Lepidopteran Model Insect Research Articles

Successful oral RNA interference efficiency in the silkworm Bombyx mori through nanoparticle-shielded dsRNA delivery.

Double-stranded RNA (dsRNA) mediated RNA interference (RNAi) is a tool in functional gene study and pest control. However, RNAi efficiency in Lepidoptera is low compared to the RNAi sensitive Coleoptera. Previous studies on RNAi in the silkworm Bombyx mori, the lepidopteran model insect, were performed by injection only. Successful oral RNAi in the silkworm has never been reported yet. This study aims to develop a successful oral dsRNA delivery method to the silkworm larvae. Chitosan is an economical and biodegradable polymer. Chitosan/dsRNA nanoparticles were prepared by self-assembly. These nanoparticles were found to be stable when incubated in the midgut juice of the silkworm larvae, whereas naked dsRNA underwent complete degradation. Chitosan/dsRNA nanoparticles targeting various immune genes in oral administration to the silkworm larvae mediated significant knockdown of gene transcript. This silencing effect resulted in smaller larvae and cocoons when the silkworms were fed with Chitosan/dsRNA nanoparticles targeting BmToll9-2 gene, indicating that immune genes might be used as targets in pest control. Optimization of the chitosan/dsRNA nanoparticles maintained an RNAi effect from 3-5 days. The efficient RNAi was due to the persistence of nanoparticle-shielded dsRNA in the larvae. The above findings contribute to the first oral RNAi report in the silkworm, which facilitates the application of RNAi in insects and oral RNAi in pest control.

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.

Larval development of a parasitoid depends on host ecdysteroids

Parasitoids often exhibit high flexibility in their development depending on stages of their host at the parasitism, yet little is known about the mechanism underlying such flexibility. In the study, we evaluated the larval development time of the parasitoid Exorista sorbillans (Diptera: Tachinidae) on the lepidopteran model insect Bombyx mori (Lepidoptera: Bombycidae). We found that the development duration of E. sorbillans larvae parasitizing on the late-developmental silkworms was significantly shorter than that of the larvae parasitizing on the early-developmental hosts. Intriguingly, the 2nd-3rd instar molting of parasitoid always occurred when the ecdysteroid titers in the host were increased to higher levels. Furthermore, inhibiting the release of ecdysteroids to parasitic abdomen by thorax-abdomen ligation of the host only repressed the 2nd-instar growth and molting of E. sorbillans larvae, but had no effect on their pupation. Meanwhile, the ecdysone synthesis and 20-hydroxyecdysone (20 E) signaling in larval parasitoids were impeded after ligation treatment. Moreover, exogenous 20 E application could largely rescue the defect in 2nd instar growth and molting through stimulating ecdysone synthesis and signaling in E. sorbillans. Our results indicate that the parasitoid requires the host ecdysteroids to stimulate 20 E signaling and the subsequent 2nd-instar growth and molting. These findings will improve our understanding of the host utilization strategies of parasitoids, and contribute to the development of in vitro rearing procedures of tachinid parasitoids for biological control.

Transcriptome Analysis Reveals Antioxidant Defense Mechanisms in the Silkworm Bombyx mori after Exposure to Lead.

Lead (Pb) is a major source of heavy metal contamination, and poses a threat to biodiversity and human health. Elevated levels of Pb can hinder insect growth and development, leading to apoptosis via mechanisms like oxidative damage. The midgut of silkworms is the main organ exposed to heavy metals. As an economically important lepidopteran model insect in China, heavy metal-induced stress on silkworms causes considerable losses in sericulture, thereby causing substantial economic damage. This study aimed to investigate Pb-induced detoxification-related genes in the midgut of silkworms using high-throughput sequencing methods to achieve a deeper comprehension of the genes' reactions to lead exposure. This study identified 11,567 unigenes and 14,978 transcripts. A total of 1265 differentially expressed genes (DEGs) were screened, comprising 907 upregulated and 358 downregulated genes. Subsequently, Gene Ontology (GO) classification analysis revealed that the 1265 DEGs were distributed across biological processes, cellular components, and molecular functions. This suggests that the silkworm midgut may affect various organelle functions and biological processes, providing crucial clues for further exploration of DEG function. Additionally, the expression levels of 12 selected detoxification-related DEGs were validated using qRT-PCR, which confirmed the reliability of the RNA-seq results. This study not only provides new insights into the detoxification defense mechanisms of silkworms after Pb exposure, but also establishes a valuable foundation for further investigation into the molecular detoxification mechanisms in silkworms.

Effect of acute exposure of Hg on physiological parameters and transcriptome expression in silkworms (Bombyx mori).

Mercury (Hg) contamination poses a global threat to the environment, given its elevated ecotoxicity. Herein, we employed the lepidopteran model insect, silkworm (Bombyx mori), to systematically investigate the toxic effects of Hg-stress across its growth and development, histomorphology, antioxidant enzyme activities, and transcriptome responses. High doses of Hg exposure induced evident poisoning symptoms, markedly impeding the growth of silkworm larvae and escalating mortality in a dose-dependent manner. Under Hg exposure, the histomorphology of both the midgut and fat body exhibited impairments. Carboxylesterase (CarE) activity was increased in both midgut and fat body tissues responding to Hg treatment. Conversely, glutathione S-transferase (GST) levels increased in the fat body but decreased in the midgut. The transcriptomic analysis revealed that the response induced by Hg stress involved multiple metabolism processes. Significantly differently expressed genes (DEGs) exhibited strong associations with oxidative phosphorylation, nutrient metabolisms, insect hormone biosynthesis, lysosome, ribosome biogenesis in eukaryotes, and ribosome pathways in the midgut or the fat body. The findings implied that exposure to Hg might induce the oxidative stress response, attempting to compensate for impaired metabolism. Concurrently, disruptions in nutrient metabolism and insect hormone activity might hinder growth and development, leading to immune dysfunction in silkworms. These insights significantly advance our theoretical understanding of the potential mechanisms underlying Hg toxicity in invertebrate organisms.

ABHD5 regulates midgut-specific lipid homeostasis in Bombyx mori.

Lipids are an important energy source and are utilized as substrates for various physiological processes in insects. Comparative gene identification58 (CGI-58), also known as α/β hydrolase domain-containing5 (ABHD5), is a highly conserved and multifunctional gene involved in regulating lipid metabolism and cellular energy balance in many organisms. However, the biological functions of ABHD5 in insects are poorly understood. In the current study, we describe the identification and characterization of the ABHD5 gene in the lepidopteran model insect, Bombyx mori. The tissue expression profile investigated using quantitative reverse transcription polymerase chain reaction (RT-qPCR) reveals that BmABHD5 is widely expressed in all tissues, with particularly high levels found in the midgut and testis. A binary transgenic CRISPR/Cas9 system was employed to conduct a functional analysis of BmABHD5, with the mutation of BmABHD5 leading to the dysregulation of lipid metabolism and excessive lipid accumulation in the larval midgut. Histological and physiological analysis further reveals a significant accumulation of lipid droplets in the midgut of mutant larvae. RNA-seq and RT-qPCR analysis showed that genes related to metabolic pathways were significantly affected by the absence of BmABHD5. Altogether, our data prove that BmABHD5 plays an important role in regulating tissue-specific lipid metabolism in the silkworm midgut.

The mechanoreceptor Piezo is required for spermatogenesis in Bombyx mori

BackgroundThe animal sperm shows high diversity in morphology, components, and motility. In the lepidopteran model insect, the silkworm Bombyx mori, two types of sperm, including nucleate fertile eupyrene sperm and anucleate unfertile apyrene sperm, are generated. Apyrene sperm assists fertilization by facilitating the migration of eupyrene spermatozoa from the bursa copulatrix to the spermatheca. During spermatogenesis, eupyrene sperm bundles extrude the cytoplasm by peristaltic squeezing, while the nuclei of the apyrene sperm bundles are discarded with the same process, forming matured sperm.ResultsIn this study, we describe that a mechanoreceptor BmPiezo, the sole Piezo ortholog in B. mori, plays key roles in larval feeding behavior and, more importantly, is essential for eupyrene spermatogenesis and male fertility. CRISPR/Cas9-mediated loss of BmPiezo function decreases larval appetite and subsequent body size and weight. Immunofluorescence analyses reveal that BmPiezo is intensely localized in the inflatable point of eupyrene sperm bundle induced by peristaltic squeezing. BmPiezo is also enriched in the middle region of apyrene sperm bundle before peristaltic squeezing. Cytological analyses of dimorphic sperm reveal developmental arrest of eupyrene sperm bundles in BmPiezo mutants, while the apyrene spermatogenesis is not affected. RNA-seq analysis and q-RT-PCR analyses demonstrate that eupyrene spermatogenic arrest is associated with the dysregulation of the actin cytoskeleton. Moreover, we show that the deformed eupyrene sperm bundles fail to migrate from the testes, resulting in male infertility due to the absence of eupyrene sperm in the bursa copulatrix and spermatheca.ConclusionsIn conclusion, our studies thus uncover a new role for Piezo in regulating spermatogenesis and male fertility in insects.

GASZ is indispensable for gametogenesis in the silkworm, Bombyx mori.

The prominent role of the P-element induced wimpy testis (PIWI)-interacting RNA (piRNA) pathway in animals is to silence transposable elements and maintain genome stability, ensuring proper gametogenesis in gonads. GASZ (Germ cell protein with Ankyrin repeats, Sterile alpha motif, and leucine Zipper) is an evolutionarily conserved protein located on the outer mitochondrial membrane of germ cells and plays vital roles in the piRNA pathway and spermatogenesis in mammals. In the model insect Drosophila melanogaster, GASZ is essential for piRNA biogenesis and oogenesis, whereas its biological functions in non-drosophilid insects are still unknown. Here, we describe a comprehensive investigation of GASZ functions in the silkworm, Bombyx mori, a lepidopteran model insect, by using a binary transgenic CRISPR/Cas9 system. The BmGASZ mutation did not affect growth and development, but led to sterility in both males and females. Eupyrene sperm bundles of mutant males exhibited developmental defects, while the apyrene sperm bundles were normal, which were further confirmed through double copulation experiments with sex-lethal mutants, which males possess functional eupyrene sperm and abnormal apyrene sperm. In female mutant moths, ovarioles were severely degenerated and the eggs in ovarioles were deformed compared with that of wild type (WT). Further RNA-seq and RT-qPCR analysis revealed that amounts of piRNAs and transposon expression were dysregulated in gonads of mutants. In summary, this study has demonstrated vital roles of BmGASZ in gametogenesis through regulating the piRNA pathway in B. mori.

Uridine diphosphate glucosyltransferase is vital for fenpropathrin resistance in Bombyx mori (Lepidoptera).

The silkworm (Bombyx mori) is an important model lepidopteran insect and can be used to identify pesticide resistance-related genes of great significance for biological control of pests. Uridine diphosphate glucosyltransferases (UGTs), found in all organisms, are the main secondary enzymes involved in the metabolism of heterologous substances. However, it remains uncertain if silkworm resistance to fenpropathrin involves UGT. This study observes significant variations in BmUGT expression among B. mori strains with variable fenpropathrin resistance post-feeding, indicating BmUGT's role in fenpropathrin detoxification. Knockdown of BmUGT with RNA interference and overexpression of BmUGT significantly decreased and increased BmN cell activity, respectively, indicating that BmUGT plays an important role in the resistance of silkworms to fenpropathrin. In addition, fenpropathrin residues were significantly reduced after incubation for 12 h with different concentrations of a recombinant BmUGT fusion protein. Finally, we verified the conservation of UGT to detoxify fenpropathrin in Spodoptera exigua: Its resistance to fenpropathrin decreased significantly after knocking down SeUGT. In a word, UGT plays an important role in silkworm resistance to fenpropathrin by directly degrading the compound, a function seen across other insects. The results of this study are of great significance for breeding silkworm varieties with high resistance and for biological control of pests.

Lipid homeostasis is essential for oogenesis and embryogenesis in the silkworm, Bombyx mori

Reproduction, a fundamental feature of all known life, closely correlates with energy homeostasis. The control of synthesizing and mobilizing lipids are dynamic and well-organized processes to distribute lipid resources across tissues or generations. However, how lipid homeostasis is precisely coordinated during insect reproductive development is poorly understood. Here we describe the relations between energy metabolism and reproduction in the silkworm, Bombyx mori, a lepidopteran model insect, by using CRISPR/Cas9-mediated mutation analysis and comprehensively functional investigation on two major lipid lipases of Brummer (BmBmm) and hormone-sensitive lipase (BmHsl), and the sterol regulatory element binding protein (BmSrebp). BmBmm is a crucial regulator of lipolysis to maintain female fecundity by regulating the triglyceride (TG) storage among the midgut, the fat body, and the ovary. Lipidomics analysis reveals that defective lipolysis of females influences the composition of TG and other membrane lipids in the BmBmm mutant embryos. In contrast, BmHsl mediates embryonic development by controlling sterol metabolism rather than TG metabolism. Transcriptome analysis unveils that BmBmm deficiency significantly improves the expression of lipid synthesis-related genes including BmSrebp in the fat body. Subsequently, we identify BmSrebp as a key regulator of lipid accumulation in oocytes, which promotes oogenesis and cooperates with BmBmm to support the metabolic requirements of oocyte production. In summary, lipid homeostasis plays a vital role in supporting female reproductive success in silkworms.

CRISPR-Cas13-mediated RNA editing in the silkworm Bombyx mori.

The CRISPR-Cas13 system, an RNA-guided editing tool, has emerged as a highly efficient and stable RNA editing technique. Although the CRISPR-Cas13 system has been developed in several insect species, its application in lepidopterans has not yet been reported. In the present study, we evaluated the RNA cleavage activity of the CRISPR-Cas13 system in the silkworm ( Bombyx mori), a model lepidopteran insect, both ex vivo and in vivo. We established two stable silkworm BmE cell lines expressing PspCas13b and CasRx, respectively. Further analysis demonstrated that both PspCas13b and CasRx effectively down-regulated the transcription of exogenously-introduced target and endogenous genes in these cell lines. In addition, we generated two transgenic silkworm strains, one expressing CasRx and the other expressing RNA-guided CRISPR RNA targeting Sex combs reduced ( Scr). Further crossing experiments showed that CasRx induced a down-regulation of Scr transcription in silkworms, which impaired systemic growth of larvae. Overall, this study demonstrated that the CRISPR-Cas13 RNA editing system works efficiently in the silkworm, providing a potential alternative approach for RNA manipulation in lepidopteran insects.

Single-cell transcriptomes provide insights into expansion of glial cells in Bombyx mori.

The diversity of cell types in the brain and how these change during different developmental stages, remains largely unknown. The life cycle of insects is short and goes through 4 distinct stages including embryonic, larval, pupal, and adult stages. During postembryonic life, the larval brain transforms into a mature adult version after metamorphosis. The silkworm, Bombyx mori, is a lepidopteran model insect. Here, we characterized the brain cell repertoire of larval and adult B. mori by obtaining 50708 single-cell transcriptomes. Seventeen and 12 cell clusters from larval and adult brains were assigned based on marker genes, respectively. Identified cell types include Kenyon cells, optic lobe cells, monoaminergic neurons, surface glia, and astrocyte glia. We further assessed the cell type compositions of larval and adult brains. We found that the transition from larva to adult resulted in great expansion of glial cells. The glial cell accounted for 49.8% of adult midbrain cells. Compared to flies and ants, the mushroom body kenyon cell is insufficient in B. mori, which accounts for 5.4% and 3.6% in larval and adult brains, respectively. Analysis of neuropeptide expression showed that the abundance and specificity of expression varied among individual neuropeptides. Intriguingly, we found that ion transport peptide was specifically expressed in glial cells of larval and adult brains. The cell atlas dataset provides an important resource to explore cell diversity, neural circuits and genetic profiles.

The promoting effects of pyriproxyfen on autophagy and apoptosis in silk glands of non-target insect silkworm, Bombyx mori

Pyriproxyfen is a juvenile hormone analogue. The physiological effects of its low-concentration drift during the process of controlling agricultural and forestry pests on non-target organisms in the ecological environment are unpredictable, especially the effects on organs that play a key role in biological function are worthy of attention. The silk gland is an important organ for silk-secreting insects. Herein, we studied the effects of trace pyriproxyfen on autophagy and apoptosis of the silk gland in the lepidopteran model insect, Bombyx mori (silkworm). After treating fifth instar silkworm larvae with pyriproxyfen for 24 h, we found significant shrinkage, vacuolization, and fragmentation in the posterior silk gland (PSG). In addition, the results of autophagy-related genes of ATG8 and TUNEL assay also demonstrated that autophagy and apoptosis in the PSG of the silkworm was induced by pyriproxyfen. RNA-Seq results showed that pyriproxyfen treatment resulted in the activation of juvenile hormone signaling pathway genes and inhibition of 20-hydroxyecdysone (20E) signaling pathway genes. Among the 1808 significantly differentially expressed genes, 796 were upregulated and 1012 were downregulated. Among them, 30 genes were identified for autophagy-related signaling pathways, such as NOD-like receptor signaling pathway and mTOR signaling pathway, and 30 genes were identified for apoptosis-related signaling pathways, such as P53 signaling pathway and TNF signaling pathway. Further qRT-PCR and in vitro gland culture studies showed that the autophagy-related genes Atg5, Atg6, Atg12, Atg16 and the apoptosis-related genes Aif, Dronc, Dredd, and Caspase1 were responsive to the treatment of pyriproxyfen, with transcription levels up-regulated from 24 to 72 h. In addition, ATG5, ATG6, and Dronc genes had a more direct response to pyriproxyfen treatment. These results suggested that pyriproxyfen treatment could disrupt the hormone regulation in silkworms, promoting autophagy and apoptosis in the PSG. This study provides more evidence for the research on the damage of juvenile hormone analogues to non-target organisms or organs in the environment, and provides reference information for the scientific and rational use of juvenile hormone pesticides.

Insect growth-regulating activity of 1-benzyl-2-methylbenzimidazole derivatives on silkworms

Derivatives of 1-benzyl-2-methylbenzimidazoles (BMBIs) were synthesized to evaluate their biological activities against Bombyx mori, a lepidopteran model insect. Synthesized BMBIs exhibited two different biological activities: inhibition of development and acute lethality. From a structural perspective, the activity varied with the position of the substitutions on the 1-benzyl moiety; BMBIs with substitutions on the 2 and/or 4 positions had comparatively high activity in comparison with those with substitutions on the 3-position. There was more activity for the inhibition of development with low doses, and more for acute lethality with high doses. The activity was also affected by the applied stage, that is, application in the 4th instar mostly interfered the larval molting or pupation, whereas that in the 3rd instar caused more acute mortality. Taken together, these results suggest that BMBIs have multiple modes of action.

Gene expression and immunolocalization of chitin deacetylase BmCDA2 in silkworm

Deacetylation of chitin is closely related to insect development and metamorphosis. Chitin deacetylase (CDA) is a key enzyme in the process. However, to date, the CDAs of Bombyx mori (BmCDAs), which is a model Lepidopteran insect, were not well studied. In order to better understand the role of BmCDAs in the metamorphosis and development of silkworm, the BmCDA2 which is highly expressed in epidermis was selected to study by bioinformatics methods, protein expression purification and immunofluorescence localization. The results showed that the two mRNA splicing forms of BmCDA2, namely BmCDA2a and BmCDA2b, were highly expressed in the larval and pupal epidermis, respectively. Both genes had chitin deacetylase catalytic domain, chitin binding domain and low density lipoprotein receptor domain. Western blot showed that the BmCDA2 protein was mainly expressed in the epidermis. Moreover, fluorescence immunolocalization showed that BmCDA2 protein gradually increased and accumulated with the formation of larval new epidermis, suggesting that BmCDA2 may be involved in the formation or assembly of larval new epidermis. The results increased our understandings to the biological functions of BmCDAs, and may facilitate the CDA study of other insects.

Metabonomic Analysis of Silkworm Midgut Reveals Differences between the Physiological Effects of an Artificial and Mulberry Leaf Diet.

Bombyx mori is a model lepidopteran insect of great economic value. Mulberry leaves are its only natural food source. The development of artificial diets can not only resolve the seasonal shortage of mulberry leaves but also enable changes to be made to the feed composition according to need. Metabolomic differences between the midguts of male and female silkworms fed either on fresh mulberry leaves or an artificial diet were studied using liquid chromatography-mass spectrography (LC-MS/MS) analysis. A total of 758 differential metabolites were identified. Our analysis showed that they were mainly involved in disease resistance and immunity, silk quality, and silkworm growth and development. These experimental results provide insights into the formulation of optimized artificial feed for silkworms.

Highly accurate genome assembly of an improved high-yielding silkworm strain, Nichi01.

The silkworm (Bombyx mori) is an important lepidopteran model insect and an industrial domestic animal traditionally used for silk production. Here, we report the genome assembly of an improved Japanese strain Nichi01, in which the cocoon yield is comparable to that of commercial silkworm strains. The integration of PacBio Sequel II long-read and ddRAD-seq-based high-density genetic linkage map achieved the highest quality genome assembly of silkworms to date; 22 of the 28 pseudomolecules contained telomeric repeats at both ends, and only four gaps were present in the assembly. A total of 452 Mbp of the assembly with an N50 of 16.614 Mbp covered 99.3% of the complete orthologs of the lepidopteran core genes. Although the genome sequence of Nichi01 and that of the previously reported low-yielding tropical strain p50T assured their accuracy in most regions, we corrected several regions, misassembled in p50T, in our assembly. A total of 18,397 proteins were predicted using over 95 Gb of mRNA-seq derived from 10 different organs, covering 96.9% of the complete orthologs of the lepidopteran core genes. The final assembly and annotation files are available in KAIKObase (https://kaikobase.dna.affrc.go.jp/index.html) along with a genome browser and BLAST searching service, which would facilitate further studies and the breeding of silkworms and other insects.

CRISPR-Cas9-Mediated Mutation of Methyltransferase METTL4 Results in Embryonic Defects in Silkworm Bombyx mori

DNA N6-methyladenine (6mA) has recently been found to play regulatory roles in gene expression that links to various biological processes in eukaryotic species. The functional identification of 6mA methyltransferase will be important for understanding the underlying molecular mechanism of epigenetic 6mA methylation. It has been reported that the methyltransferase METTL4 can catalyze the methylation of 6mA; however, the function of METTL4 remains largely unknown. In this study, we aim to investigate the role of the Bombyx mori homolog METTL4 (BmMETTL4) in silkworm, a lepidopteran model insect. By using CRISPR-Cas9 system, we somatically mutated BmMETTL4 in silkworm individuates and found that disruption of BmMETTL4 caused the developmental defect of late silkworm embryo and subsequent lethality. We performed RNA-Seq and identified that there were 3192 differentially expressed genes in BmMETTL4 mutant including 1743 up-regulated and 1449 down-regulated. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes analyses showed that genes involved in molecular structure, chitin binding, and serine hydrolase activity were significantly affected by BmMETTL4 mutation. We further found that the expression of cuticular protein genes and collagens were clearly decreased while collagenases were highly increased, which had great contributions to the abnormal embryo and decreased hatchability of silkworm. Taken together, these results demonstrated a critical role of 6mA methyltransferase BmMETTL4 in regulating embryonic development of silkworm.

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.

The morphogen Hedgehog is essential for proper adult morphogenesis in Bombyx mori

The well-known morphogen Hedgehog (Hh) is indispensable for embryo patterning and organ development from invertebrates to vertebrates. The role of Hh signaling pathway has been extensively investigated in the model organism Drosophila melanogaster, whereas its biological functions are still poorly understood in non-drosophilid insects. In the current study, we describe comprehensive investigation of Hh biological roles in the model lepidopteran insect Bombyx mori by using both CRISPR/Cas9-mediated gene ablation and Gal4/UAS-mediated ectopic expression. Direct injection of Cas9 protein and Hh-specific sgRNAs into preblastoderm embryos induced complete lethality. In contrast, Hh mutants obtained by the binary transgenic CRISPR/Cas9 system showed no deleterious phenotypes during embryonic and larval stages. However, mutants showed abnormalities from the pupal stage and most of adult body appendages exhibited severe developmental defects. Molecular analysis focused on wing development reveal that Hh signaling, Imd signaling and Wnt signaling pathways were distorted in Hh mutant wings. Ectopic expression by using the binary Gal4/UAS system induce early larval lethality. On contrary, moderate overexpression of Hh by using a unitary transgenic system resulted in severe defects in adult leg and antenna development. Our data directly provide genetic evidence that Hh plays vital roles in imaginal discs development and proper adult morphogenesis in B. mori.