TakahitoWatanabe/published_papers/54122249 Shintaro Inoue Ayane Shimamura Kenichiro Koshiyama Sayuri Tomonari Taiyo Takada Yoshiyasu Ishimaru Katsuyuki Miyawaki Akira Takahashi Takeshi Nikawa Sumihare Noji Takahito Watanabe Taro Mito Gene misexpression system for functional studies of genes involved in cuticle development in the cricket Gryllus bimaculatus. The insect epidermis possesses the potential to generate remarkable diversity in exoskeletal cuticle traits, including colour, thickness and mechanical properties. Genetic manipulation is essential for investigating the molecular mechanisms; however, spatiotemporally controlled gene overexpression remains technically challenging despite the widespread use of loss-of-function approaches in many insects. In this study, we established an epidermal gene misexpression system in the model hemimetabolous insect Gryllus bimaculatus by inserting a gene expression cassette into the evolutionarily conserved yellow gene, which is involved in melanin synthesis, using genome editing. Insertion of the EGFP expression cassette into yellow gene resulted in fluorescence in the epidermis of pre-hatching embryos and post-moult individuals across developmental stages, corresponding to melanin pigmentation. Using this system, we induced the misexpression of Arylalkylamine N-acetyltransferase (aaNAT), an enzyme involved in the synthesis of N-acetyldopamine (NADA)-sclerotin, which serves as both a white pigment and a crosslinker in the cuticle. Compared to the background strain, the aaNAT misexpression strain exhibited a brighter body coloration, indicating that Gryllus aaNAT has the function of producing white NADA sclerotin and suppressing melanin pigment production. Wing thickness exhibited no substantial alteration, whereas a significant reduction in puncture resistance was observed. Collectively, these results suggest that an imbalance in the molecular components essential for cuticular crosslinking leads to alterations in the mechanical properties of the cuticle. This system serves as a gain-of-function analysis tool for cuticle research and has the potential to be applicable across a wide range of insect species, thereby helping to elucidate cuticle diversity. Insect molecular biology null null 20260517 10.1111/imb.70044 42144849 TakahitoWatanabe/published_papers/53970061 Minami Nakamura Hiroki Matsuki Kanon Hagiwara Saki Okuno Kosuke Kataoka Seiya Ashikari Kensuke Ikeda Katsuyuki Miyawaki Takahito Watanabe Shintaro Inoue Taro Mito Toru Asahi Akira Takahashi Takayuki Uchida Takeshi Nikawa Identification of Paramyosin as a Novel Allergen in Edible Cricket Protein. Edible insects have recently gained attention as a sustainable protein source. In a dietary intervention clinical trial, participants consumed one of the following for 28 d: wheat bread, soy protein bread, cricket protein bread, or a mixed bread containing both cricket and soy proteins. Although individuals with known allergies to soy, crustaceans, or wheat were excluded, several participants developed allergic dermatitis (erythema with itching) after consuming cricket protein bread. This study, therefore, aims to identify potential allergens in cricket protein that may have triggered these reactions. To do so, we analyzed serum samples from an individual who developed allergic dermatitis after consuming the cricket protein or cricket-soy protein mixed bread. Our results revealed a novel allergen candidate. In contrast, this allergen was not detected in serum from an individual who consumed the same bread but did not experience an allergic reaction. The newly identified allergen was solubilized by heating at 95ºC for 5 min in 10% SDS. Using liquid chromatography-mass spectrometry (LC-MS/MS) and database searches in the National Center for Biotechnology Information (NCBI) Gryllus protein database, paramyosin was identified as a candidate protein. Our findings suggest a new allergen candidate in cricket protein, which is regarded as a promising alternative protein source and edible insect. Furthermore, these results support the safety assessment of cricket consumption and underscore the importance of allergen screening in edible crickets. Journal of nutritional science and vitaminology 72 1 70 75 20260000 10.3177/jnsv.72.70 41765413 TakahitoWatanabe/published_papers/52454951 Aya Nakai Shiho Fukushima Kazuaki Mawatari Akira Takahashi Takeshi Nikawa Shintaro Inoue Takahito Watanabe Taro Mito Katsuyuki Miyawaki Differential Expression of Key Isoflavone Synthesis Genes in Soybean Sprouts under Two LED Treatments American Chemical Society (ACS) ACS Agricultural Science &amp; Technology 2692-1952 5 6 981 989 20250418 10.1021/acsagscitech.4c00646 TakahitoWatanabe/published_papers/49346534 Kohei Kawamoto Yoshiyasu Ishimaru Sayuri Tomonari Takahito Watanabe Sumihare Noji Taro Mito Myoglianin is a crucial factor for the transition to the juvenile hormone-dependent phase during hemimetabolous nymphal development Elsevier BV Insect Biochemistry and Molecular Biology 0965-1748 178 104274 104274 20250300 10.1016/j.ibmb.2025.104274 TakahitoWatanabe/published_papers/48681215 Shintaro Inoue Kai Fujie Taiki Hamaguchi Yoshiyasu Ishimaru Katsuyuki Miyawaki Akira Takahashi Takeshi Nikawa Sumihare Noji Takahito Watanabe Taro Mito Lineage-specific duplication and functional diversification of DOPA-decarboxylase genes in the Gryllidae family, as revealed in Gryllus bimaculatus. The DOPA-decarboxylase (DDC) gene is crucial for dopamine synthesis and influences various biological functions in insects, including body coloration, behavior, learning, and sleep. However, its evolutionary impact remains largely unexplored. This study reports on the tandem duplication of two bona fide ddc genes (ddc1 and ddc2) in the Gryllidae cricket family. We herein investigated the function of ddc1 and ddc2 using Gryllus bimaculatus (Gb) as a model. Our results revealed that Gb'ddc1 was expressed systemically, with its expression being higher immediately after molting compared to the stage following melanin pigmentation. In homozygous knockout mutants of Gb'ddc1, generated via CRISPR/Cas9, reduced body color pigmentation and had translucent cuticles, decreased dopamine levels, and over-accumulated DOPA. These mutants died shortly after hatching, likely due to cuticle defects, underscoring the essential role of dopamine, produced by Gb'ddc1, in melanin synthesis. Conversely, Gb'ddc2 expression was confined to the ovary and was not up-regulated after molting. Homozygous knockout mutants of Gb'ddc2 exhibited no body color defects, whereas hatchability and embryonic development rates were significantly reduced. Interestingly, dopamine levels in the ovaries were significantly elevated in Gb'ddc2 mutants. This suggests that normal ovarian dopamine levels, modulated by Gb'ddc2, are vital for fertility maintenance. The function of Gb'ddc2 differs from that of typical ddc, indicating neofunctionalization through evolutionary duplication. Overall, Gb'ddc1 and Gb'ddc2 have distinct functions, and precise regulation of ovarian dopamine levels using these two ddc genes may have enhanced cricket fertility. Insect biochemistry and molecular biology 104246 104246 20241207 10.1016/j.ibmb.2024.104246 39653316 TakahitoWatanabe/published_papers/43106095 Takahisa Yamashita Takahiro Ohde Taro Nakamura Yoshiyasu Ishimaru Takahito Watanabe Sayuri Tomonari Yuki Nakamura Sumihare Noji Taro Mito Involvement of the <i>scalloped</i> gene in morphogenesis of the wing margin via regulating cell growth in a hemimetabolous insect <i>Gryllus bimaculatus</i> Wiley Development, Growth &amp; Differentiation 1440-169X null null 20230623 10.1111/dgd.12869 TakahitoWatanabe/published_papers/42650353 Shintaro Inoue Takahito Watanabe Taiki Hamaguchi Yoshiyasu Ishimaru Katsuyuki Miyawaki Takeshi Nikawa Akira Takahashi Sumihare Noji Taro Mito Combinatorial expression of ebony and tan generates body color variation from nymph through adult stages in the cricket, Gryllus bimaculatus. Insect body colors and patterns change markedly during development in some species as they adapt to their surroundings. The contribution of melanin and sclerotin pigments, both of which are synthesized from dopamine, to cuticle tanning has been well studied. Nevertheless, little is known about how insects alter their body color patterns. To investigate this mechanism, the cricket Gryllus bimaculatus, whose body color patterns change during postembryonic development, was used as a model in this study. We focused on the ebony and tan genes, which encode enzymes that catalyze the synthesis and degradation, respectively, of the precursor of yellow sclerotin N-β-alanyl dopamine (NBAD). Expression of the G. bimaculatus (Gb) ebony and tan transcripts tended to be elevated just after hatching and the molting period. We found that dynamic alterations in the combined expression levels of Gb'ebony and Gb'tan correlated with the body color transition from the nymphal stages to the adult. The body color of Gb'ebony knockout mutants generated by CRISPR/Cas9 systemically darkened. Meanwhile, Gb'tan knockout mutants displayed a yellow color in certain areas and stages. The phenotypes of the Gb'ebony and Gb'tan mutants probably result from an over-production of melanin and yellow sclerotin NBAD, respectively. Overall, stage-specific body color patterns in the postembryonic stages of the cricket are governed by the combinatorial expression of Gb'ebony and Gb'tan. Our findings provide insights into the mechanism by which insects evolve adaptive body coloration at each developmental stage. PloS one 18 5 e0285934 null 20230000 10.1371/journal.pone.0285934 37200362 TakahitoWatanabe/published_papers/37822996 Yuki Nakamura Sayuri Tomonari Kohei Kawamoto Takahisa Yamashita Takahito Watanabe Yoshiyasu Ishimaru Sumihare Noji Taro Mito Evolutionarily conserved function of the even-skipped ortholog in insects revealed by gene knock-out analyses in Gryllus bimaculatus Elsevier BV Developmental Biology 0012-1606 485 1 8 20220500 10.1016/j.ydbio.2022.02.005 TakahitoWatanabe/published_papers/37862973 Taro Mito Yoshiyasu Ishimaru Takahito Watanabe Taro Nakamura Guillem Ylla Sumihare Noji Cassandra G Extavour Cricket: The third domesticated insect. Many researchers are using crickets to conduct research on various topics related to development and regeneration in addition to brain function, behavior, and biological clocks, using advanced functional and perturbational technologies such as genome editing. Recently, crickets have also been attracting attention as a food source for the next generation of humans. In addition, crickets are increasingly being used as disease models and biological factories for pharmaceuticals. Cricket research has thus evolved over the last century from use primarily in highly important basic research, to use in a variety of applications and practical uses. These insects are now a state-of-the-art model animal that can be obtained and maintained in large quantities at low cost. We therefore suggest that crickets are useful as a third domesticated insect for scientific research, after honeybees and silkworms, contributing to the achievement of global sustainable development goals. Current topics in developmental biology 147 291 306 20220000 10.1016/bs.ctdb.2022.02.003 35337452 TakahitoWatanabe/published_papers/33747306 Guillem Ylla Taro Nakamura Takehiko Itoh Rei Kajitani Atsushi Toyoda Sayuri Tomonari Tetsuya Bando Yoshiyasu Ishimaru Takahito Watanabe Masao Fuketa Yuji Matsuoka Austen A. Barnett Sumihare Noji Taro Mito Cassandra G. Extavour Insights into the genomic evolution of insects from cricket genomes <title>Abstract</title>Most of our knowledge of insect genomes comes from Holometabolous species, which undergo complete metamorphosis and have genomes typically under 2 Gb with little signs of DNA methylation. In contrast, Hemimetabolous insects undergo the presumed ancestral process of incomplete metamorphosis, and have larger genomes with high levels of DNA methylation. Hemimetabolous species from the Orthopteran order (grasshoppers and crickets) have some of the largest known insect genomes. What drives the evolution of these unusual insect genome sizes, remains unknown. Here we report the sequencing, assembly and annotation of the 1.66-Gb genome of the Mediterranean field cricket <italic>Gryllus bimaculatus</italic>, and the annotation of the 1.60-Gb genome of the Hawaiian cricket <italic>Laupala kohalensis</italic>. We compare these two cricket genomes with those of 14 additional insects and find evidence that hemimetabolous genomes expanded due to transposable element activity. Based on the ratio of observed to expected CpG sites, we find higher conservation and stronger purifying selection of methylated genes than non-methylated genes. Finally, our analysis suggests an expansion of the <italic>pickpocket</italic> class V gene family in crickets, which we speculate might play a role in the evolution of cricket courtship, including their characteristic chirping. Springer Science and Business Media LLC Communications Biology 2399-3642 4 1 733 733 20211200 10.1038/s42003-021-02197-9 34127782 TakahitoWatanabe/published_papers/34804644 Yuji Matsuoka Taro Nakamura Takahito Watanabe Austen A. Barnett Sumihare Noji Taro Mito Cassandra G. Extavour Establishment of CRISPR/Cas9-based knock-in in a hemimetabolous insect: targeted gene tagging in the cricket Gryllus bimaculatus <title>Abstract</title>Studies of traditional model organisms like the fruit fly <italic>Drosophila melanogaster</italic> have contributed immensely to our understanding of the genetic basis of developmental processes. However, the generalizability of these findings cannot be confirmed without functional genetic analyses in additional organisms. Direct genome editing using targeted nucleases has the potential to transform hitherto poorly-understood organisms into viable laboratory organisms for functional genetic study. To this end, here we present a method to induce targeted genome knock-out and knock-in of desired sequences in an insect that serves as an informative contrast to <italic>Drosophila</italic>, the cricket <italic>Gryllus bimaculatus</italic>. The efficiency of germ line transmission of induced mutations is comparable to that reported for other well-studied laboratory organisms, and knock-ins targeting introns yields viable, fertile animals in which knock-in events are directly detectable by visualization of a fluorescent marker in the expression pattern of the targeted gene. Combined with the recently assembled and annotated genome of this cricket, this knock-in/knock-out method increases the viability of <italic>G. bimaculatus</italic> as a tractable system for functional genetics in a basally branching insect. Cold Spring Harbor Laboratory null null 20210510 10.1101/2021.05.10.441399 TakahitoWatanabe/published_papers/33747327 Aya Nakai Akihito Tanaka Hitoshi Yoshihara Koji Murai Takahito Watanabe Katsuyuki Miyawaki Blue LED light promotes indican accumulation and flowering in indigo plant, Polygonum tinctorium Elsevier BV Industrial Crops and Products 0926-6690 155 112774 112774 20201100 10.1016/j.indcrop.2020.112774 TakahitoWatanabe/published_papers/30924085 Risa Ueta Chihiro Abe Takahito Watanabe Shigeo S Sugano Ryosuke Ishihara Hiroshi Ezura Yuriko Osakabe Keishi Osakabe Author Correction: Rapid breeding of parthenocarpic tomato plants using CRISPR/Cas9. An amendment to this paper has been published and can be accessed via a link at the top of the paper. Scientific reports 10 1 16776 16776 20201002 10.1038/s41598-020-71765-6 33009418 TakahitoWatanabe/published_papers/30924086 Yoshiyasu Ishimaru Sayuri Tomonari Takahito Watanabe Sumihare Noji Taro Mito Regulatory mechanisms underlying the specification of the pupal-homologous stage in a hemimetabolous insect Juvenile hormones and the genetic interaction between the transcription factors <italic>Krüppel homologue 1</italic> ( <italic>Kr-h1</italic> ) and <italic>Broad</italic> ( <italic>Br</italic> ) regulate the transformation of insects from immature to adult forms in both types of metamorphosis (holometaboly with a pupal stage versus hemimetaboly with no pupal stage); however, knowledge about the exact instar in which this occurs is limited. Using the hemimetabolous cricket <italic>Gryllus bimaculatus</italic> ( <italic>Gb</italic> ), we demonstrate that a genetic interaction occurs among <italic>Gb</italic> ′ <italic>Kr-h1</italic> , <italic>Gb</italic> ′ <italic>Br</italic> and the adult-specifier transcription factor <italic>Gb</italic> ′ <italic>E93</italic> from the sixth to final (eighth) nymphal instar. <italic>Gb</italic> ′ <italic>Kr-h1</italic> and <italic>Gb</italic> ′ <italic>Br</italic> mRNAs were strongly expressed in the abdominal tissues of sixth instar nymphs, with precocious adult moults being induced by <italic>Gb</italic> ′ <italic>Kr-h1</italic> or <italic>Gb</italic> ′ <italic>Br</italic> knockdown in the sixth instar. The depletion of <italic>Gb</italic> ′ <italic>Kr-h1</italic> or <italic>Gb</italic> ′ <italic>Br</italic> upregulates <italic>Gb</italic> ′ <italic>E93</italic> in the sixth instar. By contrast, <italic>Gb</italic> ′ <italic>E93</italic> knockdown at the sixth instar prevents nymphs transitioning to adults, instead producing supernumerary nymphs. <italic>Gb</italic> ′ <italic>E93</italic> also represses <italic>Gb</italic> ′ <italic>Kr-h1</italic> and <italic>Gb</italic> ′ <italic>Br</italic> expression in the penultimate nymphal instar, demonstrating its important role in adult differentiation. Our results suggest that the regulatory mechanisms underlying the pupal transition in holometabolous insects are evolutionarily conserved in hemimetabolous <italic>G</italic> . <italic>bimaculatus</italic> , with the penultimate and final nymphal periods being equivalent to the pupal stage. This article is part of the theme issue `The evolution of complete metamorphosis'. The Royal Society Philosophical Transactions of the Royal Society B: Biological Sciences 1471-2970 374 1783 20190225 20190225 20191014 10.1098/rstb.2019.0225 31438810 TakahitoWatanabe/published_papers/34818488 Hiroe Yasui Nao Fujiwara-Tsujii Tetsuya Yasuda Midori Fukaya Satoshi Kiriyama Akio Nakano Takahito Watanabe Kenji Mori Electroantennographic responses and field attraction of an emerging invader, the red-necked longicorn beetle Aromia bungii (Coleoptera: Cerambycidae), to the chiral and racemic forms of its male-produced aggregation-sex pheromone APPLIED ENTOMOLOGY AND ZOOLOGY 1347-605X 54 1 109 114 20190200 10.1007/s13355-018-0600-x TakahitoWatanabe/published_papers/30924087 Norio Kamemura Mayumi Sugimoto Norimasa Tamehiro Reiko Adachi Sayuri Tomonari Takahito Watanabe Taro Mito Cross-allergenicity of crustacean and the edible insect Gryllus bimaculatus in patients with shrimp allergy Food scarcity is a serious problem for many developing as well as developed countries. Edible insects have attracted attention recently as a novel food source. Crickets are especially high in nutritional value and easy to breed and harvest. In this study, we evaluated the risk of allergic reactions associated with cricket consumption in individuals with crustacean allergy. We evaluated food allergy risk in the consumption of Gryllus bimaculatus (cricket) in patients with shrimp allergy, using enzyme-linked immunosorbent assay (ELISA) and IgE crosslinking-induced luciferase expression assay (EXiLE). Sera from individuals with shrimp allergy (positive for shrimp-specific IgE by ImmunoCAP (>0.35 UA/mL; n = 9) or without shrimp allergy (negative for shrimp-specific IgE; n = 6) were obtained. There was a strong correlation between shrimp- and Gryllus-specific IgE levels obtained by ELISA (rs = 0.99; P < 0.001). The binding of shrimp-specific IgE on shrimp allergen was dose-dependently inhibited by Gryllus allergen (0-1.0 mg/mL). There was a strong correlation between shrimp- and Gryllus-specific IgE responses, as assessed by EXiLE assays (rs = 0.89; P < 0.001). We determined that a protein of approximately 40 kDa reacted with the positive, but not negative, sera for shrimp-specific IgE by ImmunoCAP. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis identified the major allergen in shrimp and Gryllus to be tropomyosin. Our data suggest that the cricket allergen has the potential to induce an allergic reaction in individuals with crustacean allergy. Therefore, allergy risk and shrimp-specific IgE levels should be considered before consumption of cricket meal. Elsevier BV Molecular Immunology 0161-5890 106 127 134 20190200 10.1016/j.molimm.2018.12.015 30597474 TakahitoWatanabe/published_papers/30924088 Tian Xu Hiroe Yasui Stephen A Teale Nao Fujiwara-Tsujii Jacob D Wickham Midori Fukaya Laura Hansen Satoshi Kiriyama Dejun Hao Akio Nakano Longwa Zhang Takahito Watanabe Masahiko Tokoro Jocelyn G Millar Identification of a male-produced sex-aggregation pheromone for a highly invasive cerambycid beetle, Aromia bungii. The longhorned beetle Aromia bungii (Coleoptera: Cerambycidae) is a major pest of stone fruit trees in the genus Prunus, including cherries, apricots, and peaches. Its native range includes China, Korea, Mongolia, and eastern Russia, but it has recently invaded and become established in several countries in Europe, and Japan, and it has been intercepted in shipments coming into North America and Australia. Here, we report the identification of its male-produced aggregation pheromone as the novel compound (E)-2-cis-6,7-epoxynonenal. In field trials in its native range in China, and in recently invaded areas of Japan, the pheromone attracted both sexes of the beetle. Thus, the pheromone should find immediate use in worldwide quarantine surveillance efforts to detect the beetle in incoming shipments. The pheromone will also be a crucial tool in ongoing efforts to eradicate the beetle from regions of the world that it has already invaded. Scientific reports 7 1 7330 7330 20170804 10.1038/s41598-017-07520-1 28779104 TakahitoWatanabe/published_papers/30924090 Risa Ueta Chihiro Abe Takahito Watanabe Shigeo S Sugano Ryosuke Ishihara Hiroshi Ezura Yuriko Osakabe Keishi Osakabe Rapid breeding of parthenocarpic tomato plants using CRISPR/Cas9. Parthenocarpy in horticultural crop plants is an important trait with agricultural value for various industrial purposes as well as direct eating quality. Here, we demonstrate a breeding strategy to generate parthenocarpic tomato plants using the CRISPR/Cas9 system. We optimized the CRISPR/Cas9 system to introduce somatic mutations effectively into SlIAA9-a key gene controlling parthenocarpy-with mutation rates of up to 100% in the T0 generation. Furthermore, analysis of off-target mutations using deep sequencing indicated that our customized gRNAs induced no additional mutations in the host genome. Regenerated mutants exhibited morphological changes in leaf shape and seedless fruit-a characteristic of parthenocarpic tomato. And the segregated next generation (T1) also showed a severe phenotype associated with the homozygous mutated genome. The system developed here could be applied to produce parthenocarpic tomato in a wide variety of cultivars, as well as other major horticultural crops, using this precise and rapid breeding technique. Scientific reports 7 1 507 507 20170330 10.1038/s41598-017-00501-4 28360425 TakahitoWatanabe/published_papers/47056940 Hadley Horch Jin Liu Taro Mito Aleksandar Popadić Takahito Watanabe Protocols in the cricket Springer Japan The Cricket as a Model Organism: Development, Regeneration, and Behavior 327 370 20170101 10.1007/978-4-431-56478-2_21 2-s2.0-85042820811 TakahitoWatanabe/published_papers/30924089 Takahito Watanabe Sumihare Noji Taro Mito Genome Editing in the Cricket, Gryllus bimaculatus. Hemimetabolous, or incompletely metamorphosing, insects are phylogenetically basal and include many beneficial and deleterious species. The cricket, Gryllus bimaculatus, is an emerging model for hemimetabolous insects, based on the success of RNA interference (RNAi)-based gene-functional analyses and transgenic technology. Taking advantage of genome editing technologies in this species would greatly promote functional genomics studies. Genome editing has proven to be an effective method for site-specific genome manipulation in various species. Here, we describe a protocol for genome editing including gene knockout and gene knockin in G. bimaculatus for functional genomics studies. Methods in molecular biology (Clifton, N.J.) 1630 219 233 20170000 10.1007/978-1-4939-7128-2_18 28643262 TakahitoWatanabe/published_papers/30924091 Yuriko Osakabe Takahito Watanabe Shigeo S Sugano Risa Ueta Ryosuke Ishihara Kazuo Shinozaki Keishi Osakabe Optimization of CRISPR/Cas9 genome editing to modify abiotic stress responses in plants. Genome editing using the CRISPR/Cas9 system can be used to modify plant genomes, however, improvements in specificity and applicability are still needed in order for the editing technique to be useful in various plant species. Here, using genome editing mediated by a truncated gRNA (tru-gRNA)/Cas9 combination, we generated new alleles for OST2, a proton pump in Arabidopsis, with no off-target effects. By following expression of Cas9 and the tru-gRNAs, newly generated mutations in CRIPSR/Cas9 transgenic plants were detected with high average mutation rates of up to 32.8% and no off-target effects using constitutive promoter. Reducing nuclear localization signals in Cas9 decreased the mutation rate. In contrast, tru-gRNA Cas9 cassettes driven by meristematic- and reproductive-tissue-specific promoters increased the heritable mutation rate in Arabidopsis, showing that high expression in the germ line can produce bi-allelic mutations. Finally, the new mutant alleles obtained for OST2 exhibited altered stomatal closing in response to environmental conditions. These results suggest further applications in molecular breeding to improve plant function using optimized plant CRISPR/Cas9 systems. Scientific reports 6 26685 26685 20160526 10.1038/srep26685 27226176 TakahitoWatanabe/published_papers/30924092 Yoshiyasu Ishimaru Sayuri Tomonari Yuji Matsuoka Takahito Watanabe Katsuyuki Miyawaki Tetsuya Bando Kenji Tomioka Hideyo Ohuchi Sumihare Noji Taro Mito TGF-β signaling in insects regulates metamorphosis via juvenile hormone biosynthesis. Although butterflies undergo a dramatic morphological transformation from larva to adult via a pupal stage (holometamorphosis), crickets undergo a metamorphosis from nymph to adult without formation of a pupa (hemimetamorphosis). Despite these differences, both processes are regulated by common mechanisms that involve 20-hydroxyecdysone (20E) and juvenile hormone (JH). JH regulates many aspects of insect physiology, such as development, reproduction, diapause, and metamorphosis. Consequently, strict regulation of JH levels is crucial throughout an insect's life cycle. However, it remains unclear how JH synthesis is regulated. Here, we report that in the corpora allata of the cricket, Gryllus bimaculatus, Myoglianin (Gb'Myo), a homolog of Drosophila Myoglianin/vertebrate GDF8/11, is involved in the down-regulation of JH production by suppressing the expression of a gene encoding JH acid O-methyltransferase, Gb'jhamt In contrast, JH production is up-regulated by Decapentaplegic (Gb'Dpp) and Glass-bottom boat/60A (Gb'Gbb) signaling that occurs as part of the transcriptional activation of Gb'jhamt Gb'Myo defines the nature of each developmental transition by regulating JH titer and the interactions between JH and 20E. When Gb'myo expression is suppressed, the activation of Gb'jhamt expression and secretion of 20E induce molting, thereby leading to the next instar before the last nymphal instar. Conversely, high Gb'myo expression induces metamorphosis during the last nymphal instar through the cessation of JH synthesis. Gb'myo also regulates final insect size. Because Myo/GDF8/11 and Dpp/bone morphogenetic protein (BMP)2/4-Gbb/BMP5-8 are conserved in both invertebrates and vertebrates, the present findings provide common regulatory mechanisms for endocrine control of animal development. Proceedings of the National Academy of Sciences of the United States of America 113 20 5634 9 20160517 10.1073/pnas.1600612113 27140602 TakahitoWatanabe/published_papers/30924094 Takahito Watanabe Sumihare Noji Taro Mito GeneKnockout by Targeted Mutagenesis in a Hemimetabolous Insect, the Two-Spotted Cricket Gryllus bimaculatus, using TALENs. Hemimetabolous, or incompletely metamorphosing, insects are phylogenetically basal. These insects include many deleterious species. The cricket, Gryllus bimaculatus, is an emerging model for hemimetabolous insects, based on the success of RNA interference (RNAi)-based gene-functional analyses and transgenic technology. Taking advantage of genome-editing technologies in this species would greatly promote functional genomics studies. Genome editing using transcription activator-like effector nucleases (TALENs) has proven to be an effective method for site-specific genome manipulation in various species. TALENs are artificial nucleases that are capable of inducing DNA double-strand breaks into specified target sequences. Here, we describe a protocol for TALEN-based gene knockout in G. bimaculatus, including a mutant selection scheme via mutation detection assays, for generating homozygous knockout organisms. Methods in molecular biology (Clifton, N.J.) 1338 143 55 20160000 10.1007/978-1-4939-2932-0_12 26443220 TakahitoWatanabe/published_papers/35115791 上田 梨紗 石原 諒典 阿部 千尋 渡辺 崇人 菅野 茂夫 宮脇 克行 野地 澄晴 刑部 祐里子 刑部 敬史 CRISPR/Cas9によるトマトIAA9遺伝子を標的としたゲノム編集技術の確立 (公社)日本生化学会 日本生化学会大会・日本分子生物学会年会合同大会講演要旨集 88·38 [4T21L 08(3P0837)] 20151200 TakahitoWatanabe/published_papers/30924093 Hiroko Awata Takahito Watanabe Yoshitaka Hamanaka Taro Mito Sumihare Noji Makoto Mizunami Knockout crickets for the study of learning and memory: Dopamine receptor Dop1 mediates aversive but not appetitive reinforcement in crickets. Elucidation of reinforcement mechanisms in associative learning is an important subject in neuroscience. In mammals, dopamine neurons are thought to play critical roles in mediating both appetitive and aversive reinforcement. Our pharmacological studies suggested that octopamine and dopamine neurons mediate reward and punishment, respectively, in crickets, but recent studies in fruit-flies concluded that dopamine neurons mediates both reward and punishment, via the type 1 dopamine receptor Dop1. To resolve the discrepancy between studies in different insect species, we produced Dop1 knockout crickets using the CRISPR/Cas9 system and found that they are defective in aversive learning with sodium chloride punishment but not appetitive learning with water or sucrose reward. The results suggest that dopamine and octopamine neurons mediate aversive and appetitive reinforcement, respectively, in crickets. We suggest unexpected diversity in neurotransmitters mediating appetitive reinforcement between crickets and fruit-flies, although the neurotransmitter mediating aversive reinforcement is conserved. This study demonstrates usefulness of the CRISPR/Cas9 system for producing knockout animals for the study of learning and memory. Scientific reports 5 15885 15885 20151102 10.1038/srep15885 26521965 TakahitoWatanabe/published_papers/30924095 Yuji Matsuoka Tetsuya Bando Takahito Watanabe Yoshiyasu Ishimaru Sumihare Noji Aleksandar Popadić Taro Mito Short germ insects utilize both the ancestral and derived mode of Polycomb group-mediated epigenetic silencing of Hox genes. In insect species that undergo long germ segmentation, such as Drosophila, all segments are specified simultaneously at the early blastoderm stage. As embryogenesis progresses, the expression boundaries of Hox genes are established by repression of gap genes, which is subsequently replaced by Polycomb group (PcG) silencing. At present, however, it is not known whether patterning occurs this way in a more ancestral (short germ) mode of embryogenesis, where segments are added gradually during posterior elongation. In this study, two members of the PcG family, Enhancer of zeste (E(z)) and Suppressor of zeste 12 (Su(z)12), were analyzed in the short germ cricket, Gryllus bimaculatus. Results suggest that although stepwise negative regulation by gap and PcG genes is present in anterior members of the Hox cluster, it does not account for regulation of two posterior Hox genes, abdominal-A (abd-A) and Abdominal-B (Abd-B). Instead, abd-A and Abd-B are predominantly regulated by PcG genes, which is the mode present in vertebrates. These findings suggest that an intriguing transition of the PcG-mediated silencing of Hox genes may have occurred during animal evolution. The ancestral bilaterian state may have resembled the current vertebrate mode of regulation, where PcG-mediated silencing of Hox genes occurs before their expression is initiated and is responsible for the establishment of individual expression domains. Then, during insect evolution, the repression by transcription factors may have been acquired in anterior Hox genes of short germ insects, while PcG silencing was maintained in posterior Hox genes. Biology open 4 6 702 9 20150506 10.1242/bio.201411064 25948756 TakahitoWatanabe/published_papers/30924097 Takahito Watanabe Sumihare Noji Taro Mito Gene knockout by targeted mutagenesis in a hemimetabolous insect, the two-spotted cricket Gryllus bimaculatus, using TALENs. Hemimetabolous, or incompletely metamorphosing, insects are phylogenetically basal. These insects include many deleterious species. The cricket, Gryllus bimaculatus, is an emerging model for hemimetabolous insects, based on the success of RNA interference (RNAi)-based gene-functional analyses and transgenic technology. Taking advantage of genome-editing technologies in this species would greatly promote functional genomics studies. Genome editing using transcription activator-like effector nucleases (TALENs) has proven to be an effective method for site-specific genome manipulation in various species. TALENs are artificial nucleases that are capable of inducing DNA double-strand breaks into specified target sequences. Here, we describe a protocol for TALEN-based gene knockout in G. bimaculatus, including a mutant selection scheme via mutation detection assays, for generating homozygous knockout organisms. Methods (San Diego, Calif.) 69 1 17 21 20140815 10.1016/j.ymeth.2014.05.006 24874787 TakahitoWatanabe/published_papers/30924096 Akihiro Yasue Silvia Naomi Mitsui Takahito Watanabe Tetsushi Sakuma Seiichi Oyadomari Takashi Yamamoto Sumihare Noji Taro Mito Eiji Tanaka Highly efficient targeted mutagenesis in one-cell mouse embryos mediated by the TALEN and CRISPR/Cas systems. Since the establishment of embryonic stem (ES) cell lines, the combined use of gene targeting with homologous recombination has aided in elucidating the functions of various genes. However, the ES cell technique is inefficient and time-consuming. Recently, two new gene-targeting technologies have been developed: the transcription activator-like effector nuclease (TALEN) system, and the clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein (Cas) system. In addition to aiding researchers in solving conventional problems, these technologies can be used to induce site-specific mutations in various species for which ES cells have not been established. Here, by targeting the Fgf10 gene through RNA microinjection in one-cell mouse embryos with the TALEN and CRISPR/Cas systems, we produced the known limb-defect phenotypes of Fgf10-deficient embryos at the F0 generation. Compared to the TALEN system, the CRISPR/Cas system induced the limb-defect phenotypes with a strikingly higher efficiency. Our results demonstrate that although both gene-targeting technologies are useful, the CRISPR/Cas system more effectively elicits single-step biallelic mutations in mice. Scientific reports 4 5705 5705 20140716 10.1038/srep05705 25027812 TakahitoWatanabe/published_papers/30924098 Takahito Watanabe Hiroshi Ochiai Tetsushi Sakuma Hadley W Horch Naoya Hamaguchi Taro Nakamura Tetsuya Bando Hideyo Ohuchi Takashi Yamamoto Sumihare Noji Taro Mito Non-transgenic genome modifications in a hemimetabolous insect using zinc-finger and TAL effector nucleases. Hemimetabolous, or incompletely metamorphosing, insects are phylogenetically relatively basal and comprise many pests. However, the absence of a sophisticated genetic model system, or targeted gene-manipulation system, has limited research on hemimetabolous species. Here we use zinc-finger nuclease and transcription activator-like effector nuclease technologies to produce genetic knockouts in the hemimetabolous insect Gryllus bimaculatus. Following the microinjection of mRNAs encoding zinc-finger nucleases or transcription activator-like effector nucleases into cricket embryos, targeting of a transgene or endogenous gene results in sequence-specific mutations. Up to 48% of founder animals transmit disrupted gene alleles after zinc-finger nucleases microinjection compared with 17% after microinjection of transcription activator-like effector nucleases. Heterozygous offspring is selected using mutation detection assays that use a Surveyor (Cel-I) nuclease, and subsequent sibling crosses create homozygous knockout crickets. This approach is independent from a mutant phenotype or the genetic tractability of the organism of interest and can potentially be applied to manage insect pests using a non-transgenic strategy. Nature communications 3 1017 1017 20120000 10.1038/ncomms2020 22910363 TakahitoWatanabe/published_papers/30924099 Tetsuya Bando Taro Mito Yuko Maeda Taro Nakamura Fumiaki Ito Takahito Watanabe Hideyo Ohuchi Sumihare Noji Regulation of leg size and shape by the Dachsous/Fat signalling pathway during regeneration. An amputated cricket leg regenerates all missing parts with normal size and shape, indicating that regenerating blastemal cells are aware of both their position and the normal size of the leg. However, the molecular mechanisms regulating this process remain elusive. Here, we use a cricket model to show that the Dachsous/Fat (Ds/Ft) signalling pathway is essential for leg regeneration. We found that knockdown of ft or ds transcripts by regeneration-dependent RNA interference (rdRNAi) suppressed proliferation of the regenerating cells along the proximodistal (PD) axis concomitantly with remodelling of the pre-existing stump, making the regenerated legs shorter than normal. By contrast, knockdown of the expanded (ex) or Merlin (Mer) transcripts induced over-proliferation of the regenerating cells, making the regenerated legs longer. These results are consistent with those obtained using rdRNAi during intercalary regeneration induced by leg transplantation. We present a model to explain our results in which the steepness of the Ds/Ft gradient controls growth along the PD axis of the regenerating leg. Development (Cambridge, England) 136 13 2235 45 20090700 10.1242/dev.035204 19474149 TakahitoWatanabe/misc/52454961 井上智晶 石丸善康 川本晃平 井上慎太郎 渡邉崇人 野地澄晴 三戸太郎 フタホシコオロギをモデルとした不完全変態昆虫におけるE93遺伝子の発現と成虫化に関わる機能の解析 日本分子生物学会年会プログラム・要旨集(Web) 47th null null 20240000 TakahitoWatanabe/misc/52454957 島村彩音 井上慎太郎 石丸善康 宮脇克行 高橋章 二川健 野地澄晴 渡邉崇人 三戸太郎 フタホシコオロギの体色パターン形成における白色スクレロチン合成酵素遺伝子の発現と機能の解析 日本分子生物学会年会プログラム・要旨集(Web) 47th null null 20240000 TakahitoWatanabe/misc/35424013 渡辺 崇人 三戸 太郎 野地 澄晴 Gene Knockout in the Cricket Using ZFN/TALEN ZFN/TALENを用いたコオロギの遺伝子ノックアウト (特集 ゲノム編集革命 : 遺伝子改変はZFN・TALEN・CRISPR/Cas三強時代へ) 学研メディカル秀潤社 ; 1982- 細胞工学 0287-3796 32 5 543 549 20130000 TakahitoWatanabe/misc/52323622 渡辺崇人 落合博 佐久間哲史 中井綾 中村太郎 板東哲哉 三戸太郎 山本卓 野地澄晴 ZFN及びTALENを使用したコオロギ変異体の作製 日本分子生物学会年会プログラム・要旨集(Web) 35th null null 20120000 TakahitoWatanabe/misc/39566756 Tetsuya Bando Taro Mito Yuko Maeda Taro Nakamura Fumiaki Ito Takahito Watanabe Hideyo Ohuchi Sumihare Noji Regulation of leg size and shape by the Dachsous/Fat signalling pathway during regeneration in Gryllus bimaculatus MECHANISMS OF DEVELOPMENT 0925-4773 126 S293 S294 20090800 10.1016/j.mod.2009.06.797 TakahitoWatanabe/presentations/48013285 渡邉崇人 循環型タンパク質としてのコオロギについて，フードテック啓発セミナー フードテック啓発セミナー 20240200 TakahitoWatanabe/presentations/48013402 渡邉崇人 循環型タンパク質としての食用コオロギについて 第56回 全国手話通訳問題研究集会 サマーフォーラム in とくしま， 20230800 TakahitoWatanabe/presentations/48013331 渡邉崇人 循環型タンパク質としての食用コオロギについて 第53回中国四国大学保健管理研究集会 20230800 TakahitoWatanabe/presentations/48013471 渡邉崇人 循環型食品としての食用コオロギについて 生物工学会シンポジウム，オンライン 20230700 TakahitoWatanabe/presentations/48013951 渡邉崇人 食用コオロギの社会実装とゲノム編集による品種改良について， 中国四国地区 生物系三学会合同大会 20230500 TakahitoWatanabe/presentations/48013926 渡邉崇人 循環型食品としての食用コオロギについて ifiaJAPAN2023，東京 20230500 TakahitoWatanabe/presentations/48013994 渡邉崇人 昆虫工場研究開発の現状と植物工場との統合の可能性 植物工場研究会，オンライン 20230400 TakahitoWatanabe/presentations/48013963 32. 渡邉崇人:循環型タンパク質としての食用コオロギについて 農学会公開シンポジウム 20230400 TakahitoWatanabe/presentations/48014055 渡邉崇人 新規食品 昆虫食の現状と課題 第18回 食品安全シンポジウム，東京 20230300 TakahitoWatanabe/presentations/48014030 渡邉崇人:新規食品 昆虫食の現状と課題 第18回 食品安全シンポジウム，東京 20230300 TakahitoWatanabe/presentations/48014025 渡邉崇人 コオロギ食用化に向けた取り組み ~大学での研究から起業による社会実装まで~ 第67回日本応用動物昆虫学会，大阪 20230300 TakahitoWatanabe/presentations/48014006 渡邉崇人:コオロギに関する基礎研究と， 食用コオロギという社会実装について 科学技術と経済の会シンポジウム，東京 20230300 TakahitoWatanabe/presentations/48014066 渡邉崇人 持続可能な循環型タンパク質としての コオロギの可能性 第12回CSJ化学フェスタ2022，東京 20221000 TakahitoWatanabe/presentations/48014103 渡邉崇人 循環型タンパク質としての 食用コオロギの可能性 第23回日本食品工学会，岡山 20220900 TakahitoWatanabe/presentations/48014089 渡邉 崇人，井上 慎太郎，濱口 汰暉，石丸 善康，三戸 太郎: ゲノム編集を活用した食用コオロギの育種研究 日本遺伝学会 第94回大会，札幌 20220900 TakahitoWatanabe/presentations/48014132 渡邉崇人 昆虫食を「サーキュラーフード」にアップデートするという挑戦 WIREDオンライン「THURS DAY NIGHT，オンライン 20220400 TakahitoWatanabe/presentations/48014123 渡邉崇人 コオロギ × テクノロジーが生み出す 新たな調和で実現する健康でしあわせな未来 フードテックオンラインセミナー，オンライン 20220400 TakahitoWatanabe/presentations/48014169 渡邉崇人 サーキュラーフードとしての食用コオロギビジネスの事業展開について 食品ニューテクノロジー研究会，オンライン 20220300 TakahitoWatanabe/presentations/48014158 渡邉崇人 持続可能な循環型タンパク質としての食用コオロギ 第66回バイオ・マイクロ・ナノテク研究会，オンライン 20220300 TakahitoWatanabe/presentations/48014144 井上 慎太郎，濱口 汰暉，石丸 善康，三戸 太郎，渡邉 崇人 フタホシコオロギの体色関連遺伝子のノックアウト系統作製および表現型解析 第66回日本応用動物昆虫学会大会，オンライン 20220300 TakahitoWatanabe/presentations/48014184 渡邉崇人 食品ロスを循環させる新たなタンパク源としての「食用コオロギ」 健康博覧会2022，東京 20220200 TakahitoWatanabe/presentations/48014214 渡邉崇人 循環型食品``サーキュラーフード''としての食用コオロギについて フード・フォラム・つくば 冬の例会「未来の食文化 -肉替(にくたい)改造-」，オンライン 20211200 TakahitoWatanabe/presentations/48014753 渡邉崇人 世界のタンパク質不足と食品ロス問題を解消する循環型タンパク質「食用コオロギ」の生産・販売 アグリビジネス創出フェア2021，東京 20210100 TakahitoWatanabe/presentations/48013312 渡邉崇人 サステナブルで美味しい食の選択肢としての食用コオロギ 全国高等学校長協会 20131100 TakahitoWatanabe/presentations/48013245 Mito T Watanabe T Genome modification in a hemimetabolous insect Gryllus bimaculatus, Janelia Workshop on Genomic Modification in Model and Non-Model Insects 20130300 TakahitoWatanabe/books_etc/48014865 渡邉崇人 『代替タンパク質の技術と市場』 株式会社シーエムシー出版， 20240400 TakahitoWatanabe/books_etc/48014834 Mito T Ishimaru Y Watanabe T Nakamura T Ylla G Noji S Extavour C.G 『Current Topics in Developmental Biology』 ELSEVIER, pp291-306 20220300 TakahitoWatanabe/books_etc/33747400 渡邉崇人 フードテックの最新動向 株式会社シーエムシー出版 20210700 TakahitoWatanabe/books_etc/33747388 渡邉崇人 岡部慎司 「代替プロテイン」 ∼植物肉製品の開発，昆虫・藻類の食品素材利用と培養肉の作製∼ 株式会社エヌティーエス 20210700 TakahitoWatanabe/books_etc/33747376 渡邉崇人 三戸太郎 「代替プロテイン」 ∼植物肉製品の開発，昆虫・藻類の食品素材利用と培養肉の作製∼ 株式会社エヌティーエス 20210700 TakahitoWatanabe/books_etc/33747411 Horch H.W Liu J Mito T Popadic A Watanabe T The Cricket as a Model Organism Springer Link 20170000 TakahitoWatanabe/books_etc/33747420 Watanabe T Noji S Mito T TALENs, Methods in Molecular Biology Springer Link 20160000