Trehalase Activity Research Articles

The Modulation of Trehalose Metabolism by 20-Hydroxyecdysone in Antheraea pernyi (Lepidoptera: Saturniidae) During its Diapause Termination and Post-Termination Period.

Trehalose plays a crucial role in the diapause process of many insects, serving as an energy source and a stress protectant. Trehalose accumulation has been reported in diapause pupae of Antheraea pernyi; however, trehalose metabolic regulatory mechanisms associated with diapause termination remain unclear. Here, we showed that the enhanced trehalose catabolism was associated with an increase in endogenous 20-hydroxyecdysone (20E) in hemolymph of A. pernyi pupae during their diapause termination and posttermination period. Injection of 20E increased the mRNA level of trehalase 1A (ApTre-1A) and trehalase 2 (ApTre-2) of A. pernyi diapause pupae in a dose-dependent manner but did not affect the mRNA level of trehalase 1B (ApTre-1B). Meanwhile, exogenous 20E increased the enzyme activities of soluble and membrane-bound trehalase, leading to a decline in hemolymph trehalose. Conversely, the expression of ApTre-1A and ApTre-2 were down-regulated after the ecdysone receptor gene (ApEcRB1) was silenced by RNA interference or by injection of an ecdysone receptor antagonist cucurbitacin B (CucB), which inhibits the 20E pathway. Moreover, CucB treatment delayed adult emergence, which suggests that ApEcRB1 might be involved in regulating pupal-adult development of A. pernyi by mediating ApTre-1A and ApTre-2 expressions. This study provides an overview of the changes in the expression and activity of different trehalase enzymes in A. pernyi in response to 20E, confirming the important role of 20E in controlling trehalose catabolism during A. pernyi diapause termination and posttermination period.

1H NMR‐based metabonomic evaluation of the pesticides camptothecin and matrine against larvae of Spodoptera litura

Camptothecin (CPT) and matrine (MAT) have potential as botanical pesticides against several pest species. However, the mechanisms of metabolic and physiological changes in pests induced by CPT and MAT are unknown. In this study, a toxicological test, an NMR-based metabolomic study, an enzymatic test, and an RT quantitative PCR (RT-qPCR) experiment were all conducted to examine the effect of CPT and MAT on Spodoptera litura. CPT (0.5-1%) exerted high toxicity against larvae of S. litura and caused growth stagnation and high mortality of larvae. A variety of metabolites were significantly influenced by 0.5% CPT, including several energy-related metabolites such as trehalose, lactate, succinate, citrate, malate, and fumarate. In contrast, MAT showed low toxicity against larvae and induced almost no changes in hemolymph metabolites of S. litura. Enzymatic tests showed that trehalase activity was significantly decreased in larvae after feeding with 0.5% CPT. RT-qPCR showed that the transcription levels of alanine aminotransferase, malate dehydrogenase, and isocitrate dehydrogenase were decreased while lactate dehydrogenase was increased in the 0.5% CPT-treated group. These data indicate that one of the important mechanisms of CPT against S. litura larvae is via the inhibition of trehalose hydrolysis and glycolysis. Our findings also suggest that CPT exhibits a stronger toxicological effect than MAT against S. litura, which provides basic information for the application of CPT in the control of S. litura or other lepidoptera pests.

Effects of long-term cadmium exposure on trehalose metabolism, growth, and development of Aedes albopictus (Diptera: Culicidae)

Trehalose is the major blood sugar in insects; it not only serves as an energy source but also plays important roles in physiological responses to adverse conditions. However, only a few studies have explored the effects of heavy metal exposure stress on trehalose metabolism in insects. Therefore, in this study, we examined the effects of cadmium stress on changes in trehalose metabolism in Aedes albopictus. Three concentrations of cadmium (0.005, 0.01, and 0.1 mg/L) were selected for evaluation of long-term stress in Ae. albopictus (from eggs to adults); Ae. albopictus in double-distilled water was used as the control group. The trehalose and glucose contents, trehalase activity, and trehalose metabolism-related gene expression were determined. The effects of long-term cadmium exposure on growth, development, and reproduction were also assessed. Trehalose contents were increased, whereas glucose contents and trehalase activity were decreased in Ae. albopictus following long-term exposure to low concentrations of cadmium compared with those in untreated individuals. Moreover, the expression of trehalose-6-phosphate synthase was upregulated, and that of trehalase was downregulated, indicating that Ae. albopictus may enhance trehalose synthesis to resist cadmium stress. Cadmium exposure also caused Ae. albopictus individuals to become smaller with a longer developmental duration, whereas both reproduction and hatching rates of the offspring were decreased compared with those in the control group. Our findings demonstrated that cadmium exposure affected the morphology, physiology, and biochemistry of Ae. albopictus. These findings also confirmed the role of trehalose in the response of Ae. albopictus to cadmium stress, providing insights into the effects of heavy metal stress on trehalose metabolism in an insect model.

Reduced fecundity and regulation of reproductive factors in flubendiamide-resistant strains of Plutella xylostella

Diamondback moth (DBM), Plutella xylostella, is an important pest of crucifers worldwide. The extensive use of flubendiamide has led to the development of resistance in field populations and reports of control failures. In this study, the lab-selected (Rf) and field-collected (Rb) flubendiamide-resistant strains of P. xylostella with LC50 resistance ratios of 1890-fold and 1251-fold, respectively, were used, as well as a lab-reared flubendiamide-susceptible strain (S). The results showed that the fecundity of the Rf and Rb-resistant strains was significantly lower than that of S strain. The contents of vitellin and transcripts of P. xylostella vitellogenin (PxVg) and P. xylostella vitellogenin receptor (PxVgR) genes in the Rf and Rb strains were significantly higher than those of S strains at 0–48 h after adult eclosion. At 96 h after eclosion, the content of vitellin in the Rf and Rb strains did not differ significantly from those of S strains, whereas transcripts of the PxVg and PxVgR genes in the Rf and Rb strains were significantly lower than that of the S strain. The content of the juvenile hormone III (JH III), β-ecdysone (20E), and the gene expression level of P. xylostella methoprene tolerant (PxMet) in the Rf and Rb strains were significantly higher than that of the S strain. The activity of trehalase was significantly higher in the Rf and Rb strains than that of the S strain in the first to the third instar larvae, whereas in the fourth instar larvae, there was no significantly difference in the three strains. At different times after adult eclosion, the differences in trehalase activity were erratic between the strains. The transcripts of P. xylostella trehalase (PxTre) gene in the Rf and Rb strains were significantly higher than that of the S strain in most developmental stages. Here, we report differences in fecundity between flubendiamide-resistant and susceptible strains of P. xylostella and discuss gene expression of several reproductive factors, which provides a possible explanation for the mechanism of fecundity reduction concurrent with flubendiamide-resistance in P. xylostella.

Inhibition of trehalase affects the trehalose and chitin metabolism pathways in Diaphorina citri (Hemiptera: Psyllidae).

The Asian citrus psyllid, Diaphorina citri is the principal vector of huanglongbing, which transmits Candidatus Liberibacter asiaticus. Trehalase is a key enzyme involved in trehalose hydrolysis and plays an important role in insect growth and development. The specific functions of this enzyme in D. citri have not been determined. In this study, three trehalase genes (DcTre1-1, DcTre1-2, and DcTre2) were identified based on the D. citri genome database. Bioinformatic analysis showed that DcTre1-1 and DcTre1-2 are related to soluble trehalase, whereas DcTre2 is associated with membrane-bound trehalase. Spatiotemporal expression analysis indicated that DcTre1-1 and DcTre1-2 had the highest expression levels in the head and wing, respectively, and DcTre2 had high expression levels in the fat body. Furthermore, DcTre1-1 and DcTre1-2 expression levels were induced by 20-hydroxyecdysone and juvenile hormone Ⅲ, but DcTre2 was unaffected. The expression levels of DcTre1-1, DcTre1-2, and DcTre2 were significantly upregulated, which resulted in high mortality after treatment with validamycin. Trehalase activities and glucose contents were downregulated, but the trehalose content increased after treatment with validamycin. In addition, the expression levels of chitin metabolism-related genes significantly decreased at 24 and 48 h after treatment with validamycin. Furthermore, silencing of DcTre1-1, DcTre1-2, and DcTre2 reduced the expression levels of chitin metabolism-related genes and led to a malformed phenotype of D. citri. These results indicate that D. citri trehalase plays an essential role in regulating chitin metabolism and provides a new target for control of D. citri.

SPECIFIC CARBOHYDRATE HYDROLYZING ENZYMES IN RELATION TO DIFFERENT INSECTICIDES TREATMENTS IN WHITEFLY, Bemisia tabaci

Toxicity of acetamiprid (mospilan 20% Sp), chlorpyrifos-methyl (reldan22.5% EC) and pyriproxyfen (admiral 10% Ew) against laboratory and field strains of whitefly, Bemisiatabaci was studied. Evaluation of biochemical alterations of carbohydrate hydrolyzing enzymes (amylase, trehalase and invertase)activity were recorded and discussed as biomarkers for acetamiprid, chlorpyrifos-methyl and pyriproxyfen toxicity in the both of strains of B.tabaci. Results indicated that these compounds decreased the activities of amylase and trehalase enzymes in laboratory and field strains. Regardingto invertase enzyme, acetamiprid increased the activity ofinvertase enzymein the two strains while chlorpyrifos-methyl elevated the activity of invertase enzyme in laboratory strain, pyriproxyfen reduced the activity of invertase enzyme in both strains. Field study was conducted to evaluate the efficacy of tested insecticides against the adults of B. tabacion tomato crop, during seasons 2018 and 19 at Zagazig district, Sharkia Governorate. Results showed that acetamiprid was the most effective insecticide in both seasons, which achieved 86.81 and 88.20% of reduction percentage in B. tabaci population, followed by chlorpyrifos- methyl recording77.17 and 81.82%, pyriproxyfen was the least potent compound which recorded 59.58and 63.62% of reduction percentage in the pest population in seasons 2018 and 2019 respectively. Conclusively, it was proved through this study that the neonicotined, acetamiprid was the most potent toxicant against b. tabaci, so we used acetamiprid in b. tabaci management.

Ocean acidification, hypoxia and warming impair digestive parameters of marine mussels

Global change and anthropogenic activities have driven marine environment changes dramatically during the past century, and hypoxia, acidification and warming have received much attention recently. Yet, the interactive effects among these stressors on marine organisms are extremely complex and not accurately clarified. Here, we evaluated the combined effects of low dissolved oxygen (DO), low pH and warming on the digestive enzyme activities of the mussel Mytilus coruscus. In this experiment, mussels were exposed to eight treatments, including two degrees of pH (8.1, 7.7), DO (6, 2 mg/l) and temperature (30 °C and 20 °C) for 30 days. Amylase (AMS), lipase (LPS), trypsin (TRY), trehalase (TREH) and lysozyme (LZM) activities were measured in the digestive glands of mussels. All the tested stress conditions showed significant effects on the enzymatic activities. AMS, LPS, TRY, TREH showed throughout decreased trend in their activities due to low pH, low DO, increased temperature and different combinations of these three stressors with time but LZM showed increased and then decreased trend in their activities. Hypoxia and warming showed almost similar effects on the enzymatic activities. PCA showed a positive correlation among all measured biochemical parameters. Therefore, the fitness of mussel is likely impaired by such marine environmental changes and their population may be affected under the global change scenarios.

Mechanisms of trehalose-mediated mitigation of Cd toxicity in rice seedlings

Trehalose (TR) functions as a compatible solute and plays an important physiological role in osmotic protection, thereby enhancing the tolerance of plants to the adverse effects of abiotic stress. Currently, however, there is limited information available regarding the TR-mediated mitigation of stress related to potentially toxic metals in plants. In this study, experiments were conducted to determine the effect of TR on the growth status of rice seedlings, changes in oxidative stress markers, and Cadmium (Cd) absorption in response to Cd stress. Plants were subjected to single Cd, single TR, and combined Cd and TR treatments, and the effects on biomass, Cd uptake, and selected physiological indices including reactive oxygen species (ROS) levels and antioxidant enzyme activities were examined to elucidate the protective mechanisms of TR. It was found that Cd had a concentration-dependent inhibitory effect on rice biomass production, whereas single TR treatment had only a limited effect on the biomass of rice. A remarkable decrease in chlorophyll a and b content was observed during Cd treatment, up to 21.5% and 29.0%, respectively, which improved after exogenous TR was applied. Furthermore, compared to that of Cd treatment alone, TR could effectively reduce the content of stress-related oxidants (H2O2 and O2·-) and lower the upregulation of antioxidant enzyme activity, conditions that are more conducive to the growth of rice. Given that plants can degrade excess TR by increasing the activity of trehalase, exogenous TR application has limited effects on the normal physiology of plants. The uptake of Cd by rice was considerably lower (between 30% and 70%) than that of the control group when plants were cotreated with TR. Lower pH levels tend to promote Cd absorption in rice, and the addition of TR protects rice plants mainly via the considerable reduction in Cd concentration in the shoots and roots. Moreover, it was found that the stress-related effects of Cd on rice were closely related to the prevailing form of Cd in the culture medium. When Cd and TR are added simultaneously in a quantum chemical simulation, Cd occurs primarily as a Cd-TR chelate, which effectively reduces its mobility in and toxicity to rice. TR application thus represents a potential means of mitigating Cd toxicity in plants.

Effects of Diallyl Trisulfide, an Active Substance from Garlic Essential Oil, on Energy Metabolism in Male Moth Sitotroga cerealella (Olivier).

This study investigated the effects of diallyl trisulfide (DAT), an active substance from garlic essential oil, on the metabolism of the main energy substances of pre- and postmating males of Sitotroga cerealella. Males at 12 h postemergence were fumigated with DAT at a concentration (LC10 = 0.010 µL/L) in a glass jar for 7 h. The main energy metabolites from pre- and postmating males were determined, including protein, triglyceride, glycogen, total soluble sugar, trehalose, and trehalase. The contents of total protein and total soluble sugar and the trehalase activity of premating males were significantly increased following DAT treatment, whereas the contents of protein from the accessory gland, triglyceride, glycogen, and trehalose were significantly decreased after treatment. Additionally, after mating, the total protein and soluble sugar contents were significantly increased and the glycogen content was significantly decreased in the treatment group relative to the levels in controls, but there was no significant difference observed in triglyceride, accessory gland proteins, trehalose content, or trehalase activity between the treatment and control groups. Furthermore, the changes in the main energy substances between pre- and postmating in males after the DAT treatment (∆DAT) were smaller than those in the control group (∆CK). This result indicated that DAT can accelerate the rate of metabolism in males at LC10, leading to the accumulation of greater levels of total soluble sugar to support life activities and to the increased synthesis of proteins to resist an adverse environment.

Differential carbohydrate dynamics in Arabidopsis wild-type and ntrc mutant after trehalose feeding

The trehalose sugar plays pivotal roles in plants. In Arabidopsis, trehalose supplied to the medium leads to starch accumulation through redox activation of the ADP-glucose pyrophosphorylase (AGPase) by the NADPH-dependent thioredoxin reductase C (NTRC). To gain further insight on the regulation of carbohydrate metabolism by trehalose and NTRC, Arabidopsis wild-type (wt) and ntrc plants were raised in the MS medium supplemented with or without 100-mM sucrose or trehalose and alterations in sugar and starch contents as well as the activities and expressions of some carbohydrate metabolizing enzymes were investigated. The ntrc plants displayed lower growth rate compared to wt by sugar feeding. Under trehalose treatment, the total soluble, reducing and non-reducing sugars of wt and ntrc plants decreased between end of the day and end of the night, but starch in the ntrc plants remained unaltered during the aforementioned period. The obtained data showed that trehalase activity and gene expression display diurnal rhythms. Meanwhile, under darkness, trehalase gene expression required functional NTRC. Trehalose feeding induced NTRC expression during the day, but suppressed it during the night. Moreover, AGPase expression found to be jointly regulated by carbon nutrition and the light–dark period in both wt and mutant plants. The current data also revealed that AMY3 (α-amylase 3) gene expression depends on the presence of functional NTRC in plants. It seems that cross-talk between trehalose and NTRC regulates diurnal carbohydrate metabolism in plants.

Effect of long-term cold storage on trehalose metabolism of pre-wintering Harmonia axyridis adults and changes in morphological diversity before and after wintering.

Harmonia axyridis is a major bio-control agent of pests in agriculture and forest ecosystems. It is also a globally important invasive insect species. To test whether dark elytra colour is associated with greater cold hardiness, we compared the survival rate of prolonged cold exposure in both yellow and black colour morphs of female and male H. axyridis. We determined the trehalose and glycogen content, trehalase activity, and the dynamics of genes associated with the trehalose metabolic pathway. Yellow forms predominated before winter began, however black forms increased from 11.15 to 30.46% after overwintering. There was no significant difference in trehalose content between the females and males during overwintering. Glycogen content in over-wintering yellow females and black males increased significantly, while it decreased in black females. Soluble trehalase activity increased significantly in all the insects except black females. Membrane-bound trehalase activity increased in black males, and decreased in black females. Trehalose and glycogen content and trehalase activity were regulated by differential expression of TRE and TPS genes. Female beetles weighed more than males and survived in low temperatures for longer periods of time, regardless of elytra colour, suggesting that mass is a stronger predictor of overwintering survival rather than colour morph. Our results provide a guide for comparing cold resistance in insects and a theoretical basis for cold storage of H. axyridis for use as natural enemies of pests in biological control programs.

Effect of some Insecticides on Pink Bollworm, Pectinophora gossypiella (Saunders) (Lepidoptera: Gelechiidae), Associated Predators in Cotton Field and some of their Biochemical Effects

Efficiency of profenofos, chlorpyrifos and alpha-cypermethrin against Pectinophora gossypiella and their associated predators in cotton field at Zagazig, Sharkia Governorate. Alpha-cypermethrin increased mean seasonal reduction percentages (92.92%), while, profenofos decrease it (78.38%). Alpha-cypermethrin caused the highest mean seasonal reduction percentages in the Coccineledae, Chrysoperla carnea and True spider predators (88.26, 73.67 and 85.70%, respectively). The pupal mortality percentage increased while, pupal duration, adult emergency percentage, pupal weight, pupation percentage, deposited eggs and hatchability percentage decreased. The activity of trehalase, invertase and acetylcholine esterase enzymes decreased but of amylase and AST (Aspartate aminotransferase) increased. Disturbance recorded in ALT (Alanine aminotransferase) enzyme, total lipid and total soluble protein content relative to control.

Role of phosphoglucomutase in regulating trehalose metabolism in Nilaparvata lugens

Phosphoglucomutase (PGM) is a key enzyme in glycolysis and gluconeogenesis, regulating both glycogen and trehalose metabolism in insects. In this study, we explored the potential function of phosphoglucomutase (PGM) using RNA interference technology in Nilaparvata lugens, the brown planthopper. PGM1 and PGM2 were found highly expressed in the midgut of brown planthoppers, with different expression levels in different instar nymphs. The glycogen, glucose, and trehalose levels were also significantly increased after brown planthoppers were injected with dsRNA targeting PGM1 (dsPGM1) or PGM2 (dsPGM2). In addition, injection of dsPGM1 or dsPGM2 resulted in increased membrane-bound trehalase activity but not soluble trehalase activity. Furthermore, the expression of genes related to trehalose and glycogen metabolism decreased significantly after injection with dsPGM1 and dsPGM2. The expression levels of genes involved in chitin metabolism in the brown planthopper were also significantly decreased and the insects showed wing deformities and difficulty molting following RNAi. We suggest that silencing of PGM1 and PGM2 expression directly inhibits trehalose metabolism, leading to impaired chitin synthesis.

The secreted acid trehalase encoded by the CgATH1 gene is involved in Candida glabrata virulence.

BACKGROUND Candida glabrata yeast is the second cause of candidiasis worldwide. Differs from other yeasts since assimilates only glucose and trehalose (a characteristic used in rapid identification tests for this pathogen) by secreting into the medium a highly active acid trehalase encoded by the CgATH1 gene.OBJECTIVE This study aimed to characterise the function of the acid trehalase in the physiopathology of C. glabrata.METHODS Gene deletion was performed to obtain a mutant ath1Δ strain, and the ability of the ath1Δ strain to grow in trehalase, or the presence of trehalase activity in the ath1Δ yeast cells, was verified. We also tested the virulence of the ath1Δ strain in a murine model of infection. FINDINGS The ath1Δ mutant strain grows normally in the presence of glucose, but loses its ability to grow in trehalose. Due to the high acid trehalase activity present in wild-type cells, the cytoplasmic neutral trehalase activity is only detected in the ath1Δ strain. We also observed a significantly lower virulence of the ath1Δ strain in a murine model of infection with either normal or immunocompromised mice. MAIN CONCLUSIONS The acid trehalase is involved in the hydrolysis of external trehalose by C. glabrata, and the enzyme also plays a major virulence role during infectivity.

Evaluation of the Expression and Function of the TRE2-like and TRE2 Genes in Ecdysis of Harmonia axyridis.

Harmonia axyridis is an important predatory insect and widely used in biological control of agricultural and forestry pests. Trehalose is directly involved in the energy storage of the H. axyridis and in the oxidative function of various physiological activities thereby providing an energy source for its growth and development. The aim of this study was to explore the potential function of membrane-bound-like trehalase (TRE2-like) and membrane-bound trehalase (TRE2) genes in H. axyridis by RNAi. In addition, the activity of soluble and membrane-bound trehalase and the expression of genes related to trehalose and glycogen metabolism were determined in the larvae injected with dsTRE2-like or dsTRE2. The results showed that wing abnormality and mortality appeared in adults, as well as the activity of soluble trehalase and glycogen contents increased when interfering with TRE2-like gene. However, the activity of membrane-bound trehalase, trehalose and glucose contents in the larvae decreased. The expression of glycogen synthase (GS) and glycogen phosphorylase (GP) genes were decreased after RNAi in the ecdysis stage. The expression of chitin synthase gene A (CHSA), chitin synthase gene B (CHSB), and trehalose-6-phosphate synthase genes (TPS) were decreased significantly after RNAi, especially in the ecdysis stage. These results indicated that RNA interference is capable of knocking down gene expression of TRE2-like and TRE2, thereby disrupting trehalose metabolism which affects the chitin synthesis pathway in turn and also leads to developmental defects, such as wing deformities. This study could provide some theoretical guidance for the function of TRE2 gene in other insects.

Fenoxycarb and methoxyfenozide (RH-2485) affected development and chitin synthesis through disturbing glycometabolism in Lymantria dispar larvae

Fenoxycarb as a juvenile hormone analogue and methoxyfenozide (RH-2485) as a 20-hydroxyecdysone (20E) agonist are two main insect growth regulators (IGRs) used for pest control, whose insecticidal mechanisms had been widely reported in past decades. However, there were few studies focused on their effects on the carbohydrate metabolism of insects. Here, we reported that two IGRs (fenoxycarb and RH-2485) significantly affected growth and development of L. dispar larvae and caused larval lethality. Furthermore, both contens of three sugars (glycogen, threhalose, glucose) in four tissues (fat body, midgut, hemolymph and epidermis) and trehalase activity in three tissues (fat body, midgut and hemolymph) of L. dispar larvae were markedly affected by these two IGRs. Moreover, we found that mRNA expression levels of LdTPS, LdTre1 and LdTre2 in L. dispar larvae were dramatically suppressed by two IGRs. Additionally, chitin content in both midgut and epidermis decreased significantly after L. dispar larvae treated with fenoxycarb or RH-2485. Summarily, these results indicated that these two IGRs disturbed glycometabolism in L. dispar larvae, resulting in impeding chitin synthesis, generating new epidermis failure, disrupting molting and larval lethality in the end.

SlZRT2 Encodes a ZIP Family Zn Transporter With Dual Localization in the Ectomycorrhizal Fungus Suillus luteus.

Ectomycorrhizal (ECM) fungi are important root symbionts of trees, as they can have significant effects on the nutrient status of plants. In polluted environments, particular ECM fungi can protect their host tree from Zn toxicity by restricting the transfer of Zn while securing supply of essential nutrients. However, mechanisms and regulation of cellular Zn homeostasis in ECM fungi are largely unknown, and it remains unclear how ECM fungi affect the Zn status of their host plants. This study focuses on the characterization of a ZIP (Zrt/IrtT-like protein) transporter, SlZRT2, in the ECM fungus Suillus luteus, a common root symbiont of young pine trees. SlZRT2 is predicted to encode a plasma membrane-located Zn importer. Heterologous expression of SlZRT2 in yeast mutants with impaired Zn uptake resulted in a minor impact on cellular Zn accumulation and growth. The SlZRT2 gene product showed a dual localization and was detected at the plasma membrane and perinuclear region. S. luteus ZIP-family Zn uptake transporters did not show the potential to induce trehalase activity in yeast and to function as Zn sensors. In response to excess environmental Zn, gene expression analysis demonstrated a rapid but minor and transient decrease in SlZRT2 transcript level. In ECM root tips, the gene is upregulated. Whether this regulation is due to limited Zn availability at the fungal–plant interface or to developmental processes is unclear. Altogether, our results suggest a function for SlZRT2 in cellular Zn redistribution from the ER next to a putative role in Zn uptake in S. luteus.

Glucose Utilization in the Regulation of Chitin Synthesis in Brown Planthopper.

Glucose-6-phosphatase (G6Pase) and hexokinase (HK) are two key enzymes in the glycolysis and gluconeogenesis pathways, which catalyze the synthesis and degradation of glucose in insects, respectively. G6Pase and HK play an important role in insect growth by regulating the metabolism of glucose, leading to the efficient metabolism of other macromolecules. However, it is unclear whether these genes could be investigated for pest control through their actions on chitin metabolism. We studied the potential functions of G6Pase and HK genes in the regulation of chitin metabolism pathways by RNAi technology. Interference with G6Pase expression did not affect trehalose and chitin metabolism pathways in brown planthopper, Nilaparvata lugens (Stål). However, knockdown of the HK gene resulted in a significant decrease of expression of genes associated with the trehalose metabolic pathway but had no significant effect on trehalase activity, trehalose content, or glucogen content. Additionally, HK knockdown resulting in downregulation of the genes involved in chitin metabolism in the brown planthopper. These insects also showed wing deformities and difficulty in molting to varying degrees. We suggest that the silencing of HK expression directly inhibited the decomposition of glucose, leading to impaired chitin synthesis.

Improving expression of thermostable trehalase from Myceliophthora sepedonium in Aspergillus niger mediated by the CRISPR/Cas9 tool and its purification, characterization

Trehalase catalyzes the conversion of one molecule of trehalose into two glucose molecules. The trehalase TreM from thermophilic fungus Myceliophthora sepedonium was expressed in Aspergillus niger via traditional homologous recombination with trehalase activity of 406.44 U/mL. The multi-copy knock-in expression strategy mediated by the CRISPR/Cas9 tool was used to improve the production of the TreM trehalase in Aspergillus niger, which was up to 1943.06 U/mL with a low-background of secreted proteins, 4.8-fold than the transformant obtained via the traditional method. The highest recombinant trehalase activity of the shake fermentation supernatant achieved 4268.29 U/mL when 1.5% glucose was added. Activity assaying showed that the recombinant TreM possessed a specific activity of 679.09 U/mg after gel filtration chromatography purification. The recombinant TreM displayed optimal activity at pH 5.6 and 60 °C and exhibited prominent stability under the conditions of 45–50 °C and pH 4.0–7.5. The activity of recombinant TreM was strongly enhanced by Co2+ (1, 5 mM), Cu2+ (1 mM), Mn2+ (1, 5 mM) and ATP (5 mM), and was greatly inhibited by Cu2+ (10 mM), EDTA (10 mM) and SDS (10 mM).

Sanguinarine caused larval lethality and growth inhibition by suppressing energy metabolism in silkworms, Bombyx mori

Sanguinarine (Sang) is a natural alkaloid and distributed in several plants of Papaveraceae. The antitumor, antioxidant, antimicrobial and anti-inflammatory effects of Sang were extensively reported, but its speciality and mechanism against Lepidoptera insects were still unknown. In this study, detailed toxicological parameters of Sang against silkworms, Bombyx mori (B. mori), were determined by a toxicological test. Then, a nuclear magnetic resonance-based (NMR) metabolomics method was adopted to analyze the changes in hemolymph metabolites of silkworms after feeding Sang. The growth of fourth-instar larvae was significantly ceased by the oral administration of 0.05–0.3% Sang and vast deaths appeared in 0.3% Sang group on Day 4 and Day 5. The quantitative analysis of metabolites indicated that trehalose and citrate levels in hemolymph were increased after 24 h of feeding 0.3% Sang, whereas the concentrations of pyruvate, succinate, malate and fumarate were decreased. In addition, the enzymatic determination and reverse transcription quantitative PCR (RT-qPCR) showed that the trehalase (THL) activity and the transcriptional level of one gene coding THL were uniformly weakened by 0.3% Sang. One of the important mechanisms of Sang against silkworms might be interpreted as follows. Sang impaired trehalose hydrolysis, reduced THL activity and transcription, and led to the inhibition of energy metabolism, consequent antigrowth and high lethality in larvae of B. mori. Our findings offered new insights into the insecticidal effect of Sang from the perspective of energy metabolism and provided the basis for the application of Sang in the control of Lepidoptera pests.