Search

Although genome editing techniques have made significant progress, introducing exogenous genes into the genome through knock-in remains a challenge in many organisms. In silkworm Bombyx mori , TALEN-mediated knock-in methods have been established. However, difficulties in construction and limitations of the target sequence have hindered the application of these methods. In the present study, we verified several CRISPR/Cas9-mediated knock-in methods to expand the application of gene knock-in techniques and found that the short single-stranded oligodeoxynucleotide (ssODN)-mediated method is the most effective in silkworms. Using ssODN-mediated methods, we established knock-in silkworm strains that harbor an attP sequence, a 50 bp phiC31 integrase recognition site, at either the BmHr38 ( Hormone receptor 38 ) or Bmdsx ( doublesex ) locus. Additionally, we found that the long ssODN (lsODN)-mediated method successfully introduced the GAL4 gene at the doublesex locus in embryos. The present study provides valuable information on CRISPR/Cas9-mediated knock-in methods in silkworms, expanding the utility of genome editing techniques in insects and paving the way for analyzing gene and genome function in silkworms.

Sexual dimorphisms of the brain play essential roles in successful reproduction. Silkmoth Bombyx mori exhibits extensive sexual differences in sexual behavior, as well as their morphology. Although the neural circuits that transmit information about sex pheromone in the male brain are extensively analyzed, the molecular mechanisms that regulate their development are still elusive. In the present study, we focused on the silkmoth ortholog of fruitless (fru) as a candidate gene that regulates sexual dimorphisms of the brain. fru transcripts were expressed from multiple promoters in various tissues, and brain-specific transcripts were sex-specifically spliced, in a manner similar to Drosophila. Interestingly, fru was highly expressed in the adult female brain and the male larval testis. Analysis of CRISPR/Cas9-mediated fru knockout strains revealed that fru plays important roles in survival during late larval and pupal stages, testis development, and adult sexual behavior. fru mutant males exhibited highly reduced levels of courtship and low copulation rate, indicating that fru plays significant roles in the sexual behavior of silkmoths, although it is not absolutely necessary for copulation. In the fru mutant males, sexually dimorphic pattern of the odorant receptor expression was impaired, possibly causing the defects in courtship behavior. These results provide important clues to elucidate the development of sexual dimorphisms of silkmoth brains, as well as the evolution of fruitless gene in insects., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Sexual differences in behavior are generated by sexually dimorphic neural circuits in animals. In insects, a highly conserved sex-determining gene doublesex (dsx) plays essential roles in the development of sexual dimorphisms. In the present study, to elucidate the neural basis of sexual differences in behaviors of silkmoth Bombyx mori, we investigated Bombyx mori dsx (Bmdsx) expression in the brains through development. In the brain, Bmdsx was differentially expressed in sex- and developmental stage-dependent manners. BmDSX protein-expressing cells were located in the dorsomedial region of the pupal and adult brains, and constituted two and one neural clusters in males and females, respectively. The number of BmDSX-positive cells was developmentally regulated and peaked at the early to middle pupal stages, suggesting that the sexually dimorphic neural circuits are established during this period. The detection of a neural activity marker protein BmHR38 suggested that the BmDSX-positive cells are not active during sexual behavior in both male and female moths, even though the cells in the vicinity of the BmDSX-positive cell clusters are active. These results imply that Bmdsx plays roles in the development of sexually dimorphic neural circuits, but the neural circuits are not related to sexual behavior in silkmoths., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

In the central nervous system of insects, motor patterns are generated in the thoracic ganglia under the control of brain, where sensory information is integrated and behavioral decisions are made. Previously, we established neural activity-mapping methods using an immediate early gene, BmHr38, as a neural activity marker in the brain of male silkmoth Bombyx mori. In the present study, to gain insights into neural mechanisms of motor-pattern generation in the thoracic ganglia, we investigated expression of BmHr38 in response to sex pheromone-induced courtship behavior. Levels of BmHr38 expression were strongly correlated between the brain and thoracic ganglia, suggesting that neural activity in the thoracic ganglia is tightly controlled by the brain. In situ hybridization of BmHr38 revealed that 20-30% of thoracic neurons are activated by courtship behavior. Using serial sections, we constructed a comprehensive map of courtship behaviorinduced activity in the thoracic ganglia. These results provide important clues into how complex courtship behavior is generated in the neural circuits of thoracic ganglia.

Juvenile hormone (JH) plays important roles in insect development and physiology. JH titer is tightly regulated to coordinately adjust systemic physiology and development. Although control of JH titer is explained by the expression of JH biosynthetic enzymes in the corpora allata (CA), molecular mechanisms that regulate the expression of these genes remain elusive. In the present study, to identify novel regulators of JH biosynthetic genes, we conducted a gene expression screen using the CA and corpora cardiaca (CC) of the silkworm, Bombyx mori, in the JH synthesis period. We identified seven candidate genes and characterized their properties through extensive expression analyses. Of these candidates, we found that a novel gene, which encodes type II phosphatidylinositol 3,4-bisphosphate [PI(3,4)P 2 ] 4-phosphatase, shows highly correlated expression with JH titer. In addition, expression of this gene was strongly upregulated by starvation, when JH biosynthetic enzyme genes are concurrently upregulated. These results, for the first time, imply possible involvement of phosphoinositol signal in regulation of JH biosynthesis, providing novel insights into molecular mechanisms of nutrition-dependent regulation of JH biosynthesis.

Silkmoth, Bombyx mori, is one of the important model insects in which transgenic techniques and the GAL4/UAS system are applicable. However, due to cytotoxicity and low transactivation activity of GAL4, effectiveness of the GAL4/UAS system and its application in B. mori are still limited. In the present study, we refined the previously reported UAS vector by exploiting transcriptional and translational enhancers, and achieved 200-fold enhancement of reporter GFP fluorescence in the GAL4/UAS system. Enhanced protein expression of membrane-targeted GFP and calcium indicator protein (GCaMP5G) drastically improved visualization of fine neurite structures and neural activity, respectively. Also, with the refined system, we generated a transgenic strain that expresses tetanus toxin light chain (TeTxLC), which blocks synaptic transmission, under the control of GAL4. Ectopic TeTxLC expression in the sex pheromone receptor neurons inhibited male courtship behavior, proving effectiveness of TeTxLC on loss-of-function analyses of neural circuits. In addition, suppression of prothoracicotropic hormone (PTTH) or insulin-like peptide (bombyxin) secretion impaired developmental timing and growth rate, respectively. Furthermore, we revealed that larval growth is sex-differentially regulated by these peptide hormones. The present study provides important technical underpinnings of transgenic approaches in silkmoths and insights into mechanisms of postembryonic development in insects.

Background: Silkmoth, Bombyx mori, is an ideal model insect for investigating the neural mechanisms underlying sex pheromone-induced innate behavior. Although transgenic techniques and the GAL4/UAS system are well established in the silkmoth, genetic tools useful for investigating brain function at the neural circuit level have been lacking., Results: In the present study, we established silkmoth strains in which we could visualize neural projections (UAS-mCD8GFP) and cell nucleus positions (UAS-GFP.nls), and manipulate neural excitability by thermal stimulation (UAS-dTrpA1). In these strains, neural projections and nucleus position were reliably labeled with green fluorescent protein in a GAL4-dependent manner. Further, the behavior of silkworm larvae and adults could be controlled by GAL4-dependent misexpression of dTrpA1. Ubiquitous dTrpA1 misexpression led both silkmoth larvae and adults to exhibit seizure-like phenotypes in a heat stimulation-dependent manner. Furthermore, dTrpA1 misexpression in the sex pheromone receptor neurons of male silkmoths allowed us to control male sexual behavior by changing the temperature. Thermally stimulated male silkmoths exhibited full sexual behavior, including wing-flapping, orientation, and attempted copulation, and precisely approached a thermal source in a manner similar to male silkmoths stimulated with the sex pheromone., Conclusion: These findings indicate that a thermogenetic approach using dTrpA1 is feasible in Lepidopteran insects and thermogenetic analysis of innate behavior is applicable in the silkmoth. These tools are essential for elucidating the relationships between neural circuits and function using neurogenetic methods.

Many insects exhibit stereotypic instinctive behavior [1-3], but the underlying neural mechanisms are not well understood due to difficulties in detecting brain activity in freely moving animals. Immediate early genes (IEGs), such as c-fos, whose expression is transiently and rapidly upregulated upon neural activity, are powerful tools for detecting behavior-related neural activity in vertebrates [4, 5]. In insects, however, this powerful approach has not been realized because no conserved IEGs have been identified. Here, we identified Hr38 as a novel IEG that is transiently expressed in the male silkmoth Bombyx mori by female odor stimulation. Using Hr38 expression as an indicator of neural activity, we mapped comprehensive activity patterns of the silkmoth brain in response to female sex pheromones. We found that Hr38 can also be used as a neural activity marker in the fly Drosophila melanogaster. Using Hr38, we constructed a neural activity map of the fly brain that partially overlaps with fruitless (fru)-expressing neurons in response to female stimulation. These findings indicate that Hr38 is a novel and conserved insect neural activity marker gene that will be useful for a wide variety of neuroethologic studies., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

The insect brain secretes prothoracicotropic hormone (PTTH), which stimulates the prothoracic gland to synthesize ecdysone. The active metabolite of ecdysone, 20-hydroxyecdysone (20E), works through ecdysone receptor (EcR) and ultraspiracle (USP) to initiate molting and metamorphosis by regulating downstream genes. Previously, we found that EcR was expressed in the PTTH-producing neurosecretory cells (PTPCs) in larval brain of the silkworm Bombyx mori, suggesting that PTPCs function as the master cells of development under the regulation of 20E. To gain a better understanding of the molecular mechanism of the 20E control of PTPCs, we performed a comprehensive screening of genes induced by 20E using DNA microarray with brains of day-2 fifth instar silkworm larvae. Forty-one genes showed greater than twofold changes caused by artificial application of 20E. A subsequent semiquantitative screening identified ten genes upregulated by 20E, four of which were novel or not previously identified as 20E-response genes. Developmental profiling determined that two genes, UP4 and UP5, were correlated with the endogenous ecdysteroid titer. Whole-mount in situ hybridization showed exclusive expression of these two genes in two pairs of cells in the larval brain in response to 20E-induction, suggesting that the cells are PTPCs. BLAST searches revealed that UP4 and UP5 are Bombyx homologs of vrille and tarsal-less, respectively. The present study identifies 20E-induced genes that may be involved in the ecdysone signal hierarchies underlying pupal-adult development and/or the 20E regulation of PTPCs.

Sexually dimorphic neural circuits are essential for reproductive behaviour. The molecular basis of sexual dimorphism in the silkworm moth (Bombyx mori) brain, however, is unclear. We conducted cDNA subtraction screening and identified nervous wreck (Bmnwk), a synaptic growth regulatory gene, whose expression is higher in the male brain than in the female brain of the silkworm. Bmnwk was preferentially expressed in the brain at the late pupae and adult stages. In situ hybridization revealed that Bmnwk is highly expressed in the optic lobe of the male moth brain. These findings suggest that Bmnwk has a role in the development and/or maintenance of the optic lobe in the male silkworm brain., (© 2011 The Authors. Insect Molecular Biology © 2011 The Royal Entomological Society.)

Insulin family peptide members play key roles in regulating growth, metabolism, and reproduction. Bombyxin is an insulin-related peptide of the silkmoth Bombyx mori. We analyzed the full genome of B. mori and identified five novel bombyxin families, V to Z. We characterized the genomic organization and chromosomal location of the novel bombyxin family genes. In contrast to previously identified bombyxin genes, bombyxin-V and -Z genes had intervening introns at almost the same positions as vertebrate insulin genes. We performed reverse transcription-polymerase chain reaction and in situ hybridization in different tissues and developmental stages to observe their temporal and spatial expression patterns. The newly identified bombyxin genes were expressed in diverse tissues: bombyxin-V, -W, and -Y mRNAs were expressed in the brain and bombyxin-X mRNA in fat bodies. Bombyxin-Y gene was expressed in both brain and ovary of larval stages. High level of bombyxin-Z gene expression in the follicular cells may suggest its function in reproduction. The presence of a short C-peptide domain and an extended A chain domain, and high expression of bombyxin-X gene in the fat body cells during non-feeding stages suggest its insulin-like growth factor-like function. These results suggest that the bombyxin genes originated from a common ancestral gene, similar to the vertebrate insulin gene, and evolved into a diverse gene family with multiple functions.

Sexually dimorphic neural circuits are essential for the reproductive behavior. The molecular basis underling the sexual dimorphism, however, is still elusive in the brains of insects. To identify genes with sex-differential expression in the brain of silkworm moths, we performed fluorescent differential display screening and identified a novel gene, termed Fben-1 (Female-brain expressed noncoding RNA-1), whose expression is preferential to the female brain. Fben-1 cDNA sequences contained no significant open-reading frames and comprised a sex-differential transcript composition. Expression of Fben-1 was developmentally regulated and predominant in adult female moth brains. In situ hybridization revealed that Fben-1 is mainly expressed in the cells around the mushroom bodies, a higher brain center of the insect brain. In addition, Fben-1 transcripts were localized exclusively in the nuclei of these cells. This is the first report that a long nuclear noncoding RNA is expressed in a sex-differential manner in the higher center of insect brains. Our results suggest the possible involvement of nuclear noncoding RNA in sexually dimorphic brain functions., (Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.)