A novel cascade catalysis for one-pot enzymatically modified isoquercitrin (EMIQ) conversion from rutin and sucrose using rationally designed gradient temperature control.

Enzymatically modified isoquercitrin (EMIQ) is a glyco-chemically modified flavonoid exhibiting notably high biological activity, such as antioxidant, anti-inflammatory and anti-allergic properties. However, the utilization of expensive substrates such as isoquercitrin and cyclodextrin in the conventional approach has hindered the industrial-scale production of EMIQ due to high cost and low yields. Hence, the development of a cost-effective and efficient method is crucial for the biological synthesis of EMIQ. In this study, a natural cascade catalytic reaction system was constructed with α-L-rhamnosidase and amylosucrase using the inexpensive substrates rutin and sucrose. Additionally, a novel approach integrating gradient temperature regulation into biological cascade reactions was implemented. Under the optimal conditions, the rutin conversion reached a remarkable 95.39% at 24 h. Meanwhile, the productivity of quercetin-3-O-tetraglucoside with the best bioavailability reached an impressive 41.69%. This study presents promising prospects for future mass production of EMIQ directly prepared from rutin and sucrose.

Binding characteristics of pheromone-binding protein 1 in Glyphodes pyloalis to organophosphorus insecticides: Insights from computational and experimental approaches.

Glyphodes pyloalis (Lepidoptera: Pyralidae) is one of the major pests in mulberry production in China, which has developed resistance to various insecticides. Chemoreception is one of the most crucial physiological tactics in insects, playing a pivotal role in recognizing chemical stimuli in the environment, including noxious stimuli such as insecticides. Herein, we obtained recombinant pheromone-binding protein 1 (GpylPBP1) that exhibited antennae-biased expression in G. pyloalis. Ligand-binding assays indicated that GpylPBP1 had the binding affinities to two organophosphorus insecticides, with a higher binding affinity to chlorpyrifos than to phoxim. Computational simulations showed that a mass of nonpolar amino acid residues formed the binding pocket of GpylPBP1 and contributed to the hydrophobic interactions in the bindings of GpylPBP1 to both insecticides. Furthermore, the binding affinities of three GpylPBP1 mutants (F12A, I52A, and F118A) to both insecticides were all significantly reduced compared to those of the GpylPBP1-wild type, suggesting that Phe12, Ile52, and Phe118 residues were crucial binding sites and played crucial roles in the bindings of GpylPBP1 to both insecticides. Our findings can be instrumental in elucidating the effects of insecticides on olfactory recognition in moths and facilitating the development of novel pest management strategies using PBPs as targets based on insect olfaction.

Host trehalose metabolism disruption by validamycin A results in reduced fitness of parasitoid offspring.

The general cutworm, Spodoptera litura (Lepidoptera: Noctuidae) is a worldwide destructive omnivorous pest and the endoparasitoid wasp Meteorus pulchricornis (Hymenoptera: Braconidae) is the dominant endoparasitoid of S. litura larvae. Trehalase is a key enzyme in insect trehalose metabolism and plays an important role in the growth and development of insects. However, the specific function of trehalase in parasitoid and host associations has been less reported. In this study, we obtained two trehalase genes (SlTre1 and SlTre2) from our previously constructed S. litura transcriptome database; they were highly expressed in 3rd instar larvae. SlTre1 was mainly expressed in the midgut, and SlTre2 was expressed highest in the head. SlTre1 and SlTre2 were highly expressed 5 days after parasitization by M. pulchricornis. Treatment with the trehalase inhibitor validamycin A significantly inhibited the expression levels of SlTre1 and SlTre2, and the trehalase activity. Besides, the content of trehalose was increased but the content of glucose was decreased 24 h after validamycin A treatment in parasitized S. litura larvae. In addition, the immune-related genes in phenoloxidase (PO) pathway and fatty acid synthesis-related genes in lipid metabolism were upregulated in parasitized host larvae after validamycin A treatment. Importantly, the emergence rate, proportion of normal adults, and body size of parasitoid offspring was decreased in parasitized S. litura larvae after validamycin A treatment, indicating that validamycin A disrupts the trehalose metabolism of parasitized host and thus reduces the fitness of parasitoid offspring. The present study provides a novel perspective for coordinating the application of biocontrol and antibiotics in agroecosystem.

Odorant-Binding Protein 6 Contributes High Binding Affinity to Insecticides in a Parasitic Wasp Meteorus pulchricornis (Hymenoptera: Braconidae).

Meteorus pulchricornis is a preponderant parasitic wasp of various lepidopteran pests. The extensive application of broad-spectrum insecticides usually causes serious threats to the olfactory recognition of nontarget insects such as parasitoid wasps. However, the binding mechanism of odorant-binding proteins (OBPs) to insecticides in parasitoid wasps remains unknown. Herein, we find that the MpulOBP6 protein had a strong binding affinity to three insecticides (phoxim, chlorpyrifos, and chlorfenapyr). Results of computational simulations revealed that the hydrophobic interaction contributed by a mass of nonpolar amino acid residues was the primary driving force in the formation and stabilization of MpulOBP6-insecticide complexes. Among them, four residues (Met75, Val84, Phe121, and Pro122) and two residues (Val84 and Phe111) play an essential role in the binding of MpulOBP6 to phoxim and chlorfenapyr, respectively. Our findings could be instrumental to elucidate the effects of insecticide application toward the olfactory recognition of nontarget insects in the processes of agricultural production.

The role of Glutathione-S-transferases in phoxim and chlorfenapyr tolerance in a major mulberry pest, Glyphodes pyloalis walker (Lepidoptera: Pyralidae).

Glyphodes pyloalis Walker is a destructive pest on mulberry trees and poses a significant threat to the sericultural industry in China. Phoxim and chlorfenapyr are two commonly used insecticides in mulberry fields. Glutathione-S-transferases (GSTs) comprise a multifunctional protein superfamily that plays important roles in the detoxification of insecticides and xenobiotic compounds in insects. However, whether GSTs participate in the tolerance of phoxim and chlorfenapyr in G. pyloalis is still unknown. To better understand the mechanism of insecticide tolerance in G. pyloalis, the enzymatic activity of GSTs was evaluated under phoxim and chlorfenapyr exposure, respectively. GST enzyme activity was significantly increased after 12, 36 and 48 h of phoxim treatment and 12, 24, 36 and 48 h of chlorfenapyr treatment. Subsequently, eighteen GST genes were identified from the larvae transcriptome of G. pyloalis. Among these, ten GpGSTs had GSH-binding sites and fifteen GpGSTs had variable hydrophobic substrate-binding sites. The expression levels of Delta-GpGST and Epsilon-GpGST genes were significantly influenced by phoxim and chlorfenapyr treatment, and by the time post insecticide application. Furthermore, after silencing GpGST-E4, the mortality rate of G. pyloalis larvae was increased when they were exposed to chlorfenapyr, but it did not significantly alter when the larvae were exposed to phoxim. Our results indicated the vital roles of GpGSTs in the tolerance of insecticides and this action depends on the categories of insecticides. The present study provides a theoretical basis for elucidating insecticide susceptibility and promotes functional research on GST genes in G. pyloalis.

Identification of chemosensory genes by antennal transcriptome analysis and expression profiles of odorant-binding proteins in parasitoid wasp Aulacocentrum confusum.

The endoparasitoid wasp, Aulacocentrum confusum (Hymenoptera: Braconidae), is a preponderant natural enemy of the larvae of Glyphodes pyloalis Walker (Lepidoptera: Pyralidae), which is a destructive pest of mulberry trees. We first constructed the antennal transcriptome database of A. confusum. In total, we obtained 48,262,304 clean reads from the dataset and assembled 24,324 unigenes. A total of 12,690 (52.17%) unigenes indicated significant similarity (E-value < 10-5) compared to known protein sequences of other species from the NCBI non-redundant protein database. Gene ontology (GO) and cluster of orthologous groups (COG) analyses were used to determine the functional categories of these genes. A total of 84 putative chemosensory genes were identified from the antennal transcriptome of A. confusum, including 11 putative odorant-binding protein (OBP) genes, six chemosensory protein (CSP) genes, 44 olfactory receptor (OR) genes (including one olfactory co-receptor, Orco), 19 ionotropic receptor (IR) genes, and four sensory neuron membrane protein (SNMP) genes. Results of qPCR assays indicated that among of 11 AconOBPs, nine AconOBP genes were significantly expressed in the antennae of A. confusum adults. AconOBP8 was significantly expressed in the abdomen and AconOBP10 was highly expressed in the thorax. These findings can build a basis for further study on the processes of chemosensory perception in A. confusum at the molecular level.

Characterization and Functional Analysis of trehalase Related to Chitin Metabolism in Glyphodes pyloalis Walker (Lepidoptera: Pyralidae).

Glyphodes pyloalis Walker (G. pyloalis) is a serious pest on mulberry. Due to the increasing pesticide resistance, the development of new and effective environmental methods to control G. pyloalis is needed. Trehalase is an essential enzyme in trehalose hydrolysis and energy supply, and it has been considered a promising target for insect pest control. However, the specific function of trehalase in G. pyloalis has not been reported. In this study, two trehalase genes (GpTre1 and GpTre2) were identified from our previous transcriptome database. The functions of the trehalase in chitin metabolism were studied by injecting larvae with dsRNAs and trehalase inhibitor, Validamycin A. The open reading frames (ORFs) of GpTre1 and GpTre2 were 1,704 bp and 1,869 bp, which encoded 567 and 622 amino acid residues, respectively. Both of GpTre1 and GpTre2 were mainly expressed in the head and midgut. The highest expression levels of them were in 5th instar during different development stages. Moreover, knockdown both of GpTre1 and GpTre2 by the dsRNAs led to significantly decreased expression of chitin metabolism pathway-related genes, including GpCHSA, GpCDA1, GpCDA2, GpCHT3a, GpCHT7, GpCHSB, GpCHT-h, GpCHT3b, GpPAGM, and GpUAP, and abnormal phenotypes. Furthermore, the trehalase inhibitor, Validamycin A, treatment increased the expressions of GpTre1 and GpTre2, increased content of trehalose, and decreased the levels of glycogen and glucose. Additionally, the inhibitor caused a significantly increased cumulative mortality of G. pyloalis larvae on the 2nd (16%) to 6th (41.3%) day, and decreased the rate of cumulative pupation (72.3%) compared with the control group (95.6%). After the activities of trehalase were suppressed, the expressions of 6 integument chitin metabolism-related genes decreased significantly at 24 h and increased at 48 h. The expressions of GpCHSB and GpCHT-h, involved in chitin metabolism pathway of peritrophic membrane in the midgut, increased at 24 h and 48 h, and there were no changes to GpCHT3b and GpPAGM. These results reveal that GpTre1 and GpTre2 play an essential role in the growth of G. pyloalis by affecting chitin metabolism, and this provides useful information for insect pest control in the future.

Lipid Dynamics, Identification, and Expression Patterns of Fatty Acid Synthase Genes in an Endoparasitoid, Meteorus pulchricornis (Hymenoptera: Braconidae).

In insect parasitoids, fatty acid synthases (FASs) have received less attention and their roles associated with lipogenesis loss are far from clear. Meteorus pulchricornis is a solitary endoparasitoid wasp of many larvae of lepidopteran pests. The lipid content during developmental stages of M. pulchricornis was measured; it was higher in the larval and pupal stages but declined from six-day-old pupae. Lipid accumulation constantly decreased in the adult stage, even after feeding on honey solutions. To investigate the roles of FASs in lipid synthesis in M. pulchricornis, four FAS genes (MpulFAS1~4) were identified from the transcriptome database of M. pulchricornis. All FAS genes included full-length open reading frames and shared 72-79% similarity with the sequences of Microplitis demolitor. qRT-PCR validation showed that all four FASs had the highest expression after the adult wasps were fed on honey diets. MpulFAS1 and MpulFAS2 reached their expression peaks at the adult stage but MpulFAS3 and MpulFAS4 peaked at the larval stage. To further study the function of FASs, dsRNA injection knocked down the expression of four MpulFASs and resulted in a significant decline of lipid content at the adult stage in M. pulchricornis. Results from this study suggest that M. pulchricornis adults cannot accumulate lipid content effectively and FASs may still contribute to lipid synthesis in the adult stage. This broadens the knowledge on the ability of lipid synthesis in parasitoid wasps and provides insight into the roles of FASs in insects with parasitic life-history traits.

Identification, Characterization, and Functional Analysis of Chitin Synthase Genes in Glyphodes pyloalis Walker (Lepidoptera: Pyralidae).

Glyphodes pyloalis Walker (G. pyloalis) causes significant damage to mulberry every year, and we currently lack effective and environmentally friendly ways to control the pest. Chitin synthase (CHS) is a critical regulatory enzyme related to chitin biosynthesis, which plays a vital role in the growth and development of insects. The function of CHS in G. pyloalis, however, has not been studied. In this study, two chitin synthase genes (GpCHSA and GpCHSB) were screened from our previously created transcriptome database. The complete coding sequences of the two genes are 5,955 bp and 5,896 bp, respectively. Expression of GpCHSA and GpCHSB could be detected throughout all developmental stages. Relatively high expression levels of GpCHSA occurred in the head and integument and GpCHSB was most highly expressed in the midgut. Moreover, silencing of GpCHSA and GpCHSB using dsRNA reduced expression of downstream chitin metabolism pathway genes and resulted in abnormal development and wings stretching, but did not affect normal pupating of larvae. Furthermore, the inhibitor of chitin synthesis diflubenzuron (DFB) was used to further validate the RNAi result. DFB treatment significantly improved expression of GpCHSA, except GpCHSB, and their downstream genes, and also effected G. Pyloali molting at 48 h (62% mortality rate) and 72 h (90% mortality rate), respectively. These results show that GpCHSA and GpCHSB play critical roles in the development and wing stretching in G. pyloalis adults, indicating that the genes are attractive potential pest control targets.

Identifications, Characteristics, and Expression Patterns of Small Heat Shock Protein Genes in a Major Mulberry Pest, Glyphodes pyloalis (Lepidoptera: Pyralidae).

Six candidate sHSP genes were identified from the Glyphodes pyloalis transcriptome. All sHSP genes included full-length open reading frames and shared high similarity with the sequences of other lepidopteran species. These sHSP genes encoded 175-191 amino acid residues, and the predicted proteins had a molecular weight from 19.5 to 21.8 kDa. All GpsHSPs were expressed at lower levels at larval stages. All GpsHSPs were expressed at higher levels at diapaused, prepupal, or pupal stages, suggesting that sHSPs may be involved in metamorphosis in G. pyloalis. In addition to the developmental stage, extreme temperatures can induce variations in the expression of sHSPs genes. All GpsHSPs were significantly upregulated in larvae following exposure to heat shock, except GpHSP21.4 which downregulated at 4 h following exposure to the cold shock treatment. Furthermore, Starvation influenced the expression patterns of GpsHSPs as a function of the duration of food deprivation. Four GpsHSPs increased their expression with time of starvation until reaching to the peak level at 6 d of starvation. Finally, parasitism by the endoparasitoid Aulacocentrum confusum He et van Achterberg (Hymenoptera: Braconidae)-induced fluctuations in the expression of all GpsHSPs, and the expression varied with time after parasitization. Our results from this study strongly suggest functional differentiation within the sHSPs subfamily in G. pyloalis. The present study would provide further insight into the roles of sHSPs in G. pyloalis and novel avenues for promoting integrated management of this pest.

Identification and Functional Study of Chitin Metabolism and Detoxification-Related Genes in Glyphodes pyloalis Walker (Lepidoptera: Pyralidae) Based on Transcriptome Analysis.

Glyphodes pyloalis Walker (Lepidoptera: Pyralididae) is a serious pest in the sericulture industry, which has caused damage and losses in recent years. With the widespread use of insecticides, the insecticide resistance of G. pyloalis has becomes increasingly apparent. In order to find other effective methods to control G. pyloalis, this study performed a transcriptome analysis of the midgut, integument, and whole larvae. Transcriptome data were annotated with KEGG and GO, and they have been shown to be of high quality by RT-qPCR. The different significant categories of differentially expressed genes between the midgut and the integument suggested that the transcriptome data could be used for next analysis. With the exception of Dda9 (GpCDA5), 19 genes were involved in chitin metabolism, most of which had close protein-protein interactions. Among them, the expression levels of 11 genes, including GpCHSA, GpCDA1, GpCDA2, GpCDA4, GPCHT1, GPCHT2a, GPCHT3a, GPCHT7, GpTre1, GpTre2, and GpRtv were higher in the integument than in the midgut, while the expression levels of the last eight genes, including GpCHSB, GpCDA5, GpCHT2b, GpCHT3b, GpCHT-h, GpPAGM, GpNAGK, and GpUAP, were higher in the midgut than in the integument. Moreover, 282 detoxification-related genes were identified and can be divided into 10 categories, including cytochrome P450, glutathione S-transferase, carboxylesterase, nicotinic acetylcholine receptor, aquaporin, chloride channel, methoprene-tolerant, serine protease inhibitor, sodium channel, and calcium channel. In order to further study the function of chitin metabolism-related genes, dsRNA injection knocked down the expression of GpCDA1 and GpCHT3a, resulting in the significant downregulation of its downstream genes. These results provide an overview of chitin metabolism and detoxification of G. pyloalis and lay the foundation for the effective control of this pest in the sericulture industry.

Identification of glutathione-S-transferase genes by transcriptome analysis in Meteorus pulchricornis (Hymenoptera: Braconidae) and their expression patterns under stress of phoxim and cypermethrin.

Meteorus pulchricornis (Wesmael) (Hymenoptera: Braconidae) is a preponderant endoparasitoid wasp, attacking the larvae of many lepidopteran pests. We present the first body transcriptome dataset for M. pulchricornis. In total, 50,781,796 clean reads were obtained and 33,144 unigenes were assembled; 15,458 unigenes showed a significant similarity (E value < 10-5) to known proteins in the NCBI non-redundant protein database. Gene ontology and cluster of orthologous group analyses were performed to classify the functions of genes. To better understand the role of glutathione-S-transferases (GSTs) in detoxification mechanism in M. pulchricornis, we identified seventeen GST genes (MpulGSTs) from the body transcriptome. Among these, fifteen MpulGSTs belonged to cytosolic GSTs and the other two belonged to microsomal classes. The cytosolic GSTs were classified into four different clades: four in delta, three in omega, seven in sigma, and one in zeta. The expression levels of these MpulGSTs after exposure to sub-lethal concentrations of phoxim and cypermethrin were determined using real-time quantitative polymerase chain reaction: seven MpulGSTs (MpulGSTD3, MpulGSTS1, MpulGSTS2, MpulGSTS4, MpulGSTS6 MpulGSTO3, and MpulGSTmic1) and 11 MpulGSTs (MpulGSTD1, MpulGSTD2, MpulGSTD3, MpulGSTO2, MpulGSTS1, MpulGSTS2, MpulGSTS3, MpulGSTS4, MpulGSTS5, MpulGSTS7, and MpulGSTmic1) were highly expressed, respectively. These results suggested that GST genes may play a pivotal role in detoxification process in M. pulchricornis. Our findings would provide a theoretical base for elucidating insecticide susceptibility and should promote functional research on specific GST genes in parasitoid wasps.