Response Mechanisms Of Tribolium Castaneum And Callosobruchus Maculatus Exposure To Hypoxia/hypercapnia,Ebeam And Methyl Benzoate

It has been estimated 5-10%losses in the worldwide caused by stored pests in stored products which threaten food security of human beings.Physical methods are environment-friendly to slow pest population reproduction speed by manipulating stored product habitat,and finally control storage pests.Physical methods mainly include heat,cold temperature,aridity,controlled atmospheres,microware and irradiation.Fumigants and chemical insecticides are the most effective and widely adopted methods to control storage pests,but it will take many years to study candidate fumigants before widely application.In addition,some taint fumigated product may leave phytotoxic residues.Hypoxic/hypercapnic environment,electron beam?eBeam?and fumigant/chemical insecticides are important physical stress agents which reduce pest fitness.Pests could respond to these physical stresses involved in signal transduction,transcription factor regulation,gene expression,protein translations,and morphological changes.In our study,we used Tribolium castaneum and Callosobruchus maculatus to study the gene expressions of stored pests in response to hypoxia/hypercapnia stress;investigate the relationship between hypoxia/hypercapnia and eBeam irradiation for C.maculatus;and build a novel strategy of using methyl benzoate under hypoxic/hypercapnic environment to control C.maculatus.The results are summarized below:1.Identify resistance genes of T.castaneum and C.maculatus in response to hypoxic/hypercapnic stressHypoxic/hypercapnic stress?2%O₂+18%CO₂+80%N₂?results in severe damages of T.castaneum and C.maculatus larvae.A relative short term of 12 or 24 h hypoxia/hypercapnia results in invisible detrimental effects on T.castaneum or C.maculatus larvae.RNA-seq approach is used to determine the fundamental mechanisms of T.castaneum and C.maculatus to cope with hypoxia/hypercapnia.Many hypoxic/hypercapnic resistance genes are identified by RNA-seq analysis.Our results indicate that limiting the available oxygen to T.castaneum increases glycolysis and inhibits the Krebs cycle by analysis gene expressions using RT-qPCR techniques,and that accumulated pyruvic acid is preferentially converted to lactic acid via anaerobic metabolism.Mitochondrial aerobic respiration is inhibited remarkably in T.castaneum under hypoxia/hypercapnia,which also might have led to mitochondrial autophagy.The enzymatic activity of citrate synthase decreases in insects under hypoxia but recovers within 12 h,suggesting the insects recover from the hypoxia/hypercapnia.Moreover,hypoxia-reperfusion results in severe oxidative damage to insects and antioxidant enzymes activities?including superoxidase dismutase,catalase and glutathione S-transferase?increase to defend against the high level of reactive oxygen species.2.The effects of hypoxic/hypercapnic environment on eBeam irradiation resistance in C.maculatus larvaeOur results showed that the adult emergence rate of 4^th instar larvae of cowpea bruchid increase if they are exposed to hypoxia/hypercapnia prior to eBeam treatment relative to eBeam irradiation treatment under normoxia.Therefore,pre-treatment of hypoxia/hypercapnia enhances radiotolerance of the bruchid larvae.eBeam irradiation causes an increase of ROS in normoxic larvae,but pre-hypoxia/hypercapnia exposure do not display this increase under eBeam irradiation.The activity of citrated synthase,a pace-making enzyme in the first step of the citric acid cycle,is inhibited under hypoxia/hypercapnia for insects.Reduced ROS production in mitochondria under hypoxia/hypercapnia benefits presumably insects under irradiation stress.In addition,GST and CAT activities are higher in pre-hypoxic/hypercapnic irradiated larvae than normoxic individuals,protecting larvae from eBeam irradiation induced oxidative damage.3.The effects of hypoxic/hypercapnic atmosphere on the toxicity of methyl benzoate to cowpea bruchidMethhyl benzoate?MB?effectively impacts on cowpea bruchids at all developmental stages.The lethal concentration 50%(LC₅₀)of eggs,larvae,pupae and adults are 4.04,7.71,3.11,3.93 mg/L,respectively.And lethal concentration 95%(LC₉₅)is 7.27,24.28,6.10,6.98 mg/L,respectively.Cowpea bruchid larvae are the most resilient to MB treatment,compared with eggs,pupae and adults.It is observed that MB inhibits feeding behavior of cowpea bruchid larvae.Our results show that gut proteolytic activity is significantly inhibited after 48 and 72 hours'fumigation.In vitro proteolytic analysis indicates that MB treatment inhibits total gut protease activity.In addition,the anti-insect activity displayed by MB treatment is further enhanced by hypoxic/hypercapnic environment,evidenced by drastically elevated mortality in the treatment of coupling MB with hypoxia/hypercapnia.The additive effect is also reflected by stronger suppression of the major digestive enzyme,cathepsin L transcripts in the hypoxia/hypercapnia plus MB treatment than in the individual treatment.Consistently,inhibition of total gut proteolysis is stronger in insects that subjected to both MB and hypoxia/hypercapnia treatments than separate individual treatment.The new efficient fumigant,MB coupled with hypoxic/hypercapnic atmosphere is a promising method to control cowpea bruchid in the near future.