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.

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.

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.

Soybean seeds and sprouts contain isoflavones, including aglycones, glucosides, and malonylated glucosides, and their contents vary across soybean varieties. During germination and photomorphogenesis, blue light-emitting diode (LED) irradiation suppressed hypocotyl growth and enhanced the expression levels of chalcone synthase 7, isoflavone synthase (IFS), and isoflavone reductase (IFR). Conversely, red LED irradiation enhanced hypocotyl growth and decreased the expression levels of IFS and IFR. The contents of some glycosides or malonylated glycosides in sprouts and young green soybeans were significantly higher under blue LED irradiation than under red LED irradiation. These results imply that blue light more effectively increased the concentrations of some isoflavones during early germination and/or hydroponics cultivation.

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.

The photoperiod is a day-length-dependent seasonal change of physiological or developmental activities, such as flowering, in many plant species. Polygonum tinctorium (P. tinctorium) is an important industrial crop producing indigo blue dyes, and indican is an important substance as a precursor of indigo. Here, we report the day-length dependent responses of growth, flowering, and indican synthesis in P. tinctorium. Indigo plants were grown in a hydroponic system under artificial light conditions in a completely-controlled plant factory. The growth parameters and indican content of leaves were measured and compared. Certain growth parameters (such as fresh weight and number of leaves) under 24-h continuous irradiation were significantly higher than those under other day-length conditions. Under 12-h photoperiod conditions, the flowering rate of plants with blue LED treatment increased six-fold compared with fluorescent white light treatment, while none of the plants flowered with red LED treatment. In the leaves, the relative expression levels of Pt'IGS and Pt'BGL were significantly higher in newer leaves compared to older ones. Indican content was greatly enhanced by blue light under 24-h continuous irradiation, which was reflected by increased expression levels of Pt'IGS. These findings demonstrate that there is a new regulatory mechanism for the indican synthesis pathway through blue light signalling. Blue light provides feasible strategy for artificially regulating indican synthesis and flowering in P. tinctorium.

The number of plant factories in which crops are cultivated in an artificial environment has been increasing every year. In cultivation techniques involving hydroponics, plants are supplied with a circulating nutrient solution, which can become contaminated by pathogens that can propagate and spread throughout plant factories. Therefore, strategies to disinfect hydroponic nutrient solutions are needed. In this study, we developed a new disinfection device equipped with an ultraviolet A (UVA) light emitting diode (LED) that can be used to disinfect hydroponic nutrient solutions in plant factories. We first evaluated the basic disinfection capability of the device and then estimated its bactericidal effect in a small scale model system. The log survival ratio was related to UVA irradiation fluence and the volume of nutrient solution. From the assay results, we devised a kinetics equation to describe the relationship between nutrient solution volume, log survival ratio, and UVA fluence. Together our results show that UVA irradiation could be used to disinfect hydroponic nutrient solutions, and the derived kinetics equations can be used to determine optimal conditions, such as nutrient solution volume, UVA irradiation, and killing activity, to develop devices that disinfect hydroponic nutrient solutions. J. Med. Invest. 65:171-176, August, 2018.

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.

Strawberry (Fragaria × ananassa) contains anthocyanins which are important secondary metabolites and key contributors to the antioxidant capacity and nutritional value of the fruit. Anthocyanin biosynthetic genes have been identified. However, the detailed mechanism responsible for anthocyanin accumulation and regulation of biosynthetic genes during strawberry fruit ripening remain unclear. In the present study, we examined the effect of a Fragaria × ananassa myeloblastosis 1 homolog, FaMYB1, on anthocyanin accumulation in the strawberry fruit receptacle. Expression analysis shows that FaMYB1 transcripts increased in response to irradiance but not to abscisic acid treatments. Down-regulation of FaMYB1 was achieved in planta using Agrobacterium-mediated RNA interference (RNAi). As a result, FaMYB1-RNAi fruits exhibited a significant increase in anthocyanin content. Conversely, overexpression of FaMYB1 resulted in a decrease in anthocyanin content. Overexpression of FaMYB1 also significantly reduced expression of genes encoding anthocyanidin synthase and flavonoid glycosyltransferase, whereas down-regulation of FaMYB1 resulted in a significant decrease in the amount of transcripts of leucoanthocyanidin reductase. These data suggest that FaMYB1 might negatively control anthocyanin biosynthesis in the strawberry fruit at the branching-point of anthocyanin/proanthocyanidin biosynthesis.

In the most commonly used in situ hybridization (ISH) procedure, a hapten-labeled antisense nucleic acid (e.g., RNA) probe is employed to hybridize a target mRNA in tissue sections. The hapten-labeled RNA is then detected by a highly specific hapten-antibody interaction; however, it requires laborious immunostaining steps for spatial coloring on tissue sections. To simplify ISH-based mRNA detection systems, we created a new RNA-(enzyme)(n) conjugate for sensitive detection of mRNA in tissue sections. In the present simple, antibody-free ISH protocol, an antisense RNA probe of interest is first modified with a specific dipeptide substrate of microbial transglutaminase (MTG) by in vitro transcription. Alkaline phosphatase from a hyperthermophile is then covalently linked to the substrate-labeled RNA by MTG-catalyzed site-specific conjugation. A robust, multi-enzyme-labeled RNA probe enables the direct labeling of a target mRNA in tissue sections with signaling enzymes under harsh hybridization conditions, leading to one-step signal amplification after hybridization. The application of the new trans glutaminase-mediated ISH (TransISH) strategy to mRNA detection in mammalian tissues was demonstrated.

Light and ABA independently regulated anthocyanin biosynthesis via activation of FaMYB10 expression. FaMYB10 accelerated anthocyanin synthesis of pelargonidin 3-glucoside and cyanidin 3-glucoside during strawberry fruit ripening.

Objects : Since serum level of fibroblast growth factor 21 (FGF21) has been implicated as a potential biomarker for the early detection of the metabolic syndrome and type 2 diabetes, we examined how FGF21 serum levels are correlated with metabolic parameters in Japanese subjects. Methods : FGF21 levels were analyzed by enzyme-linked immunosorbent assays. Spearman's correlation and multiple stepwise regression analyses were used to examine the relationship between serum FGF21 and other factors. A Mann-Whitney U test was performed between the normal and high groups for triglycerides and systolic blood pressure (BP) respectively. Results : By univariate correlation analysis, serum FGF21 levels were significantly associated with triglyceride levels, systolic BP, diastolic BP, pulse pressure, body mass index (BMI), age, fasting plasma glucose (FPG) levels, and total cholesterol levels. Multiple regression analysis (adjusted for age, gender, and BMI) showed that serum FGF21 levels were independently and significantly associated with triglyceride levels and systolic BP. Serum FGF21 levels were significantly higher in subjects with high triglyceride levels and high systolic BP compared with those who had normal triglyceride levels and normal systolic BP respectively. Conclusions : This study found that FGF21 levels might be a biomarker for some metabolic disorders associated with metabolic syndrome.

Anthocyanins are widespread, essential secondary metabolites in higher plants during color development in certain flowers and fruits. In strawberries, anthocyanins are also key contributors to fruit antioxidant capacity and nutritional value. However, the effects of different light qualities on anthocyanin accumulation in strawberry (Fragaria x ananassa, cv. Sachinoka) fruits remain elusive. In the present study, we showed the most efficient increase in anthocyanin content occurred by blue light irradiation. Light sensing at the molecular level was investigated by isolation of two phototropin (FaPHOT1 and FaPHOT2), two cryptochrome (FaCRY1 and FaCRY2), and two phytochrome (FaPHYA and FaPHYB) homologs. Expression analysis revealed only FaPHOT2 transcripts markedly increased depending on fruit developmental stage, and a corresponding increase in anthocyanin content was detected. FaPHOT2 knockdown resulted in decreased anthocyanin content; however, overexpression increased anthocyanin content. These findings suggested blue light induced anthocyanin accumulation, and FaPHOT2 may play a role in sensing blue light, and mediating anthocyanin biosynthesis in strawberry fruits. This is the first report to find a relationship between visible light sensing, and color development in strawberry fruits.

Traditional methods used to study strawberry ripening-related gene function are time-consuming, and require at least 15 months from initiating the transformation experiment until the first ripe fruits are available for analysis. To accelerate data acquisition during gene function studies, we explored a transient assay method that employs an Agrobacterium-mediated RNAi (AmRNAi) technique in post-harvest strawberry fruit, Fragaria x ananassa (Fa) cv. Sachinoka, a Japanese cultivar. Our results showed that artificial white light induced strong expression of Fa'chalcone synthase (Fa'CHS), Fa'chalcone isomerase (Fa'CHI), and Fa'flavonoid 3'-hydroxylase orthologues (Fa'F3'H) in post-harvest fruit. Fa'CHS and Fa'F3'H function was subsequently examined by performing AmRNAi with post-harvest fruit. Although reduction of light-induced Fa'F3'H expression by AmRNAi resulted in no significant change in anthocyanin content, reduction of Fa'CHS significantly decreased anthocyanin levels, and up-regulated Fa'F3'H levels. Our results are consistent with previous data indicating that while CHS is required for anthocyanin accumulation during late stage strawberry fruit maturation, Fa'F3'H is not required. The novel system described here enabled gene function data to be available within 10 days of initiating the incubation period following in filtration. therefore, we conclude our system is a valuable tool to elucidate the molecular mechanisms underlying light-induced ripening of strawberry fruit.

Detection and localization of specific DNA or RNA sequences in cells and tissues are of great importance for biological research, diagnosis, and environmental monitoring. However, the most common procedure for in situ hybridization employs laborious immunostaining techniques. In the present study, we report proof-of-concept for a new RNA enzyme conjugated probe for the detection of mRNA on tissue sections with a simple procedure. An RNA probe modified with a specific dipeptide substrate of transglutaminase was prepared. Alkaline phosphatase was then covalently and site specifically combined to the dipeptide-labeled RNA using microbial transglutaminase. The new RNA probe labeled with alkaline phosphatase-was validated by in situ hybridization (ISH) and proved to be a sensitive and sequence specific probe for mRNA detection in tissues.. The new transglutaminase-mediated ISH (TransISH) strategy is free from antigen-antibody reaction, leads to one-step signal amplification after hybridization, and thus will be widely applicable for highly sensitive nucleic acid detection.

A new synthetic strategy for DNA-enzyme conjugates with a novel architecture was explored using a natural cross-linking catalyst, microbial transglutaminase (MTG). A glutamine-donor substrate peptide of MTG was introduced at the 5-position on the pyrimidine of deoxyuridine triphosphate to prepare a DNA strand with multiple glutamine-donor sites by polymerase chain reaction (PCR). A substrate peptide that contained an MTG-reactive lysine residue was fused to the N terminus of a thermostable alkaline phoshatase from Pyrococcus furiosus (PfuAP) by genetic engineering. By combining enzymatically the substrate moieties of MTG introduced to the DNA template and the recombinant enzyme, a DNA-(enzyme)(n) conjugate with 1:n stoichiometry was successfully obtained. The enzyme/DNA ratio of the conjugate increased as the benzyloxycarbonyl-L-glutaminylglycine (Z-QG) moiety increased in the DNA template. The potential utility of the new conjugate decorated with signaling enzymes was validated in a dot blot hybridization assay. The DNA-(enzyme)(n) probe could clearly detect 10(4) copies of the target nucleic acid with the complementary sequence under harsh hybridization conditions, thereby enabling a simple detection procedure without cumbersome bound/free processes associated with a conventional hapten-antibody reaction-based DNA-detection system.

A long-standing problem of developmental biology is how body size is determined. In Drosophila melanogaster, the insulin/insulin-like growth factor (I/IGF) and target of rapamycin (TOR) signaling pathways play important roles in this process. However, the detailed mechanisms by which insect body growth is regulated are not known. Therefore, we have attempted to utilize systemic nymphal RNA interference (nyRNAi) to knockdown expression of insulin signaling components including Insulin receptor (InR), Insulin receptor substrate (chico), Phosphatase and tensin homologue (Pten), Target of rapamycin (Tor), RPS6-p70-protein kinase (S6k), Forkhead box O (FoxO) and Epidermal growth factor receptor (Egfr) and observed the effects on body size in the Gryllus bimaculatus cricket. We found that crickets treated with double-stranded RNA (dsRNA) against Gryllus InR, chico, Tor, S6k and Egfr displayed smaller body sizes, while Gryllus FoxO nyRNAi-ed crickets exhibited larger than normal body sizes. Furthermore, RNAi against Gryllus chico and Tor displayed slow growth and RNAi against Gryllus chico displayed longer lifespan than control crickets. Since no significant difference in ability of food uptake was observed between the Gryllus chico(nyRNAi) nymphs and controls, we conclude that the adult cricket body size can be altered by knockdown of expressions of Gryllus InR, chico, Tor, S6k, FoxO and Egfr by systemic RNAi. Our results suggest that the cricket is a promising model to study mechanisms underlying controls of body size and life span with RNAi methods.

Northern and Southern blots are the most commonly used techniques for the confirmation of presence and expression of target genes. Molecular tools available for this purpose include radioisotope-, enzyme-and hapten-labeled nucleic acid probes. In particular, the use of enzyme-labeled probes are easy and safe, and do not require bound/free processes after hybridization associated with an antibody-based detection system. However, there are few approaches that enable the post-transcriptional modification of RNA with enzymes or proteins. In this study, we applied the Cu(I)-catalyzed [3 + 2] azide-alkyne cycloaddition (CuAAC) reaction to the labeling of an RNA strand with enzymes. The C-5 position of UTP was modified with an alkyne group and alkyne-bearing RNA was prepared by in vitro transcription using T7 RNA polymerase. Surface amino groups of bacterial alkaline phosphatase (BAP) were randomly derivatized with azide groups at different modification ratios. The CuAAC reaction occurred selectively between the alkyne-modified RNA and the azide-modified enzyme. The RNA probe conjugated with BAP using this technique could detect a specific RNA by dot blot northern hybridization.