Insect Metamorphosis Research Articles

Chronologically inappropriate morphogenesis (Chinmo) is required for maintenance of larval stages of fall armyworm

Broad complex (Br-C) and eip93F (E93) transcription factors promote insect metamorphosis from larva to pupa and from pupa to adult, respectively. Recently, chronologically inappropriate morphogenesis (Chinmo) has been proposed as a larval specifier in Drosophila melanogaster. However, whether Chinmo is required for larval maintenance in lepidopteran insects, the underlying mechanisms involved in maintaining the larval stage, and its interactions with the JH signaling pathway are not well understood. Here, we used a binary transgenic CRISPR/Cas9 system to knockout Chinmo and Kr-h1 (primary response gene in the JH signaling pathway) in the fall armyworm (FAW). Kr-h1 knockout induced premature metamorphosis only after L5 (penultimate), whereas Chinmo and Kr-h1 double knockout induced premature metamorphosis in L3. Sequencing and differential gene expression (DEG) analysis of RNA isolated from mutants and single-cell multiome ATAC analysis of Chinmo, Kr-h1, and Chinmo and Kr-h1 double knockout Sf9 cells revealed that Chinmo participates in chromatin modifications that prevent the promoter accessibility and expression of metamorphosis promoting genes. These results suggest that Chinmo is a larval specifier that plays a major role in preventing metamorphosis in early larval stages by controlling chromatin accessibility near the promoters of genes such as Br-C and E93 required for pupal and adult development.

The JNK signalling pathway gene BmJun is involved in the regulation of egg quality and production in the silkworm, Bombyx mori.

The Jun N-terminal kinase (JNK) signalling pathway has a key role in tissue remodelling during insect metamorphosis by regulating programmed cell death. However, multiple members of the JNK pathway in Lepidoptera remain uncharacterized. In this study, two key genes of the JNK pathway, BmJun and BmFos, were cloned from the silkworm Bombyx mori, a lepidopteran model insect, and their effects on reproductive development were investigated. BmJun and BmFos encode 239 and 380 amino acids, respectively. Both proteins have typical basic leucine zipper domains and form a BmJUN-BmFOS dimer activator protein to exert transcriptional regulation. During the wandering stage of silkworm development, interference in BmJun expression had no effect on pupation, whereas B. mori vitellogenin (BmVg) expression, which is essential for egg development, was suppressed in the fat body and egg laying was significantly reduced. Additionally, numerous eggs appeared shrivelled and deformed, suggesting that they were nutritionally stunted. Inhibition of the JNK pathway caused abnormal pupal metamorphosis, an increase in shrivelled, unfertilized eggs, a decrease in fat body synthesis, and accumulation of BmVg in the ovaries of female B. mori. The results indicated that BmJUN and BmFOS can form an AP-1 dimer. Interfering with BmJun or inhibiting the phosphorylation of BmJUN leads to a reduction in the synthesis of BmVg in the fat body and its accumulation in the ovaries, thereby affecting the quality and production of the progeny eggs. These findings suggest that regulating Jun in the JNK pathway could be a potential way to inhibit female reproduction in Lepidoptera.

The miRNA-mRNA modules enhance juvenile hormone biosynthesis for insect vitellogenesis and egg production.

In addition to preventing precocious larval metamorphosis, juvenile hormone (JH), synthesized in corpora allata (CA), is known to stimulate female reproduction of insects. JH titer is extremely low or absent during metamorphosis, but thereafter rapidly increases in the previtellogenic stage and rises to a peak in the vitellogenic phase. However, the mechanisms underlying the biosynthesis of high levels of JH in adults remain unclear. We found in this study that 12 genes involved in JH synthesis pathway were highly expressed in the CA of adult locusts. By transcriptome analysis and quantitative real-time - polymerase chain reaction validation, a total of 106 evolutionary conserved micro RNAs (miRNAs) and 163 species-specific miRNAs were identified in locust CA. Dual-luciferase assay revealed that 17 miRNAs bound to 10 JH synthesis genes (JHSGs) and downregulated their expression. These miRNAs were expressed in low levels during vitellogenic stage, which was oppositive from that of targeting JHSGs. Six miRNAs including miR-971-3p, miR-31a, miR-9-5p, miR-1-3p, miR-315, and miR-282 were selected for function study. Co-application of agomiRs resulted in significantly decreased levels of targeting JHSGs, accompanied by significantly reduced vitellogenin expression as well as arrested ovarian development. The data suggest that multiple miRNAs expressed synchronously at low levels in the vitellogenic phase, thereby ensuring the high levels of JHSG expression to facilitate JH biosynthesis required for JH-dependent female reproduction. The findings provide important information for deciphering miRNA-messenger RNA modules for JH biosynthesis as well as JH regulation of insect metamorphosis and reproduction.

The role of neuropeptide prothoracicotropic hormone (PTTH) - Torso in pyriproxyfen-induced larval-pupal abnormal metamorphosis in silkworms

The neuropeptide prothoracicotropic hormone (PTTH) plays a key role in regulating ecdysone synthesis and promoting insect metamorphosis. Pyriproxyfen is a juvenile hormone analogue. We previously reported that pyriproxyfen disrupts ecdysone secretion and inhibits larval-pupal metamorphosis in silkworms. However, the specific molecular mechanisms by which pyriproxyfen interferes with ecdysone signaling remain to be elucidated. Herein, the RNA-seq analysis on the ecdysone-secretion organ prothoracic gland (PG) was conducted following pyriproxyfen exposure. A total of 3774 differentially expressed genes (DEGs) were identified, with 1667 up-regulated and 2107 down-regulated. KEGG analysis showed that DEGs were enriched in the MAPK signaling pathway, a conserved pathway activated by PTTH binding to Torso, which regulates the ecdysone synthesis. qRT-PCR results indicated a significant up-regulation in PTTH transcription level, while the transcription levels of torso and downstream MAPK pathway genes, Ras2, Raf and ERK, were down-regulated 24 h post-pyriproxyfen treatment. Consistent with these transcriptional changes, PTTH titers in the brain also increased following pyriproxyfen treatment. These results suggest that pyriproxyfen induces abnormal metamorphosis in silkworms by impairing PTTH-Torso signaling. This study enhances our understanding of the molecular mechanisms of pyriproxyfen-induced larval-pupal abnormal metamorphosis in silkworms, and also provides insights for developing detoxification strategies for juvenile hormone analog pesticides to non-target organisms.

Rapid growth and the evolution of complete metamorphosis in insects

More than 50% of all animal species are insects that undergo complete metamorphosis. The key innovation of these holometabolous insects is a pupal stage between the larva and adult when most structures are completely rebuilt. Why this extreme lifestyle evolved is unclear. Here, we test the hypothesis that a trade-off between growth and differentiation explains the evolution of this novelty. Using a comparative approach, we find that holometabolous insects grow much faster than hemimetabolous insects. Using a theoretical model, we then show how holometaboly evolves under a growth-differentiation trade-off and identify conditions under which such temporal decoupling of growth and differentiation is favored. Our work supports the notion that the holometabolous life history evolved to remove developmental constraints on fast growth, primarily under high mortality.

Evolutionary origin and distribution of leucine-rich repeat-containing G protein-coupled receptors in insects

Leucine-rich repeat-containing G protein-coupled receptors (LGRs) are crucial for animal growth and development. They were categorized into four types (A, B, C1, and C2) based on their sequence and domain structures. Despite the widespread distribution of LGRs across bilaterians, a thorough investigation of their distribution and evolutionary history remains elusive. Recent studies insect LGRs, especially the emergence of type C2 LGRs in various hemimetabolous insects, had prompted our study to address these problems. Initially, we traced the origins of LGRs by exploiting data from 99 species spanning 11 metazoan phyla, and discovered that type A and B LGRs originated from sponges, while type C LGRs originated from cnidarians. Subsequently, through comprehensive genomic and transcriptomic analyses across 565 species across 25 orders of insects, we found that both type A and C1 LGRs divided into two gene clusters. These clusters can be traced back to basal Insecta and an early ancestor of the Arthropoda, respectively. Furthermore, the absence of type B LGRs in wingless insects suggests a role in wing development, while the absence of type C2 LGRs in holometabolous insects hints at novel functions unrelated to insect metamorphosis. According to the origin of LGRs and the investigation of LGRs in insects, we speculated that type A and B LGRs appeared first among four types of LGRs, type A evolved into type C LGRs later, and type A and C1 LGRs independently duplicated during the evolutionary process. This study provides a more comprehensive view of the evolution of LGR genes than previously available, and sheds light on the evolutionary history and significance of LGRs in insect biology.

Insect hormone PTTH regulates lifespan through temporal and spatial activation of NF-κB signaling during metamorphosis.

The prothoracicotropic hormone (PTTH) is a well-known neuropeptide that regulates insect metamorphosis (the juvenile-to-adult transition) by inducing the biosynthesis of steroid hormones. However, the role of PTTH in adult physiology and longevity is largely unexplored. Here, we show that Ptth loss-of-function mutants are long-lived and exhibit increased resistance to oxidative stress in Drosophila. Intriguingly, we find that loss of Ptth blunt age-dependent upregulation of NF-κB signaling specifically in fly hepatocytes (oenocytes). We further show that oenocyte-specific overexpression of Relish/NF-κB blocks the lifespan extension of Ptth mutants, suggesting that PTTH regulates lifespan through oenocyte-specific NF-κB signaling. Surprisingly, adult-specific knockdown of Ptth did not prolong lifespan, indicating that PTTH controls longevity through developmental programs. Indeed, knockdown of PTTH receptor Torso in prothoracic gland (PG) during fly development prolongs lifespan. To uncover the developmental processes underlying PTTH-regulated lifespan, we perform a developmental transcriptomic analysis and identify an unexpected activation of NF-κB signaling in developing oenocytes during fly metamorphosis, which is blocked in Ptth mutants. Importantly, knockdown of Relish/NF-κB specifically in oenocytes during early pupal stages significantly prolongs the lifespan of adult flies. Thus, our findings uncover an unexpected role of PTTH in controlling adult lifespan through temporal and spatial activation of NF-κB signaling in developing hepatocytes and highlight the vital role of developmental NF-κB signaling in shaping adult physiology.

Lipases are differentially regulated by hormones to maintain free fatty acid homeostasis for insect brain development

BackgroundFree fatty acids (FFAs) play vital roles as energy sources and substrates in organisms; however, the molecular mechanism regulating the homeostasis of FFA levels in various circumstances, such as feeding and nonfeeding stages, is not fully clarified. Holometabolous insects digest dietary triglycerides (TAGs) during larval feeding stages and degrade stored TAGs in the fat body during metamorphosis after feeding cessation, which presents a suitable model for this study.ResultsThis study reported that two lipases are differentially regulated by hormones to maintain the homeostasis of FFA levels during the feeding and nonfeeding stages using the lepidopteran insect cotton bollworm Helicoverpa armigera as a model. Lipase member H-A-like (Lha-like), related to human pancreatic lipase (PTL), was abundantly expressed in the midgut during the feeding stage, while the monoacylglycerol lipase ABHD12-like (Abhd12-like), related to human monoacylglycerol lipase (MGL), was abundantly expressed in the fat body during the nonfeeding stage. Lha-like was upregulated by juvenile hormone (JH) via the JH intracellular receptor methoprene-tolerant 1 (MET1), and Abhd12-like was upregulated by 20-hydroxyecdysone (20E) via forkhead box O (FOXO) transcription factor. Knockdown of Lha-like decreased FFA levels in the hemolymph and reduced TAG levels in the fat body. Moreover, lipid droplets (LDs) were small, the brain morphology was abnormal, the size of the brain was small, and the larvae showed the phenotype of delayed pupation, small pupae, and delayed tissue remodeling. Knockdown of Abhd12-like decreased FFA levels in the hemolymph; however, TAG levels increased in the fat body, and LDs remained large. The development of the brain was arrested at the larval stage, and the larvae showed a delayed pupation phenotype and delayed tissue remodeling.ConclusionsThe differential regulation of lipases expression by different hormones determines FFAs homeostasis and different TAG levels in the fat body during the feeding larval growth and nonfeeding stages of metamorphosis in the insect. The homeostasis of FFAs supports insect growth, brain development, and metamorphosis.

Dynamics of Redox Metabolism during Complete Metamorphosis of Insects: Insights from the Sunflower Caterpillar Chlosyne lacinia (Lepidoptera).

Complete insect metamorphosis requires substantial metabolic and physiological adjustments. Although oxidative stress has been implicated in metamorphosis, details on redox metabolism during larva-to-pupa and pupa-to-adult remain scarce. This study explores redox metabolism during metamorphosis of a lepidopteran (Chlosyne lacinia), focusing on core metabolism, antioxidant systems and oxidative stress. The larva-to-pupa transition was characterized by increased lactate dehydrogenase and glutathione peroxidase (GPX) activities, coupled with depletion of reduced glutathione (GSH), high disulfide-to-total-glutathione ratio (GSSG/tGSH), and increased lipid peroxidation. As metamorphosis progressed, metabolic enzyme activities, citrate synthase and glucose 6-phosphate dehydrogenase increased, indicating heightened oxidative metabolism associated with adult development. Concurrently, GSH and GPX levels returned to larval levels and GSSG/tGSH reached its most reduced state right before adult emergence. Adult emergence was marked by a further increase in oxidative metabolism, accompanied by redox imbalance and enhanced antioxidant mechanisms. These findings highlight a fluctuation in redox balance throughout metamorphosis, with periods of oxidative eustress followed by compensatory antioxidant responses. This study is the first to identify concurrent changes in metabolism, antioxidants, redox balance and oxidative stress throughout metamorphosis. Our findings extend knowledge on redox metabolism adjustments and highlight redox adaptations and oxidative stress as natural components of complete insect metamorphosis.

Insights into the activation mechanism of Bm-CPA: Implications for insect molting regulation

Carboxypeptidase A has been found across various animal species, yet its activation mechanism during the insect molting process remains elusive. Our study specifically delved into the activation mechanism of carboxypeptidase A (Bm-CPA), identified in Bombyx mori's molting fluid during metamorphosis. Initially, western blotting identified two forms of Bm-CPA, 65 kDa and 54 kDa, in the epidermis of silkworms during the molting stage. Expressing the complete Bm-CPA sequence in Pichia pastoris allowed the identification, via mass spectrometry analysis, of a 75-amino-acid propeptide for the initial hydrolysis process. Subsequently, a 35 kDa form of Bm-CPA emerged in the molting fluid, confirmed as the active form through in vitro assays, demonstrating potent carboxypeptidase A activity and faint carboxypeptidase B activity. Four potential activation sites (including Lys158/Arg159 and Arg177/Arg178) were identified through mass spectrometry and amino acid mutation analysis. RNAi of Bm-CPA indicates its critical role in molting. Finally, the carboxypeptidase inhibitor (Bm-CPI) from silkworm molting fluid was expressed to explore its role in regulating Bm-CPA activity, demonstrating a direct interaction with the 35 kDa Bm-CPA. Our research implies Bm-CPA's potential involvement in the silkworm molting process, suggesting diverse regulatory roles. These findings highlight intricate protein regulation patterns during insect metamorphosis and development.

Juvenile hormone-induced microRNA miR-iab-8 regulates lipid homeostasis and metamorphosis in Drosophila melanogaster.

Metamorphosis plays an important role in the evolutionary success of insects. Accumulating evidence indicated that microRNAs (miRNAs) are involved in the regulation of processes associated with insect metamorphosis. However, the miRNAs coordinated with juvenile hormone (JH)-regulated metamorphosis remain poorly reported. In the present study, using high-throughput miRNA sequencing combined with Drosophila genetic approaches, we demonstrated that miR-iab-8, which primarily targets homeotic genes to modulate haltere-wing transformation and sterility was up-regulated by JH and involved in JH-mediated metamorphosis. Overexpression of miR-iab-8 in the fat body resulted in delayed development and failure of larval-pupal transition. Furthermore, metabolomic analysis results revealed that overexpression of miR-iab-8 caused severe energy metabolism defects especially the lipid metabolism, resulting in significantly reduced triacylglycerol (TG) content and glycerophospholipids but enhanced accumulation of phosphatidylcholine (PC) and phosphatidylethanolamine (PE). In line with this, Nile red staining demonstrated that during the third larval development, the TG content in the miR-iab-8 overexpression larvae was continuously decreased, which is opposite to the control. Additionally, the transcription levels of genes committed to TG synthesis and breakdown were found to be significantly increased and the expression of genes responsible for glycerophospholipids metabolism were also altered. Overall, we proposed that JH induced miR-iab-8 expression to perturb the lipid metabolism homeostasis especially the TG storage in the fat body, which in turn affected larval growth and metamorphosis.

The Baboon Gene Encodes Three Functionally Distinct Transforming Growth Factor β Type I Receptor Variants in Henosepilachna vigintioctopunctata

The Transforming Growth Factor-β (TGF-β) cascade plays a critical role in insect metamorphosis and involves cell-surface receptors known as type I and II, respectively (TβRI and TβRII). In Drosophila melanogaster, the TβRI receptor, Baboon (Babo), consists of three variants (BaboA, BaboB, and BaboC), each with isoform-specific functions. However, the isoforms and functional specifications of Babo in non-Drosophilid insects have not been established. Here, we examined babo transcripts from seven coleopteran species whose genomes have been published and found that mutually exclusive alternative splicing of the third exon produces three babo isoforms, identical to the Drosophila babo gene. The same three transcript variants were accordingly recognized from the transcriptome data of a coleopteran Henosepilachna vigintioctopunctata. RNA interference (RNAi)-mediated knockdown of all three babo transcripts at the fourth-instar larval stage hindered gut modeling and arrested larval development in H. vigintioctopunctata. All the resultant larvae became arrested prepupae; they were gradually dried and darkened and, eventually, died. Depletion of HvbaboA rather than HvbaboB or HvbaboC is similar to the phenotypic alterations caused by simultaneous RNAi of all three babo isoforms. Therefore, our results established diverged roles of the three Babo isoforms and highlighted the regulatory role of BaboA during larval-pupal transition in a non-Drosophilid insect species.

E93 is involved in regulating larval-pupal metamorphosis and the development of the wing and ovary in Helicoverpa armigera, and it shows great potential for RNAi-based pest control

The ecdysone-induced transcription factor E93 in model insects plays multiple roles in the insect metamorphosis processes, such as remodeling larval tissues and determining adult tissue formation. The knockdown of E93 in insects leads to incomplete metamorphosis, suggesting that E93 is a potential target for pest control. In this study, the HaE93 gene in the cotton bollworm Helicoverpa armigera, a polyphagous pest of various commercial crops worldwide, was identified and found to have high expression in the egg, prepupal, and pupal stages. The injection of dsHaE93 induced about 60% mortality in H. armigera at the larval-pupal stage. About 30% of the H. armigera surviving knockdown of the HaE93 gene showed delayed pupation and developed abnormal wings, and the females developed reduced ovaries. About 90% of the HaE93 knockdown individuals failed to reproduce before they died. The results of qRT-PCR showed that the expression levels of ecdysone primary-response genes, chitin synthesis-related genes, and wing and ovary development-related genes were reduced in HaE93 knockdown H. armigera. These results indicated that HaE93 plays a critical role in larva-pupa-adult metamorphosis and the development of the cuticle, wing, and ovary in female H. armigera by regulating the expression of the associated genes. Bioassays of dsHaE93 administered by either oral delivery or injection showed similar knockdown results, which suggested that HaE93 can be used as a target gene for the RNAi control of the pest H. armigera.

Micro-CT data of complete metamorphosis process in Harmonia axyridis

Insect metamorphosis involves significant changes in insect internal structure and is thus a critical focus of entomological research. Investigating the morphological transformation of internal structures is vital to understanding the origins of adult insect organs. Beetles are among the most species-rich groups in insects, but the development and transformation of their internal organs have yet to be systematically documented. In this study, we have acquired a comprehensive dataset that includes 27 detailed whole-body tomographic image sets of Harmonia axyridis, spanning from the prepupal to the pupal stages. Utilizing this data, we have created intricate 3D models of key internal organs, encompassing the brain, ventral nerve cord, digestive and excretion systems, as well as the body wall muscles. These data documented the transformation process of these critical organs and correlations between the origin of adult and larval organs and can be used to enhance the understanding of holometabolous adult organ genesis and offers a valuable reference model for investigating complete metamorphosis in insects.

E93 is indispensable for reproduction in ametabolous and hemimetabolous insects.

Ecdysone-induced protein 93 (E93), known as the 'adult-specifier' transcription factor in insects, triggers metamorphosis in both hemimetabolous and holometabolous insects. Although E93 is conserved in ametabolous insects, its spatiotemporal expression and physiological function remain poorly understood. In this study, we first discover that, in the ametabolous firebrat Thermobia domestica, the previtellogenic ovary exhibits cyclically high E93 expression, and E93 mRNA is broadly distributed in previtellogenic ovarioles. E93 homozygous mutant females of T. domestica exhibit severe fecundity deficiency due to impaired previtellogenic development of the ovarian follicles, likely because E93 induces the expression of genes involved in ECM (extracellular matrix)-receptor interactions during previtellogenesis. Moreover, we reveal that in the hemimetabolous cockroach Blattella germanica, E93 similarly promotes previtellogenic ovarian development. In addition, E93 is also essential for vitellogenesis that is necessary to guarantee ovarian maturation and promotes the vitellogenesis-previtellogenesis switch in the fat body of adult female cockroaches. Our findings deepen the understanding of the roles of E93 in controlling reproduction in insects, and of E93 expression and functional evolution, which are proposed to have made crucial contributions to the origin of insect metamorphosis.

The zona pellucida protein piopio regulates the metamorphosis and reproduction in Tribolium castaneum.

The zona pellucida domain protein piopio (Pio) was only reported to mediate the adhesion of the apical epithelial surface and the overlying apical extracellular matrix in Drosophila melanogaster, but the developmental roles of Pio were poorly understood in insects. To address this issue, we comprehensively analyzed the function of Pio in Tribolium castaneum. Phylogenetic analysis indicated that pio exhibited one-to-one orthologous relationship among insects. T. castaneum pio had a 1236-bp ORF and contained eightexons. During development pio was abundantly expressed from larva to adult and lowly expressed at the late stage of embryo and adult, while it had more transcripts in the head, epidermis, and gut but fewer in the fat body of late-stage larvae. Knockdown of pio inhibited the pupation, eclosion, and reproduction of T. castaneum. The expression of vitellogenin 1 (Vg1), Vg2, and Vg receptor (VgR) largely decreased in pio-silenced female adults. Silencing pio increased the 20-hydroxyecdysone titer by upregulating phm and spo expression but decreased the juvenile hormone (JH) titer through downregulating JHAMT3 and promoting JHE, JHEH-r4, and JHDK transcription. These results suggested that Pio might regulate the metamorphosis and reproduction via modulating the ecdysone and JH metabolism in T. castaneum. This study found the novel roles of pio in insect metamorphosis and reproduction, and provided the new insights for analyzing other zona pellucida proteins functions in insects.

The embryonic role of juvenile hormone in the firebrat, Thermobia domestica, reveals its function before its involvement in metamorphosis.

To gain insights into how juvenile hormone (JH) came to regulate insect metamorphosis, we studied its function in the ametabolous firebrat, Thermobia domestica. Highest levels of JH occur during late embryogenesis, with only low levels thereafter. Loss-of-function and gain-of-function experiments show that JH acts on embryonic tissues to suppress morphogenesis and cell determination and to promote their terminal differentiation. Similar embryonic actions of JH on hemimetabolous insects with short germ band embryos indicate that JH's embryonic role preceded its derived function as the postembryonic regulator of metamorphosis. The postembryonic expansion of JH function likely followed the evolution of flight. Archaic flying insects were considered to lack metamorphosis because tiny, movable wings were evident on the thoraces of young juveniles and their positive allometric growth eventually allowed them to support flight in late juveniles. Like in Thermobia, we assume that these juveniles lacked JH. However, a postembryonic reappearance of JH during wing morphogenesis in the young juvenile likely redirected wing development to make a wing pad rather than a wing. Maintenance of JH then allowed wing pad growth and its disappearance in the mature juvenile then allowed wing differentiation. Subsequent modification of JH action for hemi- and holometabolous lifestyles are discussed.

Identification of the cytochrome P450 gene AccCYP6A13 in Apis cerana cerana and its response to environmental stress

Cytochrome P450 plays a crucial role in regulating insect growth, development, and resisting a variety of stresses. Insect metamorphosis and response to external stress are altered by deleting CYP450 genes. In this study, we identified and analyzed a novel gene of CYP450 family, AccCYP6A13, from Apis cerana cerana, and explored its role in the response of Apis cerana cerana to adverse external stressors. It was found that the expression of AccCYP6A13 was spatiotemporal specificity. The expression level increased with age and reached its highest value in the adult stage. The primarily expressiong location were legs, brain, and epidermis of honeybees. Stress conditions can affect the expression of AccCYP6A13 depending on treatment times. RNA interference experiments have shown that knocking down AccCYP6A13 reduces antioxidant activity and deactivates detoxification enzymes, resulting in oxidative damage accumulation and a decline in detoxification capability in bees, as well as inhibiting numerous antioxidant genes. Additionally, knockdown of the AccCYP6A13 gene in Apis cerana cerana resulted in increased sensitivity to pesticides and increased mortality when treated with neonicotinoid pesticides such as thiamethoxam. AccCYP6A13 overexpression in a prokaryotic system further confirmed its role in resistance to oxidative stress. To summarize, AccCYP6A13 may play an essential role in the normal development and response to environmental stress in Apis cerana cerana. Furthermore, this study contributed to the theoretical understanding of bee resistance biology.

MicroRNA miR-7-5p targets MARK2 to control metamorphosis in Galeruca daurica

The MARK2 gene, coding microtubule affinity-regulating kinase or serine/threonine protein kinase, is an important modulator in organism microtubule generation and cell polarity. However, its role in the metamorphosis of insects remains unknown. In this study, we found a conserved miRNA, miR-7-5p, which targets MARK2 to participate in the regulation of the larval-pupal metamorphosis in Galeruca daurica. The dual luciferase reporter assay showed that miR-7-5p interacted with the 3’ UTR of MARK2 and repressed its expression. The expression profiling of miR-7-5p and MARK2 displayed an opposite trend during the larval-adult development process. In in-vivo experiments, overexpression of miR-7-5p by injecting miR-7-5p agomir in the final instar larvae down-regulated MARK2 and up-regulated main ecdysone signaling pathway genes including E74, E75, ECR, FTZ-F1 and HR3, which was similar to the results from knockdown of MARK2 by RNAi. In contrast, repression of miR-7-5p by injecting miR-7-5p antagomir obtained opposite effects. Notably, both overexpression and repression of miR-7-5p in the final instar larvae caused abnormal molting and high mortality during the larval-pupal transition, and high mortality during the pupal-adult transition. The 20-hydroxyecdysone (20E) injection experiment showed that 20E up-regulated miR-7-5p whereas down-regulated MARK2. This study reveals that the accurate regulation of miRNAs and their target genes is indispensable for insect metamorphosis.

Mamestra brassicae Multiple Nucleopolyhedroviruses Prevents Pupation of Helicoverpa armigera by Regulating Juvenile Hormone Titer.

Baculovirus infection can prevent the pupation of insects. Juvenile hormone (JH) plays a vital role in regulating insect molting and metamorphosis. However, the molecular mechanism of baculovirus preventing the pupation of larvae by regulating the Juvenile hormone (JH) pathway is still unclear. In this study, we found that the Mamestra brassicae multiple nucleopolyhedroviruses (MbMNPV) infection prolonged the larval stage of fourth instar Helicoverpa armigera (H. armigera) by 0.52 d and caused an increase in JH titer. To identify the genes that contribute to the JH increase in H. armigera-MbMNPV interaction, we analyzed mRNA expression profiles of the fat bodies of H. armigera infected by MbMNPV. A total of 3637 differentially expressed mRNAs (DE-mRNAs) were filtered out through RNA-seq analysis. These DE-mRNAs were mainly enriched in Spliceosome, Ribosome biogenesis in eukaryotes, Aminoacyl-tRNA biosynthesis, Mismatch repair, and RNA degradation signaling pathway, which are related to the virus infection. Real-time PCR was used to verify the RNA sequencing results. To find out which genes caused the increase in JH titer, we analyzed all the DE-mRNAs in the transcriptome and found that the JHE and JHEH genes, which were related to JH degradation pathway, were down-regulated. JHE and JHEH genes in the larvae of MbMNPV-infected group were significantly down-regulated compared with the control group by RT-qPCR. We further proved that the JH is degraded by JHE in H. armigera larvae by RNAi, ELISA, RT-qPCR and bioassay, while the hydrolysis of JH by JHEH in H. armigera larvae can almost be ignored. Knocking down of HaJHE promoted the expression of the JH receptor gene Met and the downstream gene Kr-h1, and the replication of MbMNPV. This study clarified that JH is mainly degraded by JHE in H. armigera larvae. The MbMNPV infection of H. armigera larvae leads to the increase of JH titer by inhibiting the expression of JHE. The increase in JH titer promotes the expression of the JH receptor gene Met and the downstream gene Kr-h1, which prevents the pupation of H. armigera, and promotes MbMNPV replication. This study provides new insights into H. armigera and MbMNPV interaction mechanisms.