| Henosepilachna vigintioctopunctata damages solanaceae and cucurbit crops,especially the potato.The ladybird has been listed as one of the ten most serious potato pests and diseases by the Chinese National Agricultural Technology Center.The beetle is a typical holometamorphic pest,and the transition between larval-pupal-adult is regulated by 20-hydroxyecdysone(20E).Ecdysone receptor(EcR)is a key factor necessary for 20E to activate downstream genes cascade signaling.In insects with more than 5 larval(nymph)instars,E93 has a triple role in insects:determining the number of larval molts,terminating larval morphology and determining adult morphology.However,its function varies among insect species.In Drosophila melanogaster,the larvae only have three instars,E93 only exerts the last function,acting as a specifier of adult shape.All E93 knockout strains can pupate normally but form deformed adults.The larvae of H.vigintioctopunctata only have 4 instars.Studying the important function of E93 in the growth and development of H.vigintioctopunctata is expected to provide strong evidence for the functional evolution of insect E93.FTZ-F1 is the most downstream transcription factor in the 20E signal transduction pathway,its iso form-specific functions have not been explored in non-Drosophilid insects.For application of RNAi to control pests,ingestion dsRNA is considered the most practical option.Spraying dsRNA on plant leaves is expected to be applied as a species-specific and environmentally friendly control method of H.vigintioctopunctata.In the present paper,we intended to clarify the roles of three critical players in 20E signaling network,EcR,E93 and Ftz-fl.Moreover,we planed to determine the RNAi efficacy of spraying dsHvEcR on potato leaves.Our results will be of great theoretical significance and potential practical application value.The main results are as follows.1.RNA interference targeting ecdysone receptor blocks the larval-pupal transitionBy mining the transcriptomic data,two variants of HvEcR named as HvEcR-A and HvEcR-B1 respectively were identified.The lengths of the ORFs of HvEcR-A and HvEcR-B1 were 1491 and 1713 bp,encoding 496 and 570 amino acids respectively.Seqence alignment revealed that HvEcR had typical conserved nuclear receptor domains,including DN A binding domain(C domain)and ligand binding domain(E domain).HvEcR was widely expressed in all development stages and in different tissues of the fourth-instar larvae.The highest expression level was found at the larval stage,followed by that at the pupal period.The spatiotemporal expression data of HvEcR are compatible with the common idea that EcR functions in 20E signaling.In a laboratory bioassay,dsHvEcR targeting the common sequence of both variants was designed.The dsHvEcR solution expressed by Escherichia coli was used to immerse the potato leaves,and the dsHvEcR-treated foliage was used to feed the test larvae.After the fourth instar larvae fed the dsHvEcR-treated foliage for three days,the target transcript was successfully knocked down and disrupted by 57%.85%of the tested larvae died in the prepupal stage or formed abnormal pupae;these abnormal pupae failed to emerge.The remaining 15%of the larvae became pupae,but all died within one week after emergence.Ingestion of dsHvEcR impregnated leaves by the third instar larvae produced a similar RNAi response and resulted in more serious defects.Depletion of HvEcR completely stopped larval metamorphosis.The resultant larvae arrested in the prepupal stage for a long time and eventually died.Quantitative detection showed that RNA interfering of HvEcR significantly reduced the expression levels of 20E signaling pathway genes HvE93,HvE74,HvE75 and HvHR3,blocked 20E signal transduction.Injection of in vitro synthesized dsHvEcR produced similar RNA interference results as feeding.The injection successfully knocked down the target genes,with an interference efficiency of 75%for the third instar and 80%for the fourth instar,respectively,and all the resultant larvae failed to pupate.In the greenhouse experiment,the E.coli bacteria solution expressing dsHvEcR was sprayed on the leaves of potato plants and the third-and fourth-instar larvae were transferred to the leaves.High RNAi efficacy was obtained and more severe RNAi phenotypes were observed in treated larvae.100%of the RNAi larvae arrested developing at the prepupa stage.They stopped molting and moving,gradually withered,dried and darkened,and finally died.Moreover,spraying dsHvEcR significantly reduced larval feeding and reduced the damage degree of potato leaves.Therefore,ingestion of dsHvEcR severely impaired larval pupation,resulting in high larval mortality.Spraying dsHvEcR on plant leaves can be applied as a species-specific and environmentally friendly control method for H.vigintioctopunctata larvae.2.Involvement of HvE93 in the regulation of pupation and pupal-adult transformationTwo isoforms of HvEcR were identified and were designated as HvE93Xl and HvE93X2 respectively.The lengths of the ORFs of HvE93Xl and HvE93X2 were 2976 and 2988 bp,coding for 991 and 995 amino acids respectively.Seqence alignment displayed that HvE93 possessed two typical psq-type HTH domains.HvE93Xl and HvE93X2 were abundantly expressed at the prepupal and pupal stages.A precocious inhibition of JH signal by RNAi of HvKr-hl or HvHairy at the penultimate instar larval stage increased the expression of HvE93,but did not drive premature entry into metamorphosis.RNAi of HvE93 at the penultimate instar larval stage failed to prevent larvae to undergo normal development and to initiate a timely metamorphic transition by the end of the fourth larval stage.It appears that larval identify is default in H.vigintioctopunctata.Moreover,depletion of HvE93 at the third-instar larval,the fourth-instar larval and the prepupal stages caused a larval-pupal mixed phenotype:pupal spines and larval scoli were simultaneously present on the cuticle of the resultant pupae.Furthermore,RNAi of HvE93 at the third-instar larval,the fourth-instar larval,the prepupal and the pupal stages severely impaired pupal development.Only a few adults emerged,with separated elytra,abnormally-folded hindwings,and small adult appendages such as antennae and legs.These indicate that high levels of E93 during the pupal stage are required for the elimination of the larval organs and for the differentiation of the adult structures in H.vigintioctopunctata.Taken together,HvE93 has dual functions in H.vigintioctopunctata:terminating larval morphology and determining adult structures.Different from the triple functions of E93 in Tribolium castaneum and Blattella germanica,and the single role of E93 in D.melanogaster,the dual functions of E93 in H.vigintioctopunctata belong to the intermediate type.Therefore,our results provide a missing link in the evolutionary process of the roles of E93.3.Dissecting the isoform-specific roles of FTZ-F1 in the larval-larval and larval-pupal ecdyses in H.vigintioctopunctataIn order to confirm the hypothesis proposed by Brunet:the ecdysozoan FTZ-F1 gene(with the exception of nematode)encodes two transcripts that generate isoforms with unique N-terminal parts,the gene structures of FTZ-F1 sequences were analyzed from representative Diptera,Coleoptera,Lepidoptera and Hymenoptera insect species.We confirmed that the FTZ-F1 genes encode two splicing isoforms and that the βFTZ-F1 originates from an intron of aFTZ-F1 upstream of the DBD encoding exon,generating a transcript with an alternative 5’end.Accordingly,two variants of HvFTZ-F1 in H.vigintioctopunctata were named as HvαFTZ-F1 and HvβFTZ-F1 respectively.The lengths of ORFs of HvαFTZ-F1 and HvβFTZ-F1 were 1713 and 1827 bp,encoding 570 and 608 amino acids respectively.Both HvFTZ-F1 isoforms had typical conserved nuclear receptor domains,including DNA binding domain(C domain)and ligand binding domain(E domain).HvaFTZ-F1 and HvβFTZ-F1 were broadly expressed from embryo(egg)to adult.The highest expression level of HvaFTZ-F1 was found in eggs,while HvβFTZ-F1 was highly expressed immediately before the molt in the first and second larval instars,in the prepupae and pupae.Either HvaFTZ-F1 or HvβFTZ-F1 was highly expressed in the epidermis and guts of the fourth instar larvae,and lowly transcribed in the Malpighian tubules and fat body.In order to explore the iso form-specific roles of two HvFTZ-F1 iso forms in larval development,a dsHvFTZ-F1 targeting a common region of both variants,and two iso form-specific dsRNAs,dsHvαFTZ-F1 and dsHvβFTZ-F1,were injected respectively into the larvae.K nockdown of both HvFTZ-F1 iso forms at the fourth-instar larval stage arrested larval developing at the prepupal stage.The resultant beetles gradually withered,dried and darkened,they never moved until death.Depletion of HvβFTZ-F1 at the fourth-instar larval stage completely arrested larval development.Most of them(95%)remained as prepupae before death.Only 5%of the HvβFTZ-F1 hypomorphic prepupae formed misshapen pupae,partially wrapped in the larval exuviae.Conversely,depletion of HvαFTZ-F1 does not brought about obvious defective phenotypes during larval development.The same RNAi experiment was performed using the newly-molted third instar larvae.Quantitative qPCR results showed that dsRNA targeting both iso forms successfully depleted either variant.Whereas RNAi of HvαFTZ-F1 enhanced the expression of HvβFTZ-F1,knockdown of HvβFTZ-F1 had no influence on the level of HvαFTZ-F1.Depletion of both HvFTZ-F1 variants completely repressed the molt of the resultant larvae.All the RNAi larvae failed to molt to the fourth instar,even though the new cuticles had been formed under old larval exuviae.During the elongated third-instar larval period,the HvFTZ-F1 hypomorphic larvae consumed most of the nutrients,the size of fat body and the content of triglyceride were significantly decreased and the larval body bent into a half-moon shape.No HvFTZ-F1 RNAi deformed pupae emerged as adults.Comparably,when knockdown of HvβFTZ-F1 at the third-larval instar stage,all the resultant larvae passed third-fourth instar ecdysis and arrested at the fourth larval instar stage.However,all the HvβFTZ-F1 RNAi larvae failed to pupate.Conversely,all the HvαFTZ-F1 RNAi larvae successfully underwent the larval-larval,larval-pupal and pupal-adult transitions.In summary,both HvFTZ-F1 iso forms are essential for larval-larval molting,while HvβFTZ-F1 is necessary for larval-pupal transition and sufficient to exert the role of both HvFTZ-F1s during larval-pupal metamorphosis in H.vigintioctopunctata. |
