Molecular Characterization Of Rab Family Genes And Their Effects On Efficiency Of RNA Interference In Locusta Migratoria

Over the years,insect pests are being managed using a variety of synthetic chemicals.Excessive use of synthetic chemicals to control insect pest cause a tremendous harm to the environment,which subsequently exerts negative impact on human health.These adverse effects are mainly attributed to the highly toxic and residual nature of the synthetic chemicals.Therefore,during the recent decades researchers have been formulating the more advanced methods to control insect pest population.In modern biology,RNA interference(RNAi)has emerged as a technological breakthrough.It is an evolutionary conserved mechanism mainly triggered by the introduction of double stranded RNA(dsRNA)and causes gene silencing in a sequence specific manner.This advanced molecular tool can be used for the crop protection as fourth generation pesticide.In the present study,we aimed to understand the underlying mechanism(s)of RNAi targeting the Rab family genes and to elucidate their possible functions in transportation of exogenous double stranded RNA in model insect Locusta migratoria.Rab proteins constitute the largest family of small GTPases and play a pivotal role in intracellular membrane trafficking in all eukaryotes.A number of Rab genes have been identified in eukaryotes;however,a very little information about Rab family genes has been reported in insects.In the current study,for the first time we identified and characterized 27 Rab genes from Locusta migratoria,a notorious hemimetabolous insect pest.Phylogenetic analysis and comparison of domain architecture indicated that Rab family members are highly conserved among insect species.Tissue-dependent expression profiles indicated that expression of Rab genes was highest in the ovary,except for LmRab3 which was most highly expressed in hemolymph.The biological function of each Rab gene was determined using RNA interference(RNAi).Double-stranded RNA targeting each Rab gene was injected into the hemocoel of locust nymphs,the results exhibited the suppression of only two Rab genes(LmRab5 and LmRab11A)caused 100% mortality.Furthermore,RNAi of RNAi approach was carried out to study the role of the LmRab gene in the RNAi pathway of migratory locust.The results provided important theoretical and practical foundations to develop innovative molecular targets and devise new plant protection strategies.The key findings are as follows:1.Identification and sequence analysis of LmRab family genesA total of 27 putative Rab genes were identified based on transcriptomic and genomic database in L.migratoria.PCR amplification and sequencing were performed to validate all LmRab sequences.The number of amino acid residues of 27 deduced LmRab proteins ranged from 193 to 301.Domain analysis showed that almost all candidate LmRab proteins contained one Rab domain,except LmRab40 that had SOCS Box domain in addition to the Rab domain.Furthermore,to determine the phylogenetic relationship among Rab proteins from L.migratoria and orhologes from other insect species,a phylogenetic tree was constructed using full length Rab protein sequences of Tribolium castaneum(20 TcRab protein sequences),Drosophila melanogaster(20 DmRab protein sequences)and Cryptotermes secundus(27 CsRab protein sequences).Interestingly,each LmRab formed a clade with the corresponding Rab orthologs of other insect species.2.Expression patterns of LmRab family genesTissue specific expression profile of LmRab genes was analyzed.For this purpose,eleven different tissues including integument,foregut,midgut,gastric caeca,hindgut,fat body,Malpighian tubules,hemolymph,wing pad,testis and ovary were dissected to extract total RNA.RT-qPCR was performed to detect expression of different LmRabs in different tissues of migratory locust.Tissue-specific expression profiles revealed that all27 LmRab genes were expressed in all the tissues investigated.The majority of LmRab genes exhibited high expression in the ovary,except for LmRab3 which had significantly higher expression in the hemolymph,indicating that these LmRab genes may play a fundamental role in the reproductive system of migratory locust.3.Biological functions of LmRab genesThe biological functions of LmRab family genes were studied by RNAi technique.Ds RNA of each LmRab was injected into the hemocoel of two-day-old third instar nymphs of migratory locust.Developmental changes in migratory locust were observed and recorded.Our results revealed that only ds LmRab5 and ds LmRab11A were lethal for locust nymphs.Further study showed that suppression of target gene LmRab5 resulted in severe defects in gastric caeca and midgut and exhibited rapid death in the nymphs of L.migratoria,while suppression of LmRab11A impaired molting process.This indicates that both LmRab5 and LmRab11A are important in nymphal development and could be molecular candidates used for wide spectrum pest control,specifically against locust.4.Involvement of LmRab family genes in RNAi pathwayRab proteins constitute the largest family of small GTP binding proteins and are mainly involved in endocytic trafficking.To this end,we tried to determine that upon entrance of an exogenous dsRNA into the cell of migratory locust,whether Rab GTPase participate in intracellular transport of dsRNA and affect RNAi efficiency.To sort out the above mentioned query,we conducted RNAi of RNAi experiment and tried to investigate the role of LmRabs in the RNAi pathway.Two-day-old fifth-instar(N5D2)nymphs of L.migratoria were selected,and a dose of 10 μg of dsRNA specific for each LmRab gene or EGFP was injected into the hemocoel through the second abdominal segment of each nymph using a micro-injector.After 24 h each nymph was re-injected with 2 μg of ds LmLgl.At 24 h after the second injection,each nymph was dissected,and hemolymph,integuments and midguts were collected for the extraction of total RNA and synthesis of cDNA.RT-qPCR was performed to assess whether the inhibition of LmRabs affected the RNAi efficiency of LmLgl.Analysis of transcript levels after RNAi experiments revealed that except LmRab9 remaining all 26 LmRab genes were silenced efficiently in hemolymph,integument,and midgut following injection of their respective dsRNAs.Furthermore,LmLgl transcript levels were detected compared with control,LmLgl showed high expression after suppression of each of eight LmRab genes including LmRab5,LmRab7,LmRab14,LmRab30,LmRab33,LmRab39 B,LmRab40 and LmRab43,while LmLgl transcript level exhibited a significant decrease in different tissue types after silencing a set of ten LmRab genes including LmRab2,LmRab3,LmRab11A,LmRab24,LmRab27 A,LmRab28,LmRab31,LmRab32,LmRab35 and LmRab37.However,LmLgl tanscript levels remained stable and no substantial changes were observed after each of eight LmRab including LmRab1 A,LmRab4,LmRab6,LmRab8 A,LmRab10,LmRab18,LmRab21 and LmRab23 was suppressed.Our results suggested that not all Rabs were involved in the RNAi pathway of migratory locusts.Based on previous literature,we speculated that different LmRab genes may be involved in the intracellular transport of dsRNA in different tissues of migratory locust,some LmRabs may be involved in the trafficking of dsRNA from plasma membrane to early endosome while some may be implicated in recycling and exocytosis of endosome with dsRNA.To the great extent of our knowledge,our results revealed for the first time that different LmRab genes are involved in the trafficking of dsRNA in different tissues and their silencing affects the RNAi efficiency.In summary,a total of 27 Rab family genes in the model insect L.migratoria were identified and their sequences were analyzed in the present study.Tissue specific expression of all identified LmRab genes was performed by RT-qPCR.Further,biological functions of all candidate LmRab family genes were determined by RNAi experiment.In addition,RNAi of RNAi approach was carried out to determine the influence of Rab family genes on RNA efficiency of Locusta migratoria.Our findings have important theoretical significance for the development of new and efficient molecular targets,understand RNAi mechanism and application of RNAi technology for pest control.