beneficial to the growth and development of Megalurothrips usitatus. |
