Study On The Defense Mechanism Of Exoembryonic Tissues Of Locusta Eggs Against Pesticides And Fungi

Locusta migratoria is a serious worldwide pest and a model species for the study of metamorphosed insects.The development of locusta goes through egg stage,nymph stage and adult stage.The development of locusta eggs,which accounts for about 25%of the total development period,is characterized by a long duration,relatively static and harmless.Control of locusta is mainly based on spraying pesticides in the severely damaged nymphal or adult stages,which are prone to resistance,and the eggs that are not killed can hatch quickly and form secondary outbreaks.The control of locusta during the egg stage can effectively reduce the production of nymphs,reduce the population density,reduce the harm caused by the feeding of nymphs,and prevent the secondary hatching of eggs to reduce the secondary harm.Therefore,the study of the extra-embryonic protective structure and the mechanism of egg resistance to external stimuli will provide the basis for egg development and prevention.Locusta eggs have multiple protective structures,including maternal ones:ootheca,eggshell,and yolk membrane;Self-formed protective structures:serous cell and serous cuticle,which may be involved in the resistance of locusta eggs to environmental stimuli.In this study,HE staining,scanning electron microscopy（SEM）and transmission electron microscopy（TEM）were used to observe the morphology and ultrastructure of the extra-embryonic protective structure of locusta.Then,diafenthiuron standards and Metarhizium anisopliae suspensions were selected to treat the locusta eggs.By using RNAi,embryo microinjection,and RT-q PCR techniques,the defense mechanisms of locusta egg’s extra-embryonic protective tissue against pesticides and fungi have been explored,providing an important scientific basis for the control of locusta eggs stage.The main findings and results of this paper are as follows:1.Analysis of the characteristics of extra-embryonic protective structures in locusta eggs.Our previous study found that the serous cuticle of locusta eggs is formed between E3and E4.Therefore,E4 locusta eggs cultured at 50%RH at 30℃were selected as the research object,and optical microscopy,paraffin section,HE staining,scanning electron microscopy and transmission electron microscopy were adopted.Ultrastructure was observed in the ootheca,eggshell,yolk membrane,micropyle,caudal,serous cell and serous cuticle.The results showed the maternal protective structure consists of ootheca,eggshells and yolk membrane,as well as eggshell with micropyle and caudal.The surface of the ootheca consists of hexagonal or hexagonal-like skeleton cells with smooth surfaces and symmetric distribution.The eggshell has a smooth surface and is composed of irregular compact electron films.The yolk membrane is a thin membrane near the bottom of the eggshell;The micropyle is located at the posterior pole of the egg and consists of dense pores.The closed caudal is located opposite the micropyle and is consistent with the structure of the eggshell.Protective structures formed by locusta eggs include the serous cell and serous cuticle.The serous cell is a layer of cells that envelops the entire egg along the inside of the yolk membrane.The distance between cells is relatively long,and the nucleus is massive.The serous cuticle is secreted from the serous cell and is located above the serous cell and below the yolk membrane.The serous cuticle is composed of chitin with uneven electron density and its structure is loose and patchy.2.Defence mechanism of locusta eggs against invasion of pesticide diafenthiuronThe LC₅₀ of locusta eggs was 184μg/m L after the locusta eggs were soaked with diafenthiuron at different concentrations.But the diafenthiuron was detected in the egg by HPLC,suggesting that the diafenthiuron could have entered the egg through the egg’s extra-embryonic protective structure.HE staining,chitin staining,and transmission electron microscopy were used to investigate the mechanism by which locusta eggs become lethal.Diafenthiuron has been found to inhibit the formation of serous cuticle in locusta eggs,thus hindering embryonic development.RT-q PCR showed that at the molecular level,diafenthiuron significantly down-regulates the expression of genes involved in the formation and distribution of chitin in the serous cuticle,including Lm CHS1,Lm CDA1,Lm CDA2 and Lm Knk.Locusta eggs have the lowest survival rates when diafenthiuron is added to different parts of the egg.It was found that before the formation of the serous cuticle,the sealing of the micropyle could significantly improve the resistance of the egg to diafenthiuron,but after the formation of the serous cuticle,the sealing of the micropyle did not affect the resistance of the egg to diafenthiuron,indicating that the micropyle was the main way for diafenthiuron to invade the egg,and after the formation of the serous cuticle,the invasion of the egg by diafenthiuron through this way was significantly inhibited.The effect of locusta eggs on the invasion of diafenthiuron has been detected at various stages of serous cuticle development.It has been found that the ability of locusta eggs to resist diafenthiuron is enhanced with the formation and thickening of the serous cuticle.Furthermore,the formation of serous cuticle was inhibited by interfering with zen（Zerknullt）,a key gene for the formation of serous cuticle.Finally,the eggshell was removed by washing it with Na Cl O,and it was found to have a limited effect on the resistance of the diafenthiuron to the invading locusta eggs.Eosin staining showed that eggshells had no effect on the invasion of eosin dyes,suggesting that eggshells may not have a significant effect on the infiltration of excess diafenthiuron.In summary,diafenthiuron can enter locusta eggs and kill them.The micropyle is an important route for the diafenthiuron into the locusta eggs.The serous cuticle plays an important role in the resistance of locusta eggs to diafenthiuron invasion,while the eggshell has no significant effect on the infiltration of excess diafenthiuron.3.Defence mechanism of locusta eggs against invasion of Metarhizium anisopliaeBy soaking the locusta eggs with different concentrations of Metarhizium anisopliae suspension,the LC₅₀ of the locusta eggs lethal to Metarhizium anisopliae was 1.4 mg/m L.A large number of Metarhizium anisopliae hypha can be detected on the surface of locusta eggs by scanning electron microscopy.The content of Metarhizium anisopliae in locusta eggs was quantified using q PCR techniques.Locusta eggs were found to have the highest levels of Metarhizium anisopliae at E13,which was 11.2 times higher than in the control group.Metarhizium anisopliae infection was found to inhibit the expression of the immune-related genes Lm Dredd,Lm Casp and Lm Relish in locusta eggs.The mechanism of invasion of locusta eggs by Metarhizium anisopliae was further investigated under LC₅₀ conditions.By counting the survival rate and the amount of Metarhizium anisopliae in the eggs,it was found that locusta eggs had the lowest survival rate when the Metarhizium anisopliae was suspended up to the micropyle,and the highest levels of Metarhizium anisopliae in locusta eggs.At the same time,experiments with Na Cl O washings showed no significant effect of locusta on the infiltration of excess Metarhizium anisopliae,but a protective effect of low doses of Metarhizium anisopliae on the invasion of locusta eggs.Further using RNAi,microinjection,RT-q PCR and other technologies,it was found that the serous cuticle could resist the invasion of Metarhizium anisopliae,while the survival rate of locusta eggs without serous cuticle was significantly decreased after Metarhizium anisopliae treatment,and the content of Metarhizium anisopliae in the eggs was increased,and the immune genes were significantly down-regulated.Finally,we prepared a soil of Metarhizium anisopliae by mixing the suspension with a sandy soil.Using statistical survival rates,it was found that the soil of Metarhizium anisopliae can kill locusta eggs before the serous cuticle is formed.In summary,Metarhizium anisopliae is able to enter and kill locusta eggs,causing that survival rate of the eggs decreases.The amount of Metarhizium anisopliae increases in eggs and the expression of immune genes is significantly down-regulated.The micropyle is an important route for Metarhizium anisopliae into the locusta eggs.The eggshell and serous cuticle play an important role in resisting the invasion of locusta eggs by Metarhizium anisopliae.Metarhizium anisopliae soil is effective in killing locusta eggs and has potential farmland utilization value.