Role of juvenile hormone receptor Methoprene-tolerant 1 in silkworm larval brain development and domestication

The insect brain is the central part of the neurosecretory system,which controls morphology,physiology,and behavior during the insect’s lifecycle.Lepidoptera are holometabolous insects,and their brains develop during the larval period and metamorphosis into the adult form.As the only fully domesticated insect,the Lepidoptera silkworm Bombyx mori experienced changes in larval brain morphology and certain behaviors during the domestication process.Hormonal regulation in insects is a key factor in multiple processes.However,how juvenile hormone(JH)signals regulate brain development in Lepidoptera species,especially in the larval stage,remains elusive.We recently identified the JH receptor Methoprene tolerant 1(Met1)as a putative domestication gene.How artificial selection on Met1 impacts brain and behavioral domestication is another important issue addressing Darwin’s theory on domestication.Here,CRISPR/Cas9-mediated knockout of Bombyx Met1 caused developmental retardation in the brain,unlike precocious pupation of the cuticle.At the whole transcriptomelevel,theecdysteroid(20-hydroxyecdysone,20 E)signaling and downstream pathways were overactivated in the mutant cuticle but not in the brain.Pathways related to cell proliferation and specialization processes,such as extracellular matrix(ECM)-receptor interaction and tyrosine metabolism pathways,were suppressed in the brain.Molecular evolutionary analysis and in vitro assay identified an amino acid replacement located in a novel motif under positive selection in B.mori,which decreased transcriptional binding activity.The B.mori MET1 protein showed a changed structure and dynamic features,as well as a weakened co-expression gene network,compared with B.mandarina.Based on comparative transcriptomic analyses,we proposed a pathway downstream of JH signaling(i.e.,tyrosine metabolism pathway)that likely contributed to silkworm larval brain development and domestication and highlighted the importance of the biogenic amine system in larval evolution during silkworm domestication.

Identification and functional analysis of CG3526 in spermatogenesis of Drosophila melanogaster

Spermatogenesis is a critical part of reproduction in insects;however,its molecular mechanism is still largely unknown.In this study,we identified a testis-specific gene CG3526 in Drosophila melanogaster.Bioinformatics analysis showed that CG3526 contains a zinc binding domain and 2 C2H2 type zinc fingers,and it is clustered to the vertebrate really interesting new gene(RING)family E3 ubiquitin-protein ligases.When CG3526 was knocked down by RNA interference(RNAi),the testis became much smaller in size,and the apical tip exhibited a sharp and thin end instead of the blunt and round shape in the control testis.More importantly,compared to the control flies,only a few mature sperm were present in the seminal vesicle of C587-Gal4>CG3526 RNAi flies.Immunofluorescence staining of the testis from CG3526 RNAi flies showed that the homeostasis of testis stem cell niche was disrupted,cell distribution in the apical tip was scattered,and the process of spermatogenesis was not completed.Furthermore,we found that the phenotype of CG3526 RNAi flies’testis was similar to that of testis of Stat92E RNAi flies,the expression level of CG3526 was significantly downregulated in the Stat92EF06346 mutant flies,and the promoter activity of CG3526 was upregulated by STAT92E.Taken together,our results indicated that CG3526 is a downstream effector gene in the JAK-STAT signaling pathway that plays a key role in the spermatogenesis of Drosophila.

Comprehensive silk gland multi-omics comparison illuminates two alternative mechanisms in silkworm heterosis

Heterosis is a common phenomenon in plants and animals with diverse underlying mechanisms.Here,we applied two widely used silkworm hybrid systems and performed multi-omics analysis to identify possible intrinsic associations between different hybrid strategies and epigenetic mechanisms with silkworm heterosis.We found significant differences in the silk gland transcriptomic landscape between the two systems,including differentially expressed genes and expression patterns in the hybrid offspring compared to their parents.In the quaternary hybrid system,hybrid vigor was primarily due to upregulated genes and the parent-dominant upregulated expression pattern,involving multiple transport processes,cellular nitrogen compound catabolism,glucose metabolism,and tricarboxylic acid cycle.In the binary system,hybrid vigor was mainly due to the down-regulated genes and transgressively down-regulated expression pattern,mainly involving basic nitrogen synthesis metabolism and body function.We also demonstrated that DNA methylation may affect hybrid vigor by regulating the expression of several heterosis-related genes.Thus,this study revealed two alternative mechanisms that may contribute to silkworm heterosis,both of which facilitate the efficient utilization of energy and nitrogen for silk production.

Alternative splicing of POUM2 regulates embryonic cuticular formation and tanning in Bombyx mori

Insect cuticle is an apical extracellular matrix produced by the epidermis,tracheal,hind-and foregut epithelia during embryogenesis and renewed during molting and metamorphosis.However,the underlying regulatory mechanism for embryonic cuticle formation remains largely unclear.Here,we investigate the function of the transcription factor POUM2 in the embryonic cuticular formation in Bombyx mori,a model lepidopteran insect.Clustered regularly interspaced palindromic repeats(CRISPR)/CRISPR-associated protein-9-mediated knockout of POUM2 resulted in the defect of cuticular deposition,pigmentation,and sclerotization in the embryos.Differentially expressed transcripts analysis of 7-d-old embryos identified 174 up-or downregulated cuticular protein transcripts,8 upregulated chitin degradation transcripts,2 downregulated chitin synthesis transcripts and 48 up-or downregulated transcription factor transcripts in the POUM2−/−embryos.The expression levels of the key factors of the tyrosine metabolic pathway,such as tyrosine hydroxylase(Th),Dopa decarboxylase(DDC),and arylalkylamine N-acetyltransferase(aaNAT),were significantly decreased in the POUM2−/−embryos.POUM2 isoform POUM2-L specifically bound the POU cis-regulatory element(CRE)in the Th promoter and increased the transcription of Th,whereas POUM2-S could not bind the POU CRE,although it also increased the transcription of Th.Heterogeneous nuclear ribonucleoprotein Squid-1 directly bound the POUM2 pre-mRNA(messenger RNA)and inhibited the alternative splicing of POUM2-L to POUM2-S mRNA.These results suggest that POUM2 participates in the cuticular formation by regulating the chitin and cuticular protein synthesis and metabolism,and the cuticular pigmentation and sclerotization by regulating tyrosine metabolism during embryogenesis.This study provides new insights into novel function of POUM2 in embryogenesis.

Methyl-CpG binding domain(MBD)2/3 specifically recognizes and binds to the genomic mCpG site with a β-sheet in the MBD to affect embryonic development in Bombyx mori

Methyl-CpG(mCpG)binding domain(MBD)proteins especially bind with methylated DNA,and are involved in many important biological processes;however,the binding mechanism between insect MBD2/3 and mCpG remains unclear.In this study,we identified 2 isoforms of the MBD2/3 gene in Bombyx mori,MBD2/3-S and MBD2/3-L.Binding analysis of MBD2/3-L,MBD2/3-S,and 7 mutant MBD2/3-L proteins deficient inβ1−β6 orα1 in the MBD showed thatβ2−β3-turns in theβ-sheet of the MBD are necessary for the formation of the MBD2/3–mCpG complex;furthermore,other secondary structures,namely,β4−β6 and anα-helix,play a role in stabilizing theβ-sheet structure to ensure that the MBD is able to bind mCpG.In addition,sequence alignment and binding analyses of different insect MBD2/3s indicated that insect MBD2/3s have an intact and conserved MBD that binds to the mCpG of target genes.Furthermore,MBD2/3 RNA interference results showed that MBD2/3-L plays a role in regulating B.mori embryonic development,similar to that of DNA methylation;however,MBD2/3-S withoutβ4−β6 andα-helix does not alter embryonic development.These results suggest that MBD2/3-L recognizes and binds to mCpG through the intactβ-sheet structure in its MBD,thus ensuring silkworm embryonic development.

Identification of binding domains and key amino acids involved in the interaction between BmLARK and G4 structure in the BmPOUM2 promoter in Bombyx mori

It has been found that the non-B form DNA structures,like G-quadruplex(G4)and i-motif,are involved in many important biological processes.Our previous study showed that the silkworm transcription factor BmLARK binds to the G4 structure in the promoter of the transcription factor BmPOUM2 and regulates its promoter activity.However,the binding mechanism between BmLARK and BmPOUM2 G4 structure remains unclear.In this study,binding domains and key amino acid residues involved in the interaction between BmLARK and BmPOUM2 G4 were studied.The electrophoretic mobility shift assay results indicated that the two RNA-recognition motifs(RRM)of BmLARK are simultaneously required for the binding with the G4 structure.Either RRM1 or RRM2 alone could not bind with the G4 structure.The zinc-finger motif was not involved in the binding.A series of mutant proteins with specific amino acid mutations were expressed and used to identify the key amino acid residues involving the interaction.The results indicated thatβsheets,especially theβ1 andβ3 sheets,in the RRM domains of BmLARK played critical roles in the binding with the G4 structure.Several amino acid mutations of RRM1/2 in ribonucleoprotein domain 1(RNP1)(motif inβ3 strand)and RNP2(motif inβ1 strand)caused loss of binding ability,indicating that these amino acids are the key sites for the binding.All the results suggest that RRM domains,particularly their the RNP1 and RNP2 motifs,play important roles not only in RNA recognition,but also in the G4 structure binding.

Transcriptomic analysis reveals the regulation network of BmKruppel homolog 1 in the oocyte development of Bombyx mori

Kriippel homolog 1(Kr-hl),a zinc finger transcription factor,is involved in the metamorphosis and adult reproduction of insects.However,the role of Kr-hl in reproduction of holometabolic insects remains to be elucidated.The regulation network of 7-associated genes in the reproduction in Bombyx mori was investigated in this study.The higher expression level of BmKr-hl in the ovaries was detected during the late pupal stage and adults.RNA interference(RNAi)-mediated depletion of BmKr-hl in the female at day 6 of pupae resulted in abnormal oocytes at 48 h post-double-stranded RNA treatment,which showed less yolk protein deposition and partially transparent chorion.RNA-seq and subsequent differentially expressed transcripts analysis showed that knockdown of BmKr-hl caused a decrease in the expression of 2882 genes and an increase in the expression of 2565 genes in the oocytes at day 8 of pupae.Totally,27 genes coding for transcription factors were down-regulated,while six genes coding for other transcription factors were up-regulated.BmKr-hl bound to the Kr-hl binding site of the transcription factors AP-1(activating protein-1)and FOXG1 to increase their messenger RNA tran・scripts in the BmN cells,respectively.Gene Ontology enrichment and Kyoto Encyclopedia of Genes and Genomes pathway analyses of that positively co-expressed with AP-1 and FOXG1 transcripts showed mainly enrichment in the metabolic-related pathways,the nu・tri ent absorption and the yolk protein absorption processes.These data suggested that BmKr-h 1 might directly regulate the metabolic-related pathways,the nutrient absorption and the yolk protein absorption processes or probably through AP-1 and/or FOXG1 to regulate oocyte development.

Analysis of expression and chitin-binding activity of the wing disc cuticle protein BmWCP4 in the silkworm, Bombyx moil

The insect exoskeleton is mainly composed of chitin filaments linked by cuticle proteins. When insects molt, the cuticle of the exoskeleton is renewed by degrading the old chitin and cuticle proteins and synthesizing new ones. In this study, chitin-binding activity of the wing disc cuticle protein BmWCP4 in Bombyx mori was studied. Sequence analysis showed that the protein had a conservative hydrophilic ＂R＆R＂ chitin-binding domain （CBD）. Western blotting showed that BmWCP4 was predominately expressed in the wing disc-containing epidermis during the late wandering and early pupal stages. The immunohistochemistry result showed that the BmWCP4 was mainly present in the wing disc tissues containing wing bud and trachea blast during day 2 of wandering stage. Recombinant full-length BmWCP4 protein, ＂R＆R＂ CBD peptide （CBD）, non-CBD peptide （BmWCP4-CBD^-）, four single site-directed mutated peptides （M1, M2, M3 and M4） and four-sites-mutated peptide （MF） were generated and purified, respectively, for in vitro chitin-binding assay. The results indicated that both the full-length protein and the ＂R＆R＂ CBD peptide could bind with chitin, whereas the BmWCP4-CBD- could not bind with chitin. The single residue mutants M1, M2, M3 and M4 reduced but did not completely abolish the chitin-binding activity, while four-sites-mutated protein MF completely lost the chitin-binding activity. These data indicate that BmWCP4 protein plays a critical role by binding to the chitin filaments in the wing during larva-to-pupa transformation. The conserved aromatic amino acids are critical in the interaction between chitin and the cuticle protein.

Cloning and structural characterization of juvenile hormone diol kinase in Spodoptera litura

Juvenile hormone （JH） is one of the key insect hormones that regulate metamorphosis. Juvenile hormone diol kinase （JHDK） is an enzyme involved in JH metabolism and catalyzes JH diol to form a polar end product, JH diol phosphate that has no JH activity. In this study, a JHDK complementary DNA （cDNA） was cloned from Spodoptera litura and the structure and expression of the gene was characterized. The cDNA was 714 base pairs in length and encoded a protein of 183 amino acids with a molecular mass of 21 kDa and an isoelectric point of 4.55. Based on the structure, three putative calcium binding motifs and guanosine triphosphate-binding motifs were predicted in the protein. Modeling of the 3-D structure showed that the protein consisted of eight α-helixes linked with loops, with no β-sheets. The gene was expressed in the epidermis, fat body and midgut of fifth and sixth instar larvae. The expression level in the epidermis was lower than in the fat body and midgut. The gene was expressed at higher levels at the early stages than in the later stages of fifth and sixth instar midgut and fat body. The results suggest that this gene may be involved in the regulation of the JH titer in larvae ofS. litura.

Genome-wide identification and characterization of basic helix-loop-helix transcription factors in Spodoptera litura upon pathogen infection

Basic helix-loop-helix(bHLH)transcription factors play an important role in a wide range of metabolic and developmental processes in eukaryotes,and bHLH proteins also participate in immune responses,especially in plants.However,their roles in insects upon entomopathogen infection are unknown.In this study,54 bHLH genes in 41 families were identified in a polyphagous pest,Spodoptera litura,including a new bHLH gene in group B,which is specifically present in Lepidoptera and was thus named Lep.The conserved amino acids in the bHLH domain,structural architecture,and chromosomal distribution of bHLH genes in S.litura were analyzed.The bHLH genes in Plutella xylostella and Apis mellifera were also updated,and genome-wide comparison and phylogenetic analysis of bHLH members in 5 holometabolous insects were performed.The expression profiles of S.litura bHLH(SlbHLH)genes in 3 tissues at different developmental stages and their responses to S.litura nucleopolyhedrovirus(SpltNPV),Nomuraea rileyi(Nr),and Bacillus thuringiensis(Bt)infection were investigated.More SlbHLHs in group B were expressed and differentially expressed during pathogen infections,and SlbHLHs tended to be downregulated in the midgut of S.litura larvae after B.thuringiensis treatment.Our study provides an overview of bHLH family members in S.litura and their responses to different pathogens used for pest biocontrol.These findings on bHLH members may contribute to uncovering the mechanism of host-pathogen interaction.

IFT52 plays an essential role in sensory cilia formation and neuronal sensory function in Drosophila

Cilia are microtubule-based,hair-like organelles involved in sensory function or motility,playing critical roles in many physiological processes such as reproduction,or-gan development,and sensory perception.In insects,cilia are restricted to certain sensory neurons and sperms,being important for chemical and mechanical sensing,and fertility.Although great progress has been made regarding the mechanism of cilia assembly,the formation of insect cilia remains poorly understand,even in the insect model organism Drosophila.Intraflagellar transport(IFT)is a cilia-specific complex that traffics protein cargos bidirectionally along the ciliary axoneme and is essential for most cilia.Here we investigated the role of IFT52,a core component of IFT-B,in cilia/flagellar formation in Drosophila.We show that Drosophila IFT52 is distributed along the sensory neuronal cilia,and is essential for sensory cilia formation.Deletion of Ift52 results in severe defects in cilia-related sensory behaviors.It should be noted that IFT52 is not detected in sper-matocyte cilia or sperm flagella of Drosophila.Accordingly,ift52 mutants can produce sperms with normal motility,supporting a dispensable role of IFT in Drosophila sperm flagella formation.Altogether,IFT52 is a conserved protein essential for sensory cilia formation and sensory neuronal function in insects.

The RNase III enzyme Dicer1 is essential for larval development in Bombyx mori

MicroRNAs(miRNAs)are important regulators of nearly all aspects of biological processes in eukaryotes.During the biogenesis of miRNAs,the RNase III enzyme Dicer processes double-strand precursor miRNAs into mature miRNAs and promotes the assembly of RNA-induced silencing complexes(RISCs).Dicer has been reported to participate in a wide range of physiological processes,including development and immunity,in some insect species.However,the physiological roles of Dicer in lepidopterans remain poorly understood.In this study,we investigated the function of Bombyx mori Dicer1.We first performed sequence alignment and found that the sequence of functional domains of Dicer1 are varied among Lepidoptera,Diptera,Coleoptera,Blattaria,and Orthoptera.Using a binary clustered regularly interspaced palindromic repeats(CRISPR)/CRISPR-associated protein 9 genome editing approach,we showed that BmDicer1 mutants have arrested development from the 3rd instar into the 4th instar.RNA sequencing analysis indicated that the defects in BmDicer1 mutants are due to dysregulation of genes that encode proteins involved in metabolism,protein degradation,absorption,and renin–angiotensin pathways.Analysis using quantitative real-time polymerase chain reaction showed that mutation of BmDicer1 altered expression of miRNAs and their target genes.Therefore,our study demonstrates the critical roles of BmDicer1 in miRNA biogenesis and larval development in silkworm.

Harnessing“little mighty”cockroaches:Pest management and beneficial utilization

Rationale backs up cockroaches being“little mighty”.Cockroaches belong to the insect order Blattodea and often serve as model organisms for hemimetabolous insects.They occupy a pivotal evolutionary position in studies of insect metamorphosis.The emergence of the last common ancestor of all extant cockroaches may date back to approximately 235 million years ago.Cockroaches are a highly successful group of insects:about 5,000 living species have been recorded.Since the seismic continental drift during the ancient transition from Pangaea,cockroaches have undergone substantial invasion and distribution to all continents worldwide.1 The American cockroach(Periplaneta americana)and the German cockroach(Blattella germanica)are the most common and are closely synanthropic species,referred to as“little mighty”(a household name,xiao qiang,in China)because of their extreme vitality and adaptation.The rationale behind cockroaches’being considered“little mighty”can be attributed mostly to four biological characteristics(Figure 1).

Genomic divergences between the two polyphagous Spodoptera relatives provide cues for successful invasion of the fall armyworm

The fall armyworm Spodoptera frugiperda recently invaded China,ravaging crops in many provinces.Deciphering the possible genetic basics for its successful invasion is critical for innovative and specific control for this gluttonous pest.Here we generated comparative genomic analyses between S.frugiperda and its native relative,S.litura,which differs in host preference,locomotivity and production behavior.We demonstrated that S.frugiperda genes are enriched in taste sensory perception and nervous system,obv iously difTerent from those of S.litura.Potential host adaptation genes showed generally an elevated ratio of non-synonymous substitution rate to synonymous substitution rate,suggesting a faster evolution during the divergence of the two species.Focusing on these sets of genes,we identified 23 genes being under positive selection in S.frugiperda.Among them are two notable genes involved in sensory perception,gustatory receptor(GR)and an acetaldehyde oxidase,which are important for host detection in invasion and expansion processes.Another two genes are mitochondrial adenosine triphosphate synthase^subunit and ferritin heavy chain,which may be associated with the enhanced locomotivity and resistance,which fascinated long-distance migration needed for invasion and rapid expansion.Another interesting gene is chorion protein,in which positive selection sites in S.frugiperda were found and a replacement in one site is predicted to affect the protein function,which might be associated with competent reproductivity in S.frugiperda to ensure genetic resources for expansion.

Dual roles of juvenile hormone signaling during early oogenesis in Drosophila

Juvenile hormone(JH)signaling plays crucial roles in insect metamorphosis and reproduction.Function of JH signaling in germline stem cells(GSCs)remains largely unknown.Here,we found that the number of GSCs significantly declined in the ovaries of Met,Gee and JHAMT mutants.Then we inhibited JH signaling in selected cell types of ovaries by expressing Met and Gee or Kr-hl double-stranded RNAs(dsRNAs)using different Gal4 drivers.Blocking of JH signaling in muscle cells has no effect on GSC numbers.Blocking of JH signaling in cap cells reduced GSCs cells.Inductive expression of Met and Gee dsRNA but not Kr-hl by Nos-Gal4 increased GSC cells.These results indicate that JH signaling plays an important role in GSC maintenance.

Multiple microRNAs control ecdysone signaling in the midgut of Spodoptera litura

Metamorphosis is one of the most important physiological processes in insects.It is regulated by a serial of ecdysone cascade genes.Recently,lots o f microRNAs(miRNAs)were investigated in insects;however,their function in metamorphosis is largely unknown.In the present study,the dynamics of a small RNA population was investigated by RNA sequencing from the midgut of a lepidopteran pest Spodoptera litura during larval-pupal metamorphosis.A total of 101 miRNAs were identified,and 75 miRNAs were differentially expressed during the metamorphic process.The relationship between these differentially expressed miRNAs and 12 ecdysone cascade genes was analyzed by four classical software programs,and a multiple-to-multiple regulatory network was found to exist between these miRNAs and their targets.Among them,miR-14-3p and its two targets(EcR and E75)were chosen for further validation.MiR-14-3p had higher expression level in the 6 th instar larvae as compared with either the prepupae or pupae,which was opposite to that of both EcR and E75,two ecdysone cascade genes.Luciferase reporter assay confirmed that both EcR and E75 were regulated by miR-14-3p.Interestingly,the 3'untranslated regions are nearly identical to each other among different transcript variants of the ecdysone cascade genes,including EcR,USP,E75,E74,E78,E93,Hr3,Hr4,Hr39,K rhl and F tzfl.Thus,different transcript variants of one ecdysone cascade gene could be regulated by the same mi RNA.The above data suggest that the ecdysone signaling pathway is under the tight control of miRNA.These findings expand our understanding of the mechanism of insect metamorphosis and may also provide a novel possibility for the control of pest insects in the future.

Juvenile hormone signaling-a mini review

Since it was first postulated by Wigglesworth in 1934, juvenile hormone (JH) is considered a status quo hormone in insects because it prevents metamorphosis that is initiated by the molting hormone 20-hydroxyecdysone (20E). During the last decade, significant advances have been made regarding JH signaling. First, the bHLH-PAS transcription factor Met/Gce was identified as the JH intracellular receptor. In the presence of JH, with the assistance of Hsp83, and through physical association with a bHLH?PAS transcriptional co-activator, Met/Gce enters the nucleus and binds to E-box-like motifs in promoter regions of JH primary?response genes for inducing gene expression. Second, the zinc finger transcription factor Kr-hl was identified as the anti-metamorphic factor which transduces JH signaling. Via Kr-hl binding sites, Kr-hl represses expression of 20E primary?response genes (i.e. Bi\ E93 and E5) to prevent 20E-induced metamorphosis. Third, through the intracellular signaling, JH promotes differ ent aspects of female reproduction. Nevertheless, this action varies greatly from species to species. Last, a hypothetical JH membrane receptor has been predicted to be either a GPCR or a tyrosine kinase receptor. In future, it will be a great challenge to understand how the JH intracellular receptor Met/Gce and the yet unidentified JH membrane receptor coordinate to regulate metamorphosis and reproduction in insects.