Springer Science and Business Media LLC Acta Medica Okayama 2662-9984 7 1 2026 Diversity of hymenopteran assemblages prevailed during reproductive stage of cotton—implications for natural control 1039 EN Md Mizanur Rahman Insect Biotechnology and Biopesticide Laboratory, Department of Entomology, Bangladesh Agricultural University Tohru Hayakawa Graduate School of Interdisciplinary Science and Engineering in Health Systems, Okayama University Mohammad Tofazzal Hossain Howlader Insect Biotechnology and Biopesticide Laboratory, Department of Entomology, Bangladesh Agricultural University Hymenoptera is one of the most important insect orders contributes to pollination, biocontrol and natural control largely unexplored from Bangladesh. We, therefore, assess the diversity and relative abundance of hymenopteran insects in cotton fields from five locations of Bangladesh using five types of traps (Malaise, Yellow Pan, Fluorescent Yellow Pan, Pitfall, and Sweep Net). Analysis of samples revealed presence of diverse type of hymenopteran insects from the studied locations, with a total of 1642 insects collected, representing 28 morphospecies and 16 families of the Hymenoptera. Formicidae, Braconidae, Diapridae, Figitidae, Ichneumonidae, Scelionidae constitutes the major families comprises of 36.37, 25.09, 5.36, 5.24, 5.18 and 4.93% of the populations respectively. Among the locations, cotton fields of Rangpur showed the highest species richness, diversity indices (Simpson, and Shannon values), while cotton fields located at Jashore had the lowest. Yellow and fluorescent pan traps captured insects from nearly all families, indicating broad sampling effectiveness. Malaise traps also sampled a wide range of families except for low captures of Formicidae. Pitfall traps primarily targeted Formicidae, consistent with their ground-dwelling hymenopterans. Sweep nets captured fewer families, reflecting a more selective sampling role. Among functional guilds, parasitoids were the most abundant, followed by predators, pollinators, and herbivores of hymenopteran insects. Overall, the hymenopteran community in cotton agroecosystems from Bangladesh was dominated by parasitoid families, indicating strong potential for natural biological control. Leveraging this natural enemy dominance could significantly reduce dependence on chemical insecticides. These findings support the integration of parasitoid conservation strategies into national Integrated Pest Management (IPM) programs and landscape-level pest management policies. No potential conflict of interest relevant to this article was reported.

MDPI AG Acta Medica Okayama 2079-7737 14 5 2025 Mutagenesis Targeting the S153 Residue Within the Transmembrane β-Hairpin of Mosquito-Larvicidal Mpp46Ab Affects Its Toxicity and the Synergistic Toxicity with Cry4Aa 489 EN Tohru Hayakawa Graduate School of Interdisciplinary Science and Engineering in Health Systems, Okayama University Syun Yamaoka Graduate School of Interdisciplinary Science and Engineering in Health Systems, Okayama University Mami Asakura Graduate School of Interdisciplinary Science and Engineering in Health Systems, Okayama University Minako Hirano Graduate School of Interdisciplinary Science and Engineering in Health Systems, Okayama University Toru Ide Graduate School of Interdisciplinary Science and Engineering in Health Systems, Okayama University We constructed a library of Mpp46Ab mutants, in which S153 within the transmembrane β-hairpin was randomly replaced by other amino acids. Mutagenesis and subsequent primary screening yielded 10 different Mpp46Ab mutants in addition to the wild type. Remarkably, S153 was replaced with a more hydrophobic amino acid in most of the mutants, and the S153I mutant in particular exhibited significantly increased toxicity. Electrophysiologic analysis using artificial lipid bilayers revealed that the single-channel conductance and PK/PCl permeability ratio were significantly increased for S153I pores. This suggests that the formation of highly ion-permeable and highly cation-selective toxin pores increases the influx of cations and water into cells, thereby facilitating osmotic shock. In addition, the S153F, S153L, and S153I mutants exhibited significantly reduced synergistic toxicity with Cry4Aa. Electrophysiologic analysis showed that the S153F, S153L, and S153I mutants form toxin pores with a significantly reduced PK/PNa permeability ratio and a significantly increased PK/PCa permeability ratio compared to wild-type pores. Thus, our results suggest that pore formation is central to the insecticidal activity of Mpp46Ab and that the ion permeability of toxin pores is a potential indicator correlated with both toxicity and synergistic toxicity with other toxins. No potential conflict of interest relevant to this article was reported.

Springer Science and Business Media LLC Acta Medica Okayama 0343-8651 81 3 2024 Mutational analysis of the transmembrane α4-helix of Bacillus thuringiensis mosquito-larvicidal Cry4Aa toxin 80 EN Hirokazu Takahashi Graduate School of Interdisciplinary Science and Engineering in Health Systems, Okayama University Mami Asakura Graduate School of Interdisciplinary Science and Engineering in Health Systems, Okayama University Toru Ide Graduate School of Interdisciplinary Science and Engineering in Health Systems, Okayama University Tohru Hayakawa Graduate School of Interdisciplinary Science and Engineering in Health Systems, Okayama University Cry4Aa, produced by Bacillus thuringiensis subsp. israelensis, exhibits specific toxicity to larvae of medically important mosquito genera. Cry4Aa functions as a pore-forming toxin, and a helical hairpin (α4-loop-α5) of domain I is believed to be the transmembrane domain that forms toxin pores. Pore formation is considered to be a central mode of Cry4Aa action, but the relationship between pore formation and toxicity is poorly understood. In the present study, we constructed Cry4Aa mutants in which each polar amino acid residues within the transmembrane α4 helix was replaced with glutamic acid. Bioassays using Culex pipiens mosquito larvae and subsequent ion permeability measurements using symmetric KCl solution revealed an apparent correlation between toxicity and toxin pore conductance for most of the Cry4Aa mutants. In contrast, the Cry4Aa mutant H178E was a clear exception, almost losing its toxicity but still exhibiting a moderately high conductivity of about 60% of the wild-type. Furthermore, the conductance of the pore formed by the N190E mutant (about 50% of the wild-type) was close to that of H178E, but the toxicity was significantly higher than that of H178E. Ion selectivity measurements using asymmetric KCl solution revealed a significant decrease in cation selectivity of toxin pores formed by H178E compared to N190E. Our data suggest that the toxicity of Cry4Aa is primarily pore related. The formation of toxin pores that are highly ion-permeable and also highly cation-selective may enhance the influx of cations and water into the target cell, thereby facilitating the eventual death of mosquito larvae. No potential conflict of interest relevant to this article was reported.

MDPI Acta Medica Okayama 2079-7737 12 12 2023 Random Mutational Analysis Targeting Residue K155 within the Transmembrane β-Hairpin of the Mosquitocidal Mpp46Ab Toxin 1481 EN Midoka Miyazaki Graduate School of Interdisciplinary Science and Engineering in Health Systems, Okayama University Mami Asakura Graduate School of Interdisciplinary Science and Engineering in Health Systems, Okayama University Toru Ide Graduate School of Interdisciplinary Science and Engineering in Health Systems, Okayama University Tohru Hayakawa Graduate School of Interdisciplinary Science and Engineering in Health Systems, Okayama University Mpp46Ab is a mosquito-larvicidal pore-forming toxin derived from Bacillus thuringiensis TK-E6. Pore formation is believed to be a central mode of Mpp46Ab action, and the cation selectivity of the channel pores, in particular, is closely related to its mosquito-larvicidal activity. In the present study, we constructed a mutant library in which residue K155 within the transmembrane β-hairpin was randomly replaced with other amino acid residues. Upon mutagenesis and following primary screening using Culex pipiens mosquito larvae, we obtained 15 mutants in addition to the wild-type toxin. Bioassays using purified proteins revealed that two mutants, K155E and K155I, exhibited toxicity significantly higher than that of the wild-type toxin. Although increased cation selectivity was previously reported for K155E channel pores, we demonstrated in the present study that the cation selectivity of K155I channel pores was also significantly increased. Considering the characteristics of the amino acids, the charge of residue 155 may not directly affect the cation selectivity of Mpp46Ab channel pores. Replacement of K155 with glutamic acid or isoleucine may induce a similar conformational change in the region associated with the ion selectivity of the Mpp46Ab channel pores. Mutagenesis targeting the transmembrane β-hairpin may be an effective strategy for enhancing the ion permeability of the channel pores and the resulting mosquito-larvicidal activity of Mpp46Ab. No potential conflict of interest relevant to this article was reported.

Springer Science and Business Media LLC Acta Medica Okayama 0003-6862 57 1 2021 Characteristics of channel pores formed by Bacillus thuringiensis mosquito-larvicidal Cry4Aa toxin 63 70 EN Yuri Shiraishi Graduate School of Interdisciplinary Science and Engineering in Health Systems, Okayama University Tomoya Shiozaki Graduate School of Interdisciplinary Science and Engineering in Health Systems, Okayama University Mami Asakura Graduate School of Interdisciplinary Science and Engineering in Health Systems, Okayama University Toru Ide Graduate School of Interdisciplinary Science and Engineering in Health Systems, Okayama University Tohru Hayakawa Graduate School of Interdisciplinary Science and Engineering in Health Systems, Okayama University Cry4Aa toxin produced by Bacillus thuringiensis subsp. israelensis exhibits specific toxicity to larvae of medically-important mosquito genera. In the present study, we analyzed the characteristics of channel-pores formed by recombinant Cry4Aa in a solvent-free planar lipid bilayer. Stable channel-currents were observed in electrophysiologic measurements, and the single-channel conductance was 187 ± 10 pS in symmetrical buffer containing 150 mM KCl. The channel-pores formed by Cry4Aa were cation selective, with an estimated PK/PCl permeability ratio of 4.9. In addition, Cry4Aa channel-pores exhibited apparent cation preference in the order Na+ > K+, Na+ > Ca2+, and K+ > Ca2+. Although the effect was limited, the cation preference of Cry4Aa channel-pores seemed to be correlated with toxicity. Culex pipiens mosquito larvae reared in NaCl solution exhibited greater sensitivity to Cry4Aa, particularly early period after exposure. The presence of cations that preferentially translocate through Cry4Aa channel-pores may facilitate excessive influx of water into the midgut cells, leading to colloid-osmotic lysis. Whereas CaCl2 had some effect on the mosquito-larvicidal activity of Cry4Aa, KCl had no effect. The effect of some cations may be mitigated by the variety of ion channels present on the midgut cell membrane. No potential conflict of interest relevant to this article was reported.

MDPI Acta Medica Okayama 2072-666X 11 12 2020 A Lipid Bilayer Formed on a Hydrogel Bead for Single Ion Channel Recordings 1070 EN Minako Hirano Bio Photonics Laboratory, The Graduate School for the Creation of New Photonics Industries Daiki Yamamoto Graduate School of Interdisciplinary Science and Engineering in Health Systems, Okayama University Mami Asakura Graduate School of Interdisciplinary Science and Engineering in Health Systems, Okayama University Tohru Hayakawa Graduate School of Interdisciplinary Science and Engineering in Health Systems, Okayama University Shintaro Mise Department of Molecular and Cellular Biology, Medical Institute of Bioregulation, Kyushu University Akinobu Matsumoto Department of Molecular and Cellular Biology, Medical Institute of Bioregulation, Kyushu University Toru Ide Graduate School of Interdisciplinary Science and Engineering in Health Systems, Okayama University Ion channel proteins play important roles in various cell functions, making them attractive drug targets. Artificial lipid bilayer recording is a technique used to measure the ion transport activities of channel proteins with high sensitivity and accuracy. However, the measurement efficiency is low. In order to improve the efficiency, we developed a method that allows us to form bilayers on a hydrogel bead and record channel currents promptly. We tested our system by measuring the activities of various types of channels, including gramicidin, alamethicin, alpha-hemolysin, a voltage-dependent anion channel 1 (VDAC1), a voltage- and calcium-activated large conductance potassium channel (BK channel), and a potassium channel from Streptomyces lividans (KcsA channel). We confirmed the ability for enhanced measurement efficiency and measurement system miniaturizion. No potential conflict of interest relevant to this article was reported.

Springer Acta Medica Okayama 0175-7598 104 2020 Channel-pore cation selectivity is a major determinant of Bacillus thuringiensis Cry46Ab mosquitocidal activity 8789 8799 EN Tohru Hayakawa Graduate School of Interdisciplinary Science and Engineering in Health Systems, Okayama University Midoka Miyazaki Graduate School of Interdisciplinary Science and Engineering in Health Systems, Okayama University Syoya Harada Graduate School of Interdisciplinary Science and Engineering in Health Systems, Okayama University Mami Asakura Graduate School of Interdisciplinary Science and Engineering in Health Systems, Okayama University Toru Ide Graduate School of Interdisciplinary Science and Engineering in Health Systems, Okayama University Cry46Ab from Bacillus thuringiensis TK-E6 is a new mosquitocidal toxin with an aerolysin-type architecture, and it is expected to be used as a novel bioinsecticide. Cry46Ab acts as a functional pore-forming toxin, and characteristics of the resulting channel pores, including ion selectivity, have been analyzed. However, the relationship between channel-pore ion selectivity and insecticidal activity remains to be elucidated. To clarify the effects of charged amino acid residues on the ion permeability of channel-pores and the resulting insecticidal activity, in the present study, we constructed Cry46Ab mutants in which a charged amino acid residue within a putative transmembrane β-hairpin region was replaced with an oppositely charged residue. Bioassays using Culex pipiens mosquito larvae revealed that the mosquitocidal activity was altered by the mutation. A K155E Cry46Ab mutant exhibited toxicity apparently higher than that of wild-type Cry46Ab, but the E159K and E163K mutants exhibited decreased toxicity. Ions selectivity measurements demonstrated that the channel pores formed by both wild-type and mutant Cry46Abs were cation selective, and their cation preference was also similar. However, the degree of cation selectivity was apparently higher in channel pores formed by the K155E mutant, and reduced selectivity was observed with the E159K and E163K mutants. Our data suggest that channel-pore cation selectivity is a major determinant of Cry46Ab mosquitocidal activity and that cation selectivity can be controlled via mutagenesis targeting the transmembrane β-hairpin region. No potential conflict of interest relevant to this article was reported.

Springer Acta Medica Okayama 0003-6862 54 4 2019 Characterization of the channel-pores formed by Bacillus thuringiensis Cry46Ab toxin in planar lipid bilayers 389 398 EN Akira Sakakibara Graduate School of Interdisciplinary Science and Engineering in Health Systems, Okayama University So Takebe Graduate School of Biology-Oriented Science and Technology, Kindai University Toru Ide Graduate School of Interdisciplinary Science and Engineering in Health Systems, Okayama University Tohru Hayakawa Graduate School of Interdisciplinary Science and Engineering in Health Systems, Okayama University Cry46Ab from Bacillus thuringiensis TK-E6 is a new mosquitocidal toxin with aerolysin-type architecture, and has been shown that co-administration of Cry46Ab with other mosquitocidal Cry toxins results in synergistic toxicity against Culex pipiens Coquillett (Diptera: Culicidae) mosquito larvae. Cry46Ab, therefore, is expected to find use in improving the insecticidal activity of B. thuringiensis-based bioinsecticides. In the present study, the mode of action of Cry46Ab was explored by single-channel measurements of Cry46Ab channel-pores. The single-channel conductances of channel-pores formed in planar lipid bilayers by Cry46Ab were determined to be 31.8 +/- 2.7 pS in 150 mM NaCl and 24.2 +/- 0.7 pS in 150 mM CaCl2. Ion-selectivity measurements revealed that the channel-pores formed by Cry46Ab were cation selective. The permeability ratio of K+ to Cl-was approximately 4, and the preferences for cations were K+ > Na+, K+ > Ca2+, and Ca2+ > Na+. A calcein release assay using liposomes suggested that Cry46Ab influences the integrity of membrane vesicles. Formation of cation-selective channel-pores has been observed with other insecticidal Cry toxins that have structures distinct from those of Cry46Ab; the capability of forming such pores may be a property required of insecticidal toxins. No potential conflict of interest relevant to this article was reported.