Dfd Gene Mediated Larval Mandible Development Differing Feeding Behavior Between Oriental Armyworm And Fall Armyworm

The Oriental armyworm,Mythimna separata（Walker）,and fall armyworm,Spodoptera frugiperda（J.E.Smith）,belong to the same Lepidoptera Noctuidae insect.They are important pests endangering maize and are included in the list of class I crop diseases and pests.With the continuous use of pesticides,the drug resistance of the two pests is increasing day by day.Therefore,it is urgent to find new green control methods.This study was based on the observation that the larvae of the two species showed different feeding patterns in maize,and anatomical experiments showed that the mandible development structure of the larvae of the two species was different.Therefore,it is particularly critical to further search for genes that target the development of larvae mandible and to clarify their effects on feeding behavior.Studies have shown that the Hox gene Deformed（Dfd）plays an important role in the development of insect head appendages.In this research background,this experiment successfully identified and cloned two insect Dfd genes intact open reading frame（ORF）and the temporal and spatial expression of Dfd gene in the two species was detected by RT-qPCR.The Dfd gene was silenced by establishing RNAi technology system to further clarify the functional role of Dfd gene in the development of the mandible of the two species and its influence on the feeding behavior of larvae.The results not only provide a broader cognitive basis for studying the feeding behavior of the two pests,but also provide new ideas for developing molecular targets targeting mandible development and formulating new pest control technologies around the regulation of feeding behavior of boring pests.The main research results are as follows:1.Different feeding behavior between M.separata and S.frugiperda larvaelarvae of M.separata and S.frugiperda showed different feeding patterns in maize.The feeding patterns of the 1-6th instar larvae of S.frugiperda on maize were similar to those of the low instar larvae of M.separata on maize.The mesophyll began to feed on the surface of heart leaves or young leaves,forming a window-like damage,while the older larvae of M.separata began to feed along the edge of leaves,forming a lobed damage.Biting and boring on maize leaves were tested of the two larvae showed that the 1-6th instar larvae of S.frugiperda could feed into window-like damage from the leaf surface,while the older larvae of M.separata could only feed into lobed damage along the leaf edge.Feeding results in the case of fixing the leaf edge so that the larvae cannot reach the leaf edge show that the 6th-instar S.frugiperda larvae form a hole in the leaf surface,but 6th-instar M.separata larvae cannot feed on the leaf.2.Morphological differences between M.separata and S.frugiperda larval mandiblesBoth of M.separata and S.frugiperda larvae have chewing mouthparts,with well-developed mandibles.It plays an important role in feeding behavior.First to sixth instar S.frugiperda have the“toothed shape”mandibles with four distinct sharp incisor lobes on the distal edge.Differently,first to third instar M.separata have four sharp teeth on the mandible,but only three even less teeth on the mandible of fourth to fifth instars.In the last instar of M.separata,at the last age,the mandible has no teeth and becomes relatively flat and hard.In addition,a comparison of palate morphology and feeding patterns of 18 different insects of Lepidoptera showed that incisor lobes were necessary for the larvae to perform surface-feeding,otherwise the larvae could only perform perform edge-feeding pattern.3.Cloning and sequence analysis of MsDfd and SfDfd genesDfd genes were identified and cloned from M.separata and S.frugiperda,both with an open reading frame（ORF）of 394 aa.Protein sequence alignment showed that MsDfd and SfDfd contained the Homeobox domain and shared 98.7%similarities with each other,having similar predicted protein structures.Phylogenetic analysis showed that MsDfd and SfDfd were clustered into same branch,with close relationship to other Lepidopteran Dfds.4.Analysis of the spatiotemporal expression pattern of MsDfd and SfDfd genes by RT-qPCRRT-qPCR results showed that the expression level of MsDfd was significantly higher in the head,ventral nerve cord,brain,and mandible than in the other tissues of4^th-instar M.separata.Differently,SfDfd was highly expressed in the head but lowly in the mandible in 4^th-instar S.frugiperda.Both of MsDfd and SfDfd were highly expressed in eggs/embryos.Among 3^rd-6^th instar larval mandibles,the expression level of MsDfd was significantly higher in 3^rd-instar than 4^th-6^th instars while SfDfd had similar expression levels among 3^rd-,5^th-,and 6^th-instars.5.Study on the functions of Dfds in mandible development and larval feeding by RNAiRNAi of Dfds was performed with ds RNA-microinjection for 3^rd-instar larvae.As the results,both of MsDfd and SfDfd were knocked down in 3^rd-instar larvae after injecting ds RNA,with 43%and 75%decrease at 24 h,respectively.The results showed that 4^th-instar larvae molted from ds MsDfd-injected 3^rd-instar M.separata had shorter,thinner and flatter mandibles than controls injected with ds GFP.These injected larvae were difficult to feed on the leaf surface.Differently,ds SfDfd-injected S.frugiperda larvae showed no abnormal mandibles,which could feed leaf surface to form a hole as control larvae.Moreover,effects of ds RNA-feeding on newly first instar larvae were compared between these two species.M.separata larvae had higher mortalities after feeding ds MsDfd than control feeding ds GFP for 24,48,and 72 hours,Which mortality of larvae was 23.33%,27.78%and 28.89%respectively,while the mortality of S.frugiperda larvae was similar to that of control groups.