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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.

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.

Mlr1 (Mblk-1-related protein-1) and Mlr2 are mouse homologs of transcription factor Mblk-1 (Mushroom body large-type Kenyon cell-specific protein-1), which we originally identified from the honeybee brain. In the present study, aiming at identifying coregulator(s) of Mlr1 and Mlr2 from the mouse brain, we used yeast two-hybrid screening of mouse brain cDNA library to search for interaction partners of Mlr 1 and Mlr2, respectively. We identified nucleolar protein 4 (NOL4) splicing variants as major interaction partners for both Mlr1 and Mlr2. Among the three murine NOL4 splicing variants, we further characterized NOL4-S, which lacks an N-terminal part of NOL4-L, and NOL4-SΔ, which lacks nuclear localization signal (NLS)-containing domain of NOL4-S. A GST pull-down assay revealed that Mlr1 interacts with both NOL4-S and NOL4-SΔ, whereas Mlr2 interacts with NOL4-S, but not with NOL4-SΔ. These results indicate that the NLS-containing domain of NO4-S Is necessary for in vitro binding with Mlr2, but not for that with Mlr1. Furthermore, a luciferase assay using Schneider's Line 2 cells revealed that transactivation activity of Mlr1 was significantly suppressed by both NOL4-S and NOL4-SΔ, with almost complete suppression by NOL4-SΔ. In contrast, transactivation activity of Mlr2 was significantly suppressed by NOL4-S but rather activated by NOL4-SΔ. Our findings suggest that transactivation activities of Mlr1 and Mlr2 are differentially regulated by splicing variants of NOL4, which are expressed in a tissue-selective manner.

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.

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.