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Ryoichi Sato
Graduate School of Bio-Application and Systems Engineering, Tokyo University of Agriculture and Technology, Naka-cho 2-24-16, Koganei, Tokyo 184-8588, Japan

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Journal article
Published: 27 May 2021 in Journal of Insect Physiology
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Most lepidopteran insect larvae exhibit stepwise feeding behaviors, such as palpation using the maxillary palps (MPs) followed by test biting and persistent biting. However, the purpose of palpation has been unclear. In particular, nothing is known about the neurons in the MP and their mode of recognition of undesired plants, although such neurons have been suggested to exist. In this study, we used larvae of the stenophagous insect Bombyx mori and compared the roles of palpation and test biting in the selection of feeding behavior. When the larvae were given non-host plant leaves, they did not initiate test biting, indicating that non-host plant leaves were recognized via palpation without biting, and that this behavior resulted in a lack of persistent biting, as the leaves were judged non-suitable for consumption. Surface extracts of inedible leaves significantly suppressed test biting of mulberry leaves, a host plant of B. mori, suggesting that secondary metabolites on the leaf surface of inedible leaves function as test biting suppressors, even when another conditions are suitable for test biting. The allelochemical coumarin, which is found in the inedible leaves of cherry, Cerasus speciosa, significantly suppressed test biting of mulberry leaves, suggesting that coumarin is a possible deterrent to the eating of cherry leaves. Using the electrophysiological method of tip recording and a leaf-surface extract as the test material, leaf-surface compound-responsive neurons were identified in the MP. In addition, several neurons that respond to coumarin in the attomolar range were identified, suggesting that the larvae use ultrasensitive neurons in the MP to recognize inedible leaves. In the HEK293T cell heterologous expression system, the B. mori gustatory receptors BmGr53 and BmGr19, which were previously found to be expressed in the MP and to respond to coumarin in the attomolar range, responded to a leaf-surface extract of C. speciosa, suggesting that these receptors may be present on the inedible-leaf-recognizing neurons of the MP. These findings suggest that ultrasensitive plant secondary metabolite-recognizing neurons in the MP allow for the recognition of non-host plants via palpation without risking damage caused by ingesting harmful allelochemicals.

ACS Style

Fumika Shii; Dingze Mang; Mayu Kasubuchi; Kana Tsuneto; Tomoko Toyama; Haruka Endo; Ken Sasaki; Ryoichi Sato. Ultrasensitive detection by maxillary palp neurons allows non-host recognition without consumption of harmful allelochemicals. Journal of Insect Physiology 2021, 132, 104263 .

AMA Style

Fumika Shii, Dingze Mang, Mayu Kasubuchi, Kana Tsuneto, Tomoko Toyama, Haruka Endo, Ken Sasaki, Ryoichi Sato. Ultrasensitive detection by maxillary palp neurons allows non-host recognition without consumption of harmful allelochemicals. Journal of Insect Physiology. 2021; 132 ():104263.

Chicago/Turabian Style

Fumika Shii; Dingze Mang; Mayu Kasubuchi; Kana Tsuneto; Tomoko Toyama; Haruka Endo; Ken Sasaki; Ryoichi Sato. 2021. "Ultrasensitive detection by maxillary palp neurons allows non-host recognition without consumption of harmful allelochemicals." Journal of Insect Physiology 132, no. : 104263.

Journal article
Published: 20 May 2021 in Journal of Insect Physiology
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Previously, we found that nodule formation, a cellular defense response in insects, is regulated by humoral factors called C-type lectins in the hemolymph. To elucidate the factors that elicit nodule formation following the recognition of microorganisms by C-type lectins, a reproducible quantitative in vitro assay system was constructed. Then, using this system, the inhibitory activities of antisera raised against hemolymph proteases (HPs), serine protease homologues (SPHs), and pathogen-associated molecular pattern (PAMP)-recognition proteins were assessed. Among the antisera raised against HP and SPH, only that against HP8, a terminal proteinase that activates Spätzle, consistently inhibited in-vitro nodule-like aggregate formation in all three tested microorganisms, Micrococcus luteus, Escherichia coli, and Saccharomyces cerevisiae. Antisera raised against C-type lectins, BmLBP, and BmMBP also inhibited nodule-like aggregate formation, while those against β-glucan recognition proteins and peptidoglycan recognition protein-S1 did not. Microorganisms pretreated with hemolymph, which contains HP8 and C-type lectins, also induced nodule-like aggregate formation, indicating that nodulation factors are present on microbial cells. Furthermore, antisera raised against HP8, BmLBP, and BmMBP showed inhibitory activities in the in vivo nodule formation system using Bombyx mori larvae. Thus, two humoral factors in the hemolymph of B. mori larvae, BmHP8 and C-type lectins, were found to play significant roles in eliciting the cellular defense response of nodule formation.

ACS Style

Kotomi Tokunaga; Moeko Tezuka; Shuyi Tang; Min Shu; Takayuki Yamagishi; Ryoichi Sato. A humoral factor, hemolymph proteinase 8, elicits a cellular defense response of nodule formation in Bombyx mori larvae in association with recognition by C-type lectins. Journal of Insect Physiology 2021, 132, 104252 .

AMA Style

Kotomi Tokunaga, Moeko Tezuka, Shuyi Tang, Min Shu, Takayuki Yamagishi, Ryoichi Sato. A humoral factor, hemolymph proteinase 8, elicits a cellular defense response of nodule formation in Bombyx mori larvae in association with recognition by C-type lectins. Journal of Insect Physiology. 2021; 132 ():104252.

Chicago/Turabian Style

Kotomi Tokunaga; Moeko Tezuka; Shuyi Tang; Min Shu; Takayuki Yamagishi; Ryoichi Sato. 2021. "A humoral factor, hemolymph proteinase 8, elicits a cellular defense response of nodule formation in Bombyx mori larvae in association with recognition by C-type lectins." Journal of Insect Physiology 132, no. : 104252.

Research article
Published: 16 September 2020 in PLOS Biology
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Many herbivorous insects are mono- or oligophagous, having evolved to select a limited range of host plants. They specifically identify host-plant leaves using their keen sense of taste. Plant secondary metabolites and sugars are thought to be key chemical cues that enable insects to identify host plants and evaluate their quality as food. However, the neuronal and behavioral mechanisms of host-plant recognition are poorly understood. Here, we report a two-factor host acceptance system in larvae of the silkworm Bombyx mori, a specialist on several mulberry species. The first step is controlled by a chemosensory organ, the maxillary palp (MP). During palpation at the leaf edge, the MP detects trace amounts of leaf-surface compounds, which enables host-plant recognition without biting. Chemosensory neurons in the MP are tuned with ultrahigh sensitivity (thresholds of attomolar to femtomolar) to chlorogenic acid (CGA), quercetin glycosides, and β-sitosterol (βsito). Only if these 3 compounds are detected does the larva make a test bite, which is evaluated in the second step. Low-sensitivity neurons in another chemosensory organ, the maxillary galea (MG), mainly detect sucrose in the leaf sap exuded by test biting, allowing larvae to accept the leaf and proceed to persistent biting (feeding). The two-factor host acceptance system reported here may commonly underlie stereotyped feeding behavior in many phytophagous insects and determine their feeding habits.

ACS Style

Kana Tsuneto; Haruka Endo; Fumika Shii; Ken Sasaki; Shinji Nagata; Ryoichi Sato. Diet choice: The two-factor host acceptance system of silkworm larvae. PLOS Biology 2020, 18, e3000828 .

AMA Style

Kana Tsuneto, Haruka Endo, Fumika Shii, Ken Sasaki, Shinji Nagata, Ryoichi Sato. Diet choice: The two-factor host acceptance system of silkworm larvae. PLOS Biology. 2020; 18 (9):e3000828.

Chicago/Turabian Style

Kana Tsuneto; Haruka Endo; Fumika Shii; Ken Sasaki; Shinji Nagata; Ryoichi Sato. 2020. "Diet choice: The two-factor host acceptance system of silkworm larvae." PLOS Biology 18, no. 9: e3000828.

Journal article
Published: 06 February 2020 in Toxins
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Cry toxins are insecticidal proteins produced by Bacillus thuringiensis (Bt). They are used commercially to control insect pests since they are very active in specific insects and are harmless to the environment and human health. The gene encoding ATP-binding cassette subfamily A member 2 (ABCA2) was identified in an analysis of Cry2A toxin resistance genes. However, we do not have direct evidence for the role of ABCA2 for Cry2A toxins or why Cry2A toxin resistance does not cross to other Cry toxins. Therefore, we performed two experiments. First, we edited the ABCA2 sequence in Bombyx mori using transcription activator-like effector-nucleases (TALENs) and confirmed the susceptibility-determining ability in a diet overlay bioassay. Strains with C-terminal half-deleted BmABCA2 showed strong and specific resistance to Cry2A toxins; even strains carrying a deletion of 1 to 3 amino acids showed resistance. However, the C-terminal half-deleted strains did not show cross-resistance to other toxins. Second, we conducted a cell swelling assay and confirmed the specific ability of BmABCA2 to Cry2A toxins in HEK239T cells. Those demonstrated that BmABCA2 is a functional receptor for Cry2A toxins and that BmABCA2 deficiency-dependent Cry2A resistance does not confer cross-resistance to Cry1A, Cry1Ca, Cry1Da, Cry1Fa or Cry9Aa toxins.

ACS Style

Xiaoyi Li; Kazuhisa Miyamoto; Yoko Takasu; Sanae Wada; Tetsuya Iizuka; Satomi Adegawa; Ryoichi Sato; Kenji Watanabe. ATP-Binding Cassette Subfamily a Member 2 Is a Functional Receptor for Bacillus thuringiensis Cry2A Toxins in Bombyx mori, But Not for Cry1A, Cry1C, Cry1D, Cry1F, or Cry9A Toxins. Toxins 2020, 12, 104 .

AMA Style

Xiaoyi Li, Kazuhisa Miyamoto, Yoko Takasu, Sanae Wada, Tetsuya Iizuka, Satomi Adegawa, Ryoichi Sato, Kenji Watanabe. ATP-Binding Cassette Subfamily a Member 2 Is a Functional Receptor for Bacillus thuringiensis Cry2A Toxins in Bombyx mori, But Not for Cry1A, Cry1C, Cry1D, Cry1F, or Cry9A Toxins. Toxins. 2020; 12 (2):104.

Chicago/Turabian Style

Xiaoyi Li; Kazuhisa Miyamoto; Yoko Takasu; Sanae Wada; Tetsuya Iizuka; Satomi Adegawa; Ryoichi Sato; Kenji Watanabe. 2020. "ATP-Binding Cassette Subfamily a Member 2 Is a Functional Receptor for Bacillus thuringiensis Cry2A Toxins in Bombyx mori, But Not for Cry1A, Cry1C, Cry1D, Cry1F, or Cry9A Toxins." Toxins 12, no. 2: 104.

Review
Published: 19 February 2019 in Toxins
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When ABC transporter family C2 (ABCC2) and ABC transporter family B1 (ABCB1) were heterologously expressed in non-susceptible cultured cells, the cells swelled in response to Cry1A and Cry3 toxins, respectively. Consistent with the notion that 3D-Cry toxins form cation-permeable pores, Bombyx mori ABCC2 (BmABCC2) facilitated cation-permeable pore formation by Cry1A when expressed in Xenopus oocytes. Furthermore, BmABCC2 had a high binding affinity (KD) to Cry1Aa of 3.1 × 10−10 M. These findings suggest that ABC transporters, including ABCC2 and ABCB1, are functional receptors for 3D-Cry toxins. In addition, the Cry2 toxins most distant from Cry1A toxins on the phylogenetic tree used ABC transporter A2 as a receptor. These data suggest that 3D-Cry toxins use ABC transporters as receptors. In terms of inducing cell swelling, ABCC2 has greater activity than cadherin-like receptor. The pore opening of ABC transporters was hypothesized to be linked to their receptor function, but this was repudiated by experiments using mutants deficient in export activity. The synergistic relationship between ABCC2 and cadherin-like receptor explains their ability to cause resistance in one species of insect.

ACS Style

Ryoichi Sato; Satomi Adegawa; Xiaoyi Li; Shiho Tanaka; Haruka Endo. Function and Role of ATP-Binding Cassette Transporters as Receptors for 3D-Cry Toxins. Toxins 2019, 11, 124 .

AMA Style

Ryoichi Sato, Satomi Adegawa, Xiaoyi Li, Shiho Tanaka, Haruka Endo. Function and Role of ATP-Binding Cassette Transporters as Receptors for 3D-Cry Toxins. Toxins. 2019; 11 (2):124.

Chicago/Turabian Style

Ryoichi Sato; Satomi Adegawa; Xiaoyi Li; Shiho Tanaka; Haruka Endo. 2019. "Function and Role of ATP-Binding Cassette Transporters as Receptors for 3D-Cry Toxins." Toxins 11, no. 2: 124.

Journal article
Published: 19 June 2018 in Peptides
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Enteroendocrine cells in the insect midgut are thought to secrete peptide hormones in response to the nutritional state. However, the role of dietary compounds in inducing peptide hormone secretion from enteroendocrine cells in insects remains unknown. In the present study, we demonstrated that several dietary compounds from mulberry leaves, including glucose, amino acids, and the secondary metabolite chlorogenic acid, induced significant secretion of tachykinin-related peptides from isolated silkworm midguts at the luminal concentrations measured in fed larvae. This study provides evidence that the insect midgut senses a non-nutritious secondary metabolite in addition to nutrient metabolites to monitor luminal food status and secretes a feeding regulatory hormone, suggesting that a unique dietary sensory system modulates insect feeding via enteroendocrine control.

ACS Style

Takayuki Yamagishi; Haruka Endo; Keisuke Fukumura; Shinji Nagata; Tohru Hayakawa; Satomi Adegawa; Mayu Kasubuchi; Ryoichi Sato. Glucose, some amino acids and a plant secondary metabolite, chlorogenic acid induce the secretion of a regulatory hormone, tachykinin-related peptide, from the silkworm midgut. Peptides 2018, 106, 21 -27.

AMA Style

Takayuki Yamagishi, Haruka Endo, Keisuke Fukumura, Shinji Nagata, Tohru Hayakawa, Satomi Adegawa, Mayu Kasubuchi, Ryoichi Sato. Glucose, some amino acids and a plant secondary metabolite, chlorogenic acid induce the secretion of a regulatory hormone, tachykinin-related peptide, from the silkworm midgut. Peptides. 2018; 106 ():21-27.

Chicago/Turabian Style

Takayuki Yamagishi; Haruka Endo; Keisuke Fukumura; Shinji Nagata; Tohru Hayakawa; Satomi Adegawa; Mayu Kasubuchi; Ryoichi Sato. 2018. "Glucose, some amino acids and a plant secondary metabolite, chlorogenic acid induce the secretion of a regulatory hormone, tachykinin-related peptide, from the silkworm midgut." Peptides 106, no. : 21-27.

Journal article
Published: 01 June 2018 in Journal of Biological Chemistry
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Bacillus thuringiensis Cry toxins are insecticidal proteins used widely for pest control. They are lethal to a restricted range of insects via specific interactions with insect receptors such as the ABC transporter subfamily members C2 (ABCC2) and C3 (ABCC3). However, it is still unclear how these different receptors contribute to insect susceptibility to Cry1A toxins. Here, we investigated the differences between the silkworm (Bombyx mori) ABCC2 (BmABCC2_S) and ABCC3 (BmABCC3) receptors in mediating Cry toxicity. Compared with BmABCC2_S, BmABCC3 exhibited 80- and 267-fold lower binding affinities to Cry1Aa and Cry1Ab, respectively, and these decreased affinities correlated well with the lower receptor activities of BmABCC3 for these Cry1A toxins. To identify the amino acid residues responsible for these differences, we constructed BmABCC3 variants containing a partial amino acid replacement with extracellular loops (ECLs) from BmABCC2_S. Replacing three amino acids from ECL 1 or 3 increased BmABCC3 activity toward Cry1Aa and enabled its activity toward Cry1Ab. Meanwhile, BmABCC2_S and BmABCC3 exhibited no receptor activities for Cry1Ca, Cry1Da, and Cry3Bb, correlating with markedly lower binding affinities for these Cry toxins. ABCC2 from a Cry1Ab-resistant B. mori strain (BmABCC2_R), which has a tyrosine insertion in ECL 2, displayed 93-fold lower binding affinity to Cry1Ab compared with BmABCC2_S but maintained high binding affinity to Cry1Aa. These results indicate that the Cry toxin–binding affinities of ABCC transporters are largely linked to the level of Cry susceptibility of ABCC-expressing cells and that the ABCC ECL structures determine the specificities to Cry toxins.

ACS Style

Haruka Endo; Shiho Tanaka; Satomi Adegawa; Fumika Ichino; Hiroko Tabunoki; Shingo Kikuta; Ryoichi Sato. Extracellular loop structures in silkworm ABCC transporters determine their specificities for Bacillus thuringiensis Cry toxins. Journal of Biological Chemistry 2018, 293, 8569 -8577.

AMA Style

Haruka Endo, Shiho Tanaka, Satomi Adegawa, Fumika Ichino, Hiroko Tabunoki, Shingo Kikuta, Ryoichi Sato. Extracellular loop structures in silkworm ABCC transporters determine their specificities for Bacillus thuringiensis Cry toxins. Journal of Biological Chemistry. 2018; 293 (22):8569-8577.

Chicago/Turabian Style

Haruka Endo; Shiho Tanaka; Satomi Adegawa; Fumika Ichino; Hiroko Tabunoki; Shingo Kikuta; Ryoichi Sato. 2018. "Extracellular loop structures in silkworm ABCC transporters determine their specificities for Bacillus thuringiensis Cry toxins." Journal of Biological Chemistry 293, no. 22: 8569-8577.

Short communication
Published: 07 April 2018 in Biochemical and Biophysical Research Communications
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The taste sensing system is crucial for food recognition in insects and other animals. It is commonly believed that insect gustatory receptors (Grs) expressed in gustatory organs are indispensable for host plant selection. Many behavioral studies have shown that mono- or oligo-phagous lepidopteran insects use the balance between feeding attractants and feeding deterrents in host plants and that these are sensed by taste organs for host plant recognition. However, the molecular mechanism underlying taste recognition, especially of feeding deterrents, remains to be elucidated. To better understand this mechanism, we studied orphan Grs, including Bombyx mori Gr (BmGr) 16, BmGr18, and BmGr53, from the mono-phagous insect, Bombyx mori. Using Calcium imaging in mammalian cells, we first confirmed in lepidoptera insects that three of the putative bitter Grs widely responded to structurally different feeding deterrents. Although the phylogenetic distance of these Grs was considerable, they responded to partially overlapping deterrents of plant secondary metabolites. These findings suggest that not only these three Grs but also most of the Grs that have been assigned to putative bitter Grs are feeding-deterrent receptors that play a role in host plant recognition.

ACS Style

Mayu Kasubuchi; Fumika Shii; Kana Tsuneto; Takayuki Yamagishi; Satomi Adegawa; Haruka Endo; Ryoichi Sato. Insect taste receptors relevant to host identification by recognition of secondary metabolite patterns of non-host plants. Biochemical and Biophysical Research Communications 2018, 499, 901 -906.

AMA Style

Mayu Kasubuchi, Fumika Shii, Kana Tsuneto, Takayuki Yamagishi, Satomi Adegawa, Haruka Endo, Ryoichi Sato. Insect taste receptors relevant to host identification by recognition of secondary metabolite patterns of non-host plants. Biochemical and Biophysical Research Communications. 2018; 499 (4):901-906.

Chicago/Turabian Style

Mayu Kasubuchi; Fumika Shii; Kana Tsuneto; Takayuki Yamagishi; Satomi Adegawa; Haruka Endo; Ryoichi Sato. 2018. "Insect taste receptors relevant to host identification by recognition of secondary metabolite patterns of non-host plants." Biochemical and Biophysical Research Communications 499, no. 4: 901-906.

Journal article
Published: 06 February 2018 in Insect Biochemistry and Molecular Biology
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ACS Style

Haruka Endo; Satomi Adegawa; Shingo Kikuta; Ryoichi Sato. The intracellular region of silkworm cadherin-like protein is not necessary to mediate the toxicity of Bacillus thuringiensis Cry1Aa and Cry1Ab toxins. Insect Biochemistry and Molecular Biology 2018, 94, 36 -41.

AMA Style

Haruka Endo, Satomi Adegawa, Shingo Kikuta, Ryoichi Sato. The intracellular region of silkworm cadherin-like protein is not necessary to mediate the toxicity of Bacillus thuringiensis Cry1Aa and Cry1Ab toxins. Insect Biochemistry and Molecular Biology. 2018; 94 ():36-41.

Chicago/Turabian Style

Haruka Endo; Satomi Adegawa; Shingo Kikuta; Ryoichi Sato. 2018. "The intracellular region of silkworm cadherin-like protein is not necessary to mediate the toxicity of Bacillus thuringiensis Cry1Aa and Cry1Ab toxins." Insect Biochemistry and Molecular Biology 94, no. : 36-41.

Journal article
Published: 01 January 2018 in Drug Discoveries & Therapeutics
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ACS Style

Fumika Ichino; Hidemasa Bono; Takeru Nakazato; Atsushi Toyoda; Asao Fujiyama; Kikuo Iwabuchi; Ryoichi Sato; Hiroko Tabunoki. Construction of a simple evaluation system for the intestinal absorption of an orally administered medicine using Bombyx mori larvae. Drug Discoveries & Therapeutics 2018, 12, 7 -15.

AMA Style

Fumika Ichino, Hidemasa Bono, Takeru Nakazato, Atsushi Toyoda, Asao Fujiyama, Kikuo Iwabuchi, Ryoichi Sato, Hiroko Tabunoki. Construction of a simple evaluation system for the intestinal absorption of an orally administered medicine using Bombyx mori larvae. Drug Discoveries & Therapeutics. 2018; 12 (1):7-15.

Chicago/Turabian Style

Fumika Ichino; Hidemasa Bono; Takeru Nakazato; Atsushi Toyoda; Asao Fujiyama; Kikuo Iwabuchi; Ryoichi Sato; Hiroko Tabunoki. 2018. "Construction of a simple evaluation system for the intestinal absorption of an orally administered medicine using Bombyx mori larvae." Drug Discoveries & Therapeutics 12, no. 1: 7-15.

Journal article
Published: 01 December 2017 in Insect Biochemistry and Molecular Biology
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Because Bombyx mori ABC transporter C2 (BmABCC2) has 1000-fold higher potential than B. mori cadherin-like protein as a receptor for Bacillus thuringiensis Cry1Aa toxin (Tanaka et al., 2013), the gate-opening ability of the latent pore under six extracellular loops (ECLs) of BmABCC2 was expected to be the reason for its higher potential (Heckel, 2012). In this study, cell swelling assays in Sf9 cells showed that BmABCC2 mutants lacking substrate-excreting activity retained receptor activity, indicating that the gate-opening activity of BmABCC2 is not responsible for Cry1Aa toxicity. The analysis of 29 BmABCC2 mutants demonstrated that DYWL of ECL 4 comprise a putative binding site to Cry1Aa. This suggests that specific toxicity of Cry1Aa toxin to a restricted range of lepidopteran insects is dependent on conservation and variation in the amino acid residues around DYWL of ECL 4 in the ABCC2.

ACS Style

Shiho Tanaka; Haruka Endo; Satomi Adegawa; Ami Iizuka; Kazuhiro Imamura; Shingo Kikuta; Ryoichi Sato. Bombyx mori ABC transporter C2 structures responsible for the receptor function of Bacillus thuringiensis Cry1Aa toxin. Insect Biochemistry and Molecular Biology 2017, 91, 44 -54.

AMA Style

Shiho Tanaka, Haruka Endo, Satomi Adegawa, Ami Iizuka, Kazuhiro Imamura, Shingo Kikuta, Ryoichi Sato. Bombyx mori ABC transporter C2 structures responsible for the receptor function of Bacillus thuringiensis Cry1Aa toxin. Insect Biochemistry and Molecular Biology. 2017; 91 ():44-54.

Chicago/Turabian Style

Shiho Tanaka; Haruka Endo; Satomi Adegawa; Ami Iizuka; Kazuhiro Imamura; Shingo Kikuta; Ryoichi Sato. 2017. "Bombyx mori ABC transporter C2 structures responsible for the receptor function of Bacillus thuringiensis Cry1Aa toxin." Insect Biochemistry and Molecular Biology 91, no. : 44-54.

Article
Published: 01 December 2017 in Archives of Insect Biochemistry and Physiology
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Honey bee (Apis mellifera) workers contribute to the maintenance of colonies in various ways. The primary functions of workers are divided into two types depending on age: young workers (nurses) primarily engage in such behaviors as cleaning and food handling within the hive, whereas older workers (foragers) acquire floral nutrients beyond the colony. Concomitant with this age-dependent change in activity, physiological changes occur in the tissues and organs of workers. Nurses supply younger larvae with honey containing high levels of glucose and supply older larvae with honey containing high levels of fructose. Given that nurses must determine both the concentration and type of sugar used in honey, gustatory receptors (Gr) expressed in the chemosensory organs likely play a role in distinguishing between sugars. Glucose is recognized by Gr1 in honey bees (AmGr1); however, it remains unclear which Gr are responsible for fructose recognition. This study aimed to identify fructose receptors in honey bees and reported that AmGr3, when transiently expressed in Xenopus oocytes, responded only to fructose, and to no other sugars. We analyzed expression levels of AmGr3 to identify which tissues and organs of workers are involved in fructose recognition and determined that expression of AmGr3 was particularly high in the antennae and legs of nurses. Our results suggest that nurses use their antennae and legs to recognize fructose, and that AmGr3 functions as an accurate nutrient sensor used to maintain food quality in honey bee hives.

ACS Style

Tomoyuki Takada; Taiyo Sasaki; Ryoichi Sato; Shingo Kikuta; Maki N. Inoue. Differential expression of a fructose receptor gene in honey bee workers according to age and behavioral role. Archives of Insect Biochemistry and Physiology 2017, 97, e21437 .

AMA Style

Tomoyuki Takada, Taiyo Sasaki, Ryoichi Sato, Shingo Kikuta, Maki N. Inoue. Differential expression of a fructose receptor gene in honey bee workers according to age and behavioral role. Archives of Insect Biochemistry and Physiology. 2017; 97 (2):e21437.

Chicago/Turabian Style

Tomoyuki Takada; Taiyo Sasaki; Ryoichi Sato; Shingo Kikuta; Maki N. Inoue. 2017. "Differential expression of a fructose receptor gene in honey bee workers according to age and behavioral role." Archives of Insect Biochemistry and Physiology 97, no. 2: e21437.

Journal article
Published: 01 December 2017 in Peptides
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In this study, we examined insect and human ABCC transporters closely related to the lepidopteran ABC transporter C2 (ABCC2), a powerful receptor for the Bacillus thuringiensis Cry toxin, for their responses to various Cry toxins. ABCC2 and the lepidopteran ABC transporter C3 (ABCC3) conferred cultured cells with susceptibility to a lepidopteran-specific Cry1Aa toxin but not to lepidopteran-specific Cry1Ca and Cry1Da. One coleopteran ABCC transporter specifically responded to a coleopteran-specific Cry8Ca toxin. ABCC transporters from a dipteran insect and humans did not respond to any of the tested Cry toxins that are active to lepidopteran and coleopteran insects. These results yield important information for our understanding of insect specificity of Cry toxins and provide the first demonstration of a coleopteran ABCC transporter that serves as a Cry toxin receptor.

ACS Style

Haruka Endo; Shiho Tanaka; Kazuhiro Imamura; Satomi Adegawa; Shingo Kikuta; Ryoichi Sato. Cry toxin specificities of insect ABCC transporters closely related to lepidopteran ABCC2 transporters. Peptides 2017, 98, 86 -92.

AMA Style

Haruka Endo, Shiho Tanaka, Kazuhiro Imamura, Satomi Adegawa, Shingo Kikuta, Ryoichi Sato. Cry toxin specificities of insect ABCC transporters closely related to lepidopteran ABCC2 transporters. Peptides. 2017; 98 ():86-92.

Chicago/Turabian Style

Haruka Endo; Shiho Tanaka; Kazuhiro Imamura; Satomi Adegawa; Shingo Kikuta; Ryoichi Sato. 2017. "Cry toxin specificities of insect ABCC transporters closely related to lepidopteran ABCC2 transporters." Peptides 98, no. : 86-92.

Journal article
Published: 01 October 2017 in Microbial Pathogenesis
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Leptospirosis causes severe clinical signs more frequently in men than in women, but the mechanism underlying the gender differences in leptospirosis remains unclear. In this study, petechial hemorrhage was observed in male but not in female hamster lung tissues infected with Leptospira interrogans serovar Hebdomadis at 120 h pi, demonstrating that male hamsters were more susceptible to the development of a severe disease upon Leptospira infection. No leptospiral DNA was detected in the lung tissues at 120 h pi when pulmonary hemorrhage was observed, indicating that pulmonary hemorrhage is attributable to the immune reactions of the host rather than from the direct effect of leptospires. The upregulation of nitric oxide synthase genes in the hamsters without pulmonary hemorrhage, inos and enos in female hamsters at 96 h pi and enos in male animals without hemorrhage at 120 h pi, may suggest that nitric oxide has a suppressive effect on leptospirosis-associated pulmonary hemorrhage.

ACS Style

Rina Tomizawa; Hiromu Sugiyama; Ryoichi Sato; Makoto Ohnishi; Nobuo Koizumi. Male-specific pulmonary hemorrhage and cytokine gene expression in golden hamster in early-phase Leptospira interrogans serovar Hebdomadis infection. Microbial Pathogenesis 2017, 111, 33 -40.

AMA Style

Rina Tomizawa, Hiromu Sugiyama, Ryoichi Sato, Makoto Ohnishi, Nobuo Koizumi. Male-specific pulmonary hemorrhage and cytokine gene expression in golden hamster in early-phase Leptospira interrogans serovar Hebdomadis infection. Microbial Pathogenesis. 2017; 111 ():33-40.

Chicago/Turabian Style

Rina Tomizawa; Hiromu Sugiyama; Ryoichi Sato; Makoto Ohnishi; Nobuo Koizumi. 2017. "Male-specific pulmonary hemorrhage and cytokine gene expression in golden hamster in early-phase Leptospira interrogans serovar Hebdomadis infection." Microbial Pathogenesis 111, no. : 33-40.

Preprint
Published: 24 March 2017
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Desiccation-tolerant cultured cells Pv11 derived from the anhydrobiotic Polypedilum vanderplanki embryo endure complete desiccation because of their ametabolic state and resume their metabolism after rehydration. These features led us to develop a novel dry preservation technology for enzymes as it was still unclear whether Pv11 cells preserved an exogenous enzyme in the dry state. This study shows that Pv11 cells protect an exogenous desiccation-sensitive enzyme, luciferase, preserving the enzymatic activity even after dry storage for 372 days at room temperature. A process including pre-incubation with trehalose, dehydration, storage, and rehydration allowed Pv11 (Pvll-Luc) cells stably expressing luciferase to survive desiccation and still emit luminescence caused by luciferase after rehydration. Luminescence produced by luciferase in Pvll-Luc cells after rehydration did not significantly decrease in presence of a translation inhibitor, showing that the activity did not derive from de novo enzyme synthesis following the resumption of cell metabolism. These findings indicate that the surviving Pv11 cells almost completely protect luciferase during desiccation. Lacking of the preincubation step resulted in the loss of luciferase activity after rehydration. We showed that preincubation with trehalose associated to induction of desiccation-tolerant related genes in Pv11 cells allowed effective in vivo preservation of enzymes in the dry state.

ACS Style

Shingo Kikuta; Shunsuke J. Watanabe; Ryoichi Sato; Oleg Gusev; Alexander Nesmelov; Yoichiro Sogame; Richard Cornette; Takahiro Kikawada. Towards water-free biobanks: long-term dry-preservation at room temperature of desiccation-sensitive enzyme luciferase in air-dried insect cells. 2017, 120063 .

AMA Style

Shingo Kikuta, Shunsuke J. Watanabe, Ryoichi Sato, Oleg Gusev, Alexander Nesmelov, Yoichiro Sogame, Richard Cornette, Takahiro Kikawada. Towards water-free biobanks: long-term dry-preservation at room temperature of desiccation-sensitive enzyme luciferase in air-dried insect cells. . 2017; ():120063.

Chicago/Turabian Style

Shingo Kikuta; Shunsuke J. Watanabe; Ryoichi Sato; Oleg Gusev; Alexander Nesmelov; Yoichiro Sogame; Richard Cornette; Takahiro Kikawada. 2017. "Towards water-free biobanks: long-term dry-preservation at room temperature of desiccation-sensitive enzyme luciferase in air-dried insect cells." , no. : 120063.

Journal article
Published: 01 February 2017 in Biochimica et Biophysica Acta (BBA) - Proteins and Proteomics
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Information about the receptor-interaction region of Cry toxins, insecticidal proteins produced by Bacillus thuringiensis, is needed to elucidate the mode of action of Cry toxins and improve their toxicity through protein engineering. We analyzed the interaction sites on Cry1Aa with ABC transporter C2 (ABCC2), one of the most important Cry1A toxin receptors. A competitive binding assay revealed that the Bombyx mori ABCC2 (BmABCC2) Cry1A binding site was the same as the BtR175 binding site, suggesting that the loop region of Cry1Aa domain II is a binding site. Next, we constructed several domain II loop mutant toxins and tested their binding affinity in an SPR analysis, and also performed a cell swelling assay to evaluate receptor-mediated cytotoxicity. Our results indicate that the loop regions required for BtR175 and BmABCC2 binding and the regions important for cytotoxicity partially overlap. Our results also suggest that receptor binding is necessary but not sufficient for cytotoxicity. This is the first report showing the region of interaction between ABCC2 and Cry1Aa and the cytotoxicity-relevant properties of the Cry1Aa domain II loop region.

ACS Style

Satomi Adegawa; Yui Nakama; Haruka Endo; Naoki Shinkawa; Shingo Kikuta; Ryoichi Sato. The domain II loops of Bacillus thuringiensis Cry1Aa form an overlapping interaction site for two Bombyx mori larvae functional receptors, ABC transporter C2 and cadherin-like receptor. Biochimica et Biophysica Acta (BBA) - Proteins and Proteomics 2017, 1865, 220 -231.

AMA Style

Satomi Adegawa, Yui Nakama, Haruka Endo, Naoki Shinkawa, Shingo Kikuta, Ryoichi Sato. The domain II loops of Bacillus thuringiensis Cry1Aa form an overlapping interaction site for two Bombyx mori larvae functional receptors, ABC transporter C2 and cadherin-like receptor. Biochimica et Biophysica Acta (BBA) - Proteins and Proteomics. 2017; 1865 (2):220-231.

Chicago/Turabian Style

Satomi Adegawa; Yui Nakama; Haruka Endo; Naoki Shinkawa; Shingo Kikuta; Ryoichi Sato. 2017. "The domain II loops of Bacillus thuringiensis Cry1Aa form an overlapping interaction site for two Bombyx mori larvae functional receptors, ABC transporter C2 and cadherin-like receptor." Biochimica et Biophysica Acta (BBA) - Proteins and Proteomics 1865, no. 2: 220-231.

Journal article
Published: 13 January 2017 in BMC Genomics
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Various insect species have been added to genomic databases over the years. Thus, researchers can easily obtain online genomic information on invertebrates and insects. However, many incorrectly annotated genes are included in these databases, which can prevent the correct interpretation of subsequent functional analyses. To address this problem, we used a combination of dry and wet bench processes to select functional genes from public databases. Enolase is an important glycolytic enzyme in all organisms. We used a combination of dry and wet bench processes to identify functional enolases in the silkworm Bombyx mori (BmEno). First, we detected five annotated enolases from public databases using a Hidden Markov Model (HMM) search, and then through cDNA cloning, Northern blotting, and RNA-seq analysis, we revealed three functional enolases in B. mori: BmEno1, BmEno2, and BmEnoC. BmEno1 contained a conserved key amino acid residue for metal binding and substrate binding in other species. However, BmEno2 and BmEnoC showed a change in this key amino acid. Phylogenetic analysis showed that BmEno2 and BmEnoC were distinct from BmEno1 and other enolases, and were distributed only in lepidopteran clusters. BmEno1 was expressed in all of the tissues used in our study. In contrast, BmEno2 was mainly expressed in the testis with some expression in the ovary and suboesophageal ganglion. BmEnoC was weakly expressed in the testis. Quantitative RT-PCR showed that the mRNA expression of BmEno2 and BmEnoC correlated with testis development; thus, BmEno2 and BmEnoC may be related to lepidopteran-specific spermiogenesis. We identified and characterized three functional enolases from public databases with a combination of dry and wet bench processes in the silkworm B. mori. In addition, we determined that BmEno2 and BmEnoC had species-specific functions. Our strategy could be helpful for the detection of minor genes and functional genes in non-model organisms from public databases. The online version of this article (doi:10.1186/s12864-016-3455-y) contains supplementary material, which is available to authorized users.

ACS Style

Akira Kikuchi; Takeru Nakazato; Katsuhiko Ito; Yosui Nojima; Takeshi Yokoyama; Kikuo Iwabuchi; Hidemasa Bono; Atsushi Toyoda; Asao Fujiyama; Ryoichi Sato; Hiroko Tabunoki. Identification of functional enolase genes of the silkworm Bombyx mori from public databases with a combination of dry and wet bench processes. BMC Genomics 2017, 18, 83 .

AMA Style

Akira Kikuchi, Takeru Nakazato, Katsuhiko Ito, Yosui Nojima, Takeshi Yokoyama, Kikuo Iwabuchi, Hidemasa Bono, Atsushi Toyoda, Asao Fujiyama, Ryoichi Sato, Hiroko Tabunoki. Identification of functional enolase genes of the silkworm Bombyx mori from public databases with a combination of dry and wet bench processes. BMC Genomics. 2017; 18 (1):83.

Chicago/Turabian Style

Akira Kikuchi; Takeru Nakazato; Katsuhiko Ito; Yosui Nojima; Takeshi Yokoyama; Kikuo Iwabuchi; Hidemasa Bono; Atsushi Toyoda; Asao Fujiyama; Ryoichi Sato; Hiroko Tabunoki. 2017. "Identification of functional enolase genes of the silkworm Bombyx mori from public databases with a combination of dry and wet bench processes." BMC Genomics 18, no. 1: 83.

Article
Published: 20 December 2016 in FEBS Letters
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The Bacillus thuringiensis Cry toxin causes swelling and necrosis in insect cells, but the route(s) and significance of the water influx involved in its cytotoxicity are unclear. Here, we assessed the role of aquaporins (AQPs), known as water channels, in Cry toxin intoxication. An AQP inhibitor did not interfere with any known process to form the toxin pore, but it diminished the cell swelling and loss of membrane integrity induced by the Cry toxin. Overexpression of AQPs facilitated water influx and cytotoxicity. Our results demonstrate that water influx via aquaporin directly determines necrotic cell death induced by the Cry toxin. This article is protected by copyright. All rights reserved.

ACS Style

Haruka Endo; Masaaki Azuma; Satomi Adegawa; Shingo Kikuta; Ryoichi Sato. Water influx via aquaporin directly determines necrotic cell death induced by the Bacillus thuringiensis Cry toxin. FEBS Letters 2016, 591, 56 -64.

AMA Style

Haruka Endo, Masaaki Azuma, Satomi Adegawa, Shingo Kikuta, Ryoichi Sato. Water influx via aquaporin directly determines necrotic cell death induced by the Bacillus thuringiensis Cry toxin. FEBS Letters. 2016; 591 (1):56-64.

Chicago/Turabian Style

Haruka Endo; Masaaki Azuma; Satomi Adegawa; Shingo Kikuta; Ryoichi Sato. 2016. "Water influx via aquaporin directly determines necrotic cell death induced by the Bacillus thuringiensis Cry toxin." FEBS Letters 591, no. 1: 56-64.

Journal article
Published: 29 November 2016 in The FEBS Journal
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Bacillus thuringiensis produces Cry toxins, which are used as insecticides in sprays or in transgenic crops. However, little is known about the function of Cry toxin receptors and the mechanisms that determine their binding specificity and activity. In this study, the cRNAs of Bombyx mori ABC transporter C2 (BmABCC2), the toxin-binding region of cadherin-like receptor (BtR175-TBR), or aminopeptidase N1 (BmAPN1) were injected to Xenopus oocytes, and the Cry1Aa-dependent cation selective pore-formation activities of these receptors were analyzed using a two-electrode voltage clamp. Cation current passing through the pores was detected within 25 s, which increased in a linear fashion in BmABCC2-expressing oocytes treated with 88 nM Cry1Aa. This result suggested that Cry1Aa continuously made stable pores with the help of BmABCC2. In contrast, no cation current was observed until 60-min after incubation with 500 nM Cry1Aa in BtR175TBR-expressing oocytes even though oligomerization of Cry1Aa progressed. This result indicated that, in the presence of BtR175-TBR, most of the oligomerized toxin could not enter the cell membrane. However, oocytes that simultaneously expressed both receptors demonstrated that BtR175-TBR exerted a synergistic effect with BmABCC2 on pore formation in the presence of 22 nM Cry1Aa. These results confirm that the main reason for moderate-level resistance in insects missing the cadherin-like receptor but expressing ABCC2 is the absence of a similar synergistic promotion of toxin oligomerization. Similar to results from our previous report evaluating ectopic expression in the Sf9/Baculovirus system, BmAPN1 could not by itself cause Cry1A-related pore formation, despite the fact that BmAPN1 gathered toxin on the oocytes as well as did BmABCC2. This article is protected by copyright. All rights reserved.

ACS Style

Shiho Tanaka; Haruka Endo; Satomi Adegawa; Shingo Kikuta; Ryoichi Sato. Functional characterization ofBacillus thuringiensisCry toxin receptors explains resistance in insects. The FEBS Journal 2016, 283, 4474 -4490.

AMA Style

Shiho Tanaka, Haruka Endo, Satomi Adegawa, Shingo Kikuta, Ryoichi Sato. Functional characterization ofBacillus thuringiensisCry toxin receptors explains resistance in insects. The FEBS Journal. 2016; 283 (24):4474-4490.

Chicago/Turabian Style

Shiho Tanaka; Haruka Endo; Satomi Adegawa; Shingo Kikuta; Ryoichi Sato. 2016. "Functional characterization ofBacillus thuringiensisCry toxin receptors explains resistance in insects." The FEBS Journal 283, no. 24: 4474-4490.

Journal article
Published: 01 August 2016 in Insect Biochemistry and Molecular Biology
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Insect gustatory receptors (Grs) are members of a large family of proteins with seven transmembrane domains that provide insects with the ability to detect chemical signals critical for feeding, mating, and oviposition. To date, 69 Bombyx mori Grs (BmGrs) genes have been identified via genome studies. BmGr9 has been shown to respond specifically to fructose and to function as a ligand-gated ion channel selectively activated by fructose. However, the sites where this Gr are expressed remain unclear. We demonstrated using reverse transcription (RT)-PCR that BmGr9 is widely expressed in the central nervous system (CNS), as well as oral sensory organs. Additionally, immunohistochemistry was performed using anti-BmGr9 antiserum to show that BmGr9 is expressed in cells of the oral sensory organs, including the maxillary galea, maxillary palps, labrum, and labium, as well as in putative neurosecretory cells of the CNS. Furthermore, double immunohistochemical analysis showed that most BmGr9-expressing cells co-localized with putative neuropeptide F1-expressing cells in the brain, suggesting that BmGr9 is involved in the promotion of feeding behaviors. In addition, a portion of BmGr9-expressing cells in the brain co-localized with cells expressing BmGr6, a molecule of the sugar receptor clade, suggesting that sugars other than fructose are involved in the regulation of feeding behaviors in B. mori larvae.

ACS Style

Dingze Mang; Min Shu; Shiho Tanaka; Shinji Nagata; Tomoyuki Takada; Haruka Endo; Shingo Kikuta; Hiroko Tabunoki; Kikuo Iwabuchi; Ryoichi Sato. Expression of the fructose receptor BmGr9 and its involvement in the promotion of feeding, suggested by its co-expression with neuropeptide F1 in Bombyx mori. Insect Biochemistry and Molecular Biology 2016, 75, 58 -69.

AMA Style

Dingze Mang, Min Shu, Shiho Tanaka, Shinji Nagata, Tomoyuki Takada, Haruka Endo, Shingo Kikuta, Hiroko Tabunoki, Kikuo Iwabuchi, Ryoichi Sato. Expression of the fructose receptor BmGr9 and its involvement in the promotion of feeding, suggested by its co-expression with neuropeptide F1 in Bombyx mori. Insect Biochemistry and Molecular Biology. 2016; 75 ():58-69.

Chicago/Turabian Style

Dingze Mang; Min Shu; Shiho Tanaka; Shinji Nagata; Tomoyuki Takada; Haruka Endo; Shingo Kikuta; Hiroko Tabunoki; Kikuo Iwabuchi; Ryoichi Sato. 2016. "Expression of the fructose receptor BmGr9 and its involvement in the promotion of feeding, suggested by its co-expression with neuropeptide F1 in Bombyx mori." Insect Biochemistry and Molecular Biology 75, no. : 58-69.