Identification, expression analysis, and functional verification of three opsin genes related to the phototactic behaviour of Ostrinia furnacalis.

Ostrinia furnacalis is a disreputable herbivorous pest that poses a serious threat to corn crops. Phototaxis in nocturnal moths plays a crucial role in pest prediction and control. Insect opsins are the main component of insect visual system. However, the inherent molecular relationship between phototactic behaviour and vision of insects remains a mystery. Herein, three opsin genes were identified and cloned from O. furnacalis (OfLW, OfBL, and OfUV). Bioinformatics analysis revealed that all opsin genes had visual pigment (opsin) retinal binding sites and seven transmembrane domains. Opsin genes were distributed across different developmental stages and tissues, with the highest expression in adults and compound eyes. The photoperiod-induced assay elucidated that the expression of three opsin genes in females were higher during daytime, while their expression in males tended to increase at night. Under the sustained darkness, the expression of opsin genes increased circularly, although the increasing amplitude in males was lower when compared with females. Furthermore, the expression of OfLW, OfBL, and OfUV was upregulated under green, blue, and ultraviolet light, respectively. The results of RNA interference showed that the knockout of opsin genes decreased the phototaxis efficiency of female and male moths to green, blue, and ultraviolet light. Our results reveal that opsin genes are involved in the phototactic behaviour of moths, providing a potential target gene for pest control and a basis for further investigation on the phototactic behaviour of Lepidoptera insects.

Identification of Six Small Heat Shock Protein Genes in Ostrinia furnacalis (Lepidoptera: Pyralidae) and Analysis of Their Expression Patterns in Response to Environmental Stressors.

Ostrinia furnacalis (Guenée) is a major insect pest in maize production that is highly adaptable to the environment. Small heat shock proteins (sHsps) are a class of chaperone proteins that play an important role in insect responses to various environmental stresses. The present study aimed to clarify the responses of six O. furnacalis sHsps to environmental stressors. In particular, we cloned six sHsp genes, namely, OfHsp24.2, OfHsp21.3, OfHsp20.7, OfHsp21.8, OfHsp29.7, and OfHsp19.9, from O. furnacalis. The putative proteins encoded by these genes contained a typical α-crystallin domain. Real-time quantitative polymerase chain reaction was used to analyze the differences in the expression of these genes at different developmental stages, in different tissues of male and female adults, and in O. furnacalis under UV-A and extreme temperature stresses. The six OfsHsp genes were expressed at significantly different levels based on the developmental stage and tissue type in male and female adults. Furthermore, all OfsHsp genes were significantly upregulated in both male and female adults under extreme temperature and UV-A stresses. Thus, O. furnacalis OfsHsp genes play important and unique regulatory roles in the developmental stages of the insect and in response to various environmental stressors.

Ethyl methanesulfonate mutant library construction in Gossypium hirsutum L. for allotetraploid functional genomics and germplasm innovation.

As the gene pool is exposed to both strain on land resources and a lack of diversity in elite allotetraploid cotton, the acquisition and identification of novel alleles has taken on epic importance in facilitating cotton genetic improvement and functional genomics research. Ethyl methanesulfonate (EMS) is an excellent mutagen that induces genome-wide efficient mutations to activate the mutagenic potential of plants with many advantages. The present study established, determined and verified the experimental procedure suitable for EMS-based mutant library construction as the general reference guide in allotetraploid upland cotton. This optimized method and procedure are efficient, and abundant EMS mutant libraries (approximately 12 000) in allotetraploid cotton were successfully obtained. More than 20 mutant phenotypes were observed and screened, including phenotypes of the leaf, flower, fruit, fiber and plant architecture. Through the plants mutant library, high-throughput and high-resolution melting technology-based variation evaluation detected the EMS-induced site mutation. Additionally, based on overall genome-wide mutation analyses by re-sequencing and mutant library assessment, the examination results demonstrated the ideal quality of the cotton EMS-treated mutant library constructed in this study with appropriate high mutation density and saturated genome. What is more, the collection is composed of a broad repertoire of mutants, which is the valuable resource for basic genetic research and functional genomics underlying complex allotetraploid traits, as well as cotton breeding.

Live-Cell Imaging of Survivin mRNA by Using a Dual-Color Surface-Cross-Linked Nanoquencher.

Precise detection of cancer-related mRNAs can significantly benefit the early diagnosis and potential therapy of cancers. Herein, we report organic dark quencher-encapsulated surface-cross-linked micelles (qSCMs) as a new sort of nanoquencher for construction of potential multiple-color fluorescence imaging nanosensors. Such nanoquenchers featured simple preparation (one-pot), broad-spectrum quenching (450-800 nm), high quenching efficiency (>94%), good stability, negligible cargo leakage, facile covalent surface modification, and finally excellent modularity. As a proof-of-concept demonstration, a mRNA-detecting qSCM nanosensor was generated, capable of simultaneous live-cell imaging of endogenous actin mRNA (a house-keeping gene) and cancer-related survivin mRNA. This nanosensor was found to be GSH- and DNase I-resistant, and with actin mRNA as an intrinsic reference, it was used to image the precise survivin mRNA expression across different mammalian cells through convenient normalization of the signal readouts. Moreover, the nanosensor was further used to quantitatively image the downregulation of endogenous survivin mRNA in HeLa cells upon treatment of YM155 (an imidazolium bioactive compound known to suppresses endogenous survivin mRNA expression). These results clearly demonstrated the promising application of these qSCMs as new nanoquenchers in potential multicolor imaging of various endogenous biomarkers.

Cell-Penetrating Mitochondrion-Targeting Ligands for the Universal Delivery of Small Molecules, Proteins and Nanomaterials.

Mitochondria are key organelles that perform vital cellular functions such as those related to cell survival and death. The targeted delivery of different types of cargos to mitochondria is a well-established strategy to study mitochondrial biology and diseases. Of the various existing mitochondrion-transporting vehicles, most suffer from poor cytosolic entry, low delivery efficiency, limited cargo types, and cumbersome preparation protocols, and none was known to be universally applicable for mitochondrial delivery of different types of cargos (small molecules, proteins, and nanomaterials). Herein, two new cell-penetrating, mitochondrion-targeting ligands (named MitoLigand ) that are capable of effectively "tagging" small-molecule drugs, native proteins and nanomaterials are disclosed, as well as their corresponding chemoselective conjugation chemistry. Upon successful cellular delivery and rapid endosome escape, the released native cargos were found to be predominantly localized inside mitochondria. Finally, by successfully delivering doxorubicin, a well-known anticancer drug, to the mitochondria of HeLa cells, we showed that the released drug possessed potent cell cytotoxicity, disrupted the mitochondrial membrane potential and finally led to apoptosis. Our strategy thus paves the way for future mitochondrion-targeted therapy with a variety of biologically active agents.

Expression stability of candidate RT-qPCR housekeeping genes in Spodoptera frugiperda (Lepidoptera: Noctuidae).

Reverse-transcription quantitative polymerase chain reaction (RT-qPCR) is commonly used to quantify gene expression. For normalization, the expression of each gene is compared with a reference "housekeeping" gene that is stably expressed under relevant stress. Unfortunately, there have been no reports on the stability of such reference genes under various treatments of the Spodoptera frugiperda. In this study, we used five tools (RefFinder, GeNorm, NormFinder, BestKeeper, and ΔCt methods) to evaluate the stability of 12 candidate reference genes (RPS18, ß-tubulin, GAPDH, RPS7, RPS15, RPL7, RPL32, Actin-5C, EF1-α, EF1-γ, RPL27, and ACE) in different instars, tissues, and treatments (high and low temperature, UV-A, and emamectin benzoate). Several ribosomal proteins (RPS7, RPS15, RPL32, RPS18, and RPL7), GAPDH, Actin-5C, and ß-tubulin, were relatively stable, suggesting that they are ideal housekeeping genes for various treatments. ACE was extremely unstable under various experimental treatments, rendering it unsuitable as an internal reference. This study identified the reference housekeeping genes stably expressed by S. frugiperda under different treatments, thus setting a foundation for further exploration of the physiological and biochemical mechanisms.

Transcriptome Analysis of Myzus persicae to UV-B Stress.

As an environmental stress factor, ultraviolet-B (UV-B) radiation directly affects the growth and development of Myzus persicae (Sulzer) (Homoptera: Aphididae). How M. persicae responds to UV-B stress and the molecular mechanisms underlying this adaptation remain unknown. Here, we analyzed transcriptome data for M. persicae following exposure to UV-B radiation for 30 min. We identified 758 significant differentially expressed genes (DEGs) following exposure to UV-B stress, including 423 upregulated and 335 downregulated genes. In addition, enrichment analysis using the Gene Ontology and Kyoto Encyclopedia of Genes and Genomes databases illustrated that these DEGs are associated with antioxidation and detoxification, metabolic and protein turnover, immune response, and stress signal transduction. Simultaneously, these DEGs are closely related to the adaptability to UV-B stress. Our research can raise awareness of the mechanisms of insect responses to UV-B stress.

Recent Advances in Polymeric Nanoparticles for Enhanced Fluorescence and Photoacoustic Imaging.

In recent years, polymeric nanoparticles (NPs) have become increasingly popular for in vitro and in vivo bioimaging because of their excellent versatility and exceptional optical properties. Following a brief introduction on fluorescence and photoacoustic imaging, as well as the merits of using polymeric NPs over other types of fluorescent probes, this Minireview gives a concise summary of key advances made in recent years (2017-2020) in the use of polymeric NPs for fluorescence and photoacoustic imaging. A particular focus is placed on various strategies used for enhanced bioimaging applications, including polymeric NPs that encapsulate near-infrared dyes, aggregation-induced-emission fluorogens, cationic dyes doped with bulky hydrophobic counterions, and semiconducting polymers. Next, the current limitations in some of these polymeric NP systems are summarized and potential solutions offered to overcome them. Finally, some critical considerations in regard to the design of polymeric NPs are given for future bioimaging and sensing applications.

NBD-based fluorescent probes for separate detection of cysteine and biothiols via different reactivities.

NBD-based fluorescent probes that can separately detect cysteine and biothiols via different reactivities have been rationally designed and synthesized. The probes can be applied to kinetically distinguish cysteine from other biothiols at 30 min, and to detect all biothiols at 3 h in living cells.

MicroRNA-277 targets insulin-like peptides 7 and 8 to control lipid metabolism and reproduction in Aedes aegypti mosquitoes.

Hematophagous female mosquitoes transmit numerous devastating human diseases, including malaria, dengue fever, Zika virus, and others. Because of their obligatory requirement of a vertebrate blood meal for reproduction, these mosquitoes need a lot of energy; therefore, understanding the molecular mechanisms linking metabolism and reproduction is of particular importance. Lipids are the major energy store providing the fuel required for host seeking and reproduction. They are essential components of the fat body, a metabolic tissue that is the insect analog of vertebrate liver and adipose tissue. In this study, we found that microRNA-277 (miR-277) plays an important role in regulating mosquito lipid metabolism. The genetic disruption of miR-277 using the CRISPR-Cas9 system led to failures in both lipid storage and ovary development. miR-277 mimic injection partially rescued these phenotypic manifestations. Examination of subcellular localization of FOXO protein via CRISPR-assisted, single-stranded oligodeoxynucleotide-mediated homology-directed repair revealed that insulin signaling is up-regulated in response to miR-277 depletion. In silico target prediction identified that insulin-like peptides 7 and 8 (ilp7 and ilp8) are putative targets of miR-277; RNA immunoprecipitation and a luciferase reporter assay confirmed that ilp7 and ilp8 are direct targets of this miRNA. CRISPR-Cas9 depletion of ilp7 and ilp8 led to metabolic and reproductive defects. These depletions identified differential actions of ILP7 and ILP8 in lipid homeostasis and ovarian development. Thus, miR-277 plays a critical role in mosquito lipid metabolism and reproduction by targeting ilp7 and ilp8, and serves as a monitor to control ILP7 and ILP8 mRNA levels.

Fluorogenic Assay for Acetohydroxyacid Synthase: Design and Applications.

Acetohydroxyacid synthase (AHAS) exists in plants and many microorganisms (including gut flora) but not in mammals, making it an attractive drug target. Fluorescent-based methods should be practical for high-throughput screening of inhibitors. Herein, we describe the development of the first AHAS fluorogenic assay based on an intramolecular charge transfer (ICT)-based fluorescent probe. The assay is facile, sensitive, and continuous and can be applied toward various AHASs from different species, AHAS mutants, and crude cell lysates. The fluorogenic assay was successfully applied for (1) high-throughput screening of commerical herbicides toward different AHASs for choosing matching herbicides, (2) identification of a Soybean AHAS gene with broad-spectrum herbicide resistance, and (3) identification of selective inhibitors toward intestinal-bacterial AHASs. Among the AHAS inhibitors, an active agent was found for selective inhibition of obesity-associated Ruminococcus torques growth, implying the possibility of AHAS inhibitors for the ultimate goal toward antiobesity therapeutics. The fluorogenic assay opens the door for high-throughput programs in AHAS-related fields, and the design principle might be applied for development of fluorogenic assays of other synthases.

Dual-quenching NBD-based fluorescent probes for separate detection of H2S and Cys/Hcy in living cells.

Biothiols are implicated in different physiological and pathological processes, while abnormal levels of biothiols are related to numerous diseases. It is essential to discriminatively detect these molecules and explore their inherent transformation in living biological samples. Herein, dual-reactive and dual-quenching fluorescent probes based on thiolysis of NBD (7-nitro-1,2,3-benzoxadiazole) thioether/ether/amine for separate detection of H2S and Cys/Hcy were rationally designed. Probe 2 was able to sense H2S (λex/em = 405/455 nm) and Cys/Hcy (λex/em = 470/550 nm) in the presence of GSH in aqueous buffer. Moreover, the probe not only could be applied for visualization of exogenous H2S and Cys in living cells but also successfully showcased the potential to verify the biosynthesis of endogenous H2S from Cys.

Juvenile hormone and its receptor methoprene-tolerant promote ribosomal biogenesis and vitellogenesis in the Aedes aegypti mosquito.

Juvenile hormone (JH) controls many biological activities in insects, including development, metamorphosis, and reproduction. In the Aedes aegypti mosquito, a vector of dengue, yellow fever, chikungunya, and zika viruses, the metabolic tissue (the fat body, which is an analogue of the vertebrate liver) produces yolk proteins for developing oocytes. JH is important for the fat body to acquire competence for yolk protein production. However, the molecular mechanisms of how JH promotes mosquito reproduction are not completely understood. In this study we show that stimulation of the JH receptor methoprene-tolerant (Met) activates expression of genes encoding the regulator of ribosome synthesis 1 (RRS1) and six ribosomal proteins (two ribosomal large subunit proteins, two ribosomal small subunit proteins, and two mitochondrial ribosomal proteins). Moreover, RNAi-mediated depletion of RRS1 decreased biosynthesis of the ribosomal protein L32 (RpL32). Depletion of Met, RRS1, or RpL32 led to retardation of ovarian growth and reduced mosquito fecundity, which may at least in part have resulted from decreased vitellogenin protein production in the fat body. In summary, our results indicate that JH is critical for inducing the expression of ribosomal protein genes and demonstrate that RRS1 mediates the JH signal to enhance both ribosomal biogenesis and vitellogenesis.

A highly efficient dual-diazonium reagent for protein crosslinking and construction of a virus-based gel.

A new bench-stable reagent with double diazonium sites was designed and synthesized for protein crosslinking. Based on the highly efficient diazonium-Tyr coupling reaction, a direct mixture of the reagent and tobacco mosaic virus led to the formation of a new hydrogel, which could be degraded by chemicals and could be used to encapsulate small molecules for sustained release. Because plant viruses exhibit many chemical characteristics like protein labelling and nucleic acid packaging, the virus-based hydrogel will have large chemical space for further functionalization. Besides, this dual-diazonium reagent should be a generally useful crosslinker for chemical biology and biomaterials.

Fast-Response Turn-on Fluorescent Probes Based on Thiolysis of NBD Amine for H2 S Bioimaging.

Hydrogen sulfide (H2 S) is an important endogenous signaling molecule with multiple biological functions. New selective fluorescent turn-on probes based on fast thiolyling of NBD (7-nitro-1,2,3-benzoxadiazole) amine were explored for sensing H2 S in aqueous buffer and in living cells. The syntheses of both probes are simple and quite straightforward. The probes are highly sensitive and selective toward H2 S over other biologically relevant species. The fluorescein-NBD-based probe showed 65-fold green fluorescent increase upon H2 S activation. The rhodamine-NBD-based probe reacted rapidly with H2 S (t1/2 ≈1 min) to give a 4.5-fold increase in red fluorescence. Moreover, both probes were successfully used for monitoring H2 S in living cells and in mice. Based on such probe-based tools, we could observe H2 O2 -induced H2 S biogenesis in a concentration-dependent and time-dependent fashion in living cells.

A Dual-Response Fluorescent Probe Reveals the H2 O2 -Induced H2 S Biogenesis through a Cystathionine ß-Synthase Pathway.

The two signaling molecules H2 S and H2 O2 play key roles in maintaining intracellular redox homeostasis. The biological relationship between H2 O2 and H2 S remains largely unknown in redox biology. In this study, we rationally designed and synthesized single- and dual-response fluorescent probes for detecting both H2 O2 and H2 S in living cells. The dual-response probe was shown to be capable of mono- and dual-detection of H2 O2 and H2 S selectively and sensitively. Detailed bioimaging studies based on the probes revealed that both exogenous and endogenous H2 O2 could induce H2 S biogenesis in living cells. By using gene-knockdown techniques with bioimaging, the H2 S biogenesis was found to be majorly cystathionine ß-synthase (CBS)-dependent. Our finding shows the first direct evidence on the biological communication between H2 O2 (ROS) and H2 S (RSS) in vivo.

Differential protein expression in the susceptible and resistant Myzus persicae (Sulzer) to imidacloprid.

Myzus persicae, a serious economic agricultural pest, has developed resistance to imidacloprid (IMI), which was widely used to control this aphid worldwide. To gain a better understanding of the mechanisms of IMI resistance in M. persicae, we carried out a comparative proteomic analysis. Total proteins of the IMI-susceptible and resistant strains were extracted and separated by two-dimensional gel electrophoresis. More than 1300 protein spots were reproducibly detected, including 14 that were more abundant and 14 less abundant. Mass spectrometry analysis and database searching helped us to identify 25 differentially abundant proteins. The identified proteins were categorized into several functional groups including signal transduction, RNA processing, protein processing, transport processing, stress response, metabolisms, and cytoskeleton structure, etc. This study is the first analysis of differentially expressed proteins in IMI-susceptible and resistant M. Persicae, and gives new insights into the mechanisms of IMI resistance in M. persicae.

Enzymatic activity and development of Frankliniella occidentalis (Thysanoptera: Thripidae) in response to exogenous calcium treatments of kidney bean plants.

Frankliniella occidentalis Pergande (Thysanoptera: Thripidae) is an agricultural pest threatening various horticultural crops worldwide. Inducing plant resistance is an ecologically beneficial and potentially effective method for controlling F. occidentalis. As an essential nutrient element, exogenous calcium enhances plant-induced resistance. This study investigated the effects of CaCl2 on the secondary metabolites of kidney bean plants and detoxifying and digestive enzymes in F. occidentalis. We found that treatment of plants and treatment time and also the interactions of the 2 factors significantly affected secondary metabolites contents (tannin, flavonoids, total phenol, alkaloid, and lignin) of kidney bean leaves, which indicated that that the effect of treatment of plants on secondary metabolites varied with treatment time. Moreover, when thrips fed on CaCl2-treated plants, the activities of detoxifying enzymes, enzymes glutathione S-transferase and cytochrome P450 substantially increased compared to those in which thrips fed on control plants. However, the activity of carboxylesterase significantly decreased. The detoxifying enzyme genes CL992.contig6, CYP4PN1, and CYP4PJ2 were significantly upregulated at 24 and 48 h. The activities of digestive enzymes (α-amylase, chymotrypsin, and lipase) increased substantially in F. occidentalis. The digestive enzyme gene, FoAMY-1, was significantly upregulated at 24 and 48 h after treatment. The pupation rate and pupal weight of F. occidentalis were significantly reduced. The results indicated that exogenous CaCl2-induced metabolic changes in kidney bean plants and altered the enzymatic activity and development of F. occidentalis that fed upon them.

Integrated transcriptomic and proteomic analyses reveal the molecular mechanism underlying the thermotolerant response of Spodoptera frugiperda.

Spodoptera frugiperda (Lepidoptera: Noctuidae) is a highly destructive invasive pest with remarkable adaptability to extreme climatic conditions, posing a substantial global threat. Although the effects of temperature stress on the biological and ecological properties of S. frugiperda have been elucidated, the molecular mechanisms underlying its responses remain unclear. Herein, we combined transcriptomic and proteomic analyses to explore the key genes and proteins involved in thermotolerance regulation in S. frugiperda larvae at 42 °C. Overall, 1528 differentially expressed genes (DEGs) and 154 differentially expressed proteins (DEPs) were identified in S. frugiperda larvae under heat stress, including antioxidant enzymes, heat shock proteins (Hsps), cytochrome P450s, starch and sucrose metabolism genes, and insulin signaling pathway genes, indicating their involvement in heat tolerance regulation. Correlation analysis of DEGs and DEPs revealed that seven and eight had the same and opposite expression profiles, respectively. After nanocarrier-mediated RNA interference knockdown of SfHsp29, SfHsp20.4, SfCAT, and SfGST, the body weight and mortality of S. frugiperda larvae significantly decreased and increased under heat stress, respectively. This indicates that SfHsp29, SfHsp20.4, SfCAT, and SfGST play a crucial role in the thermotolerance of S. frugiperda larvae. These results provide insight into the mechanism of heat tolerance in S. frugiperda.

Refolding, Crystallization, and Crystal Structure Analysis of a Scavenger Receptor Cysteine-Rich Domain of Human Salivary Agglutinin Expressed in Escherichia coli.

Scavenger receptors are a protein superfamily that typically consists of one or more repeats of the scavenger receptor cysteine-rich structural domain (SRCRD), which is an ancient and highly conserved protein module. The expression and purification of eukaryotic proteins containing multiple disulfide bonds has always been challenging. The expression systems that are commonly used to express SRCRD proteins mainly consist of eukaryotic protein expression systems. Herein, we established a high-level expression strategy of a Type B SRCRD unit from human salivary agglutinin using the Escherichia coli expression system, followed by a refolding and purification process. The untagged recombinant SRCRD was expressed in E. coli using the pET-32a vector, which was followed by a refolding process using the GSH/GSSG redox system. The SRCRD expressed in E. coli SHuffle T7 showed better solubility after refolding than that expressed in E. coli BL21(DE3), suggesting the importance of the disulfide bond content prior to refolding. The quality of the refolded protein was finally assessed using crystallization and crystal structure analysis. As proteins refolded from inclusion bodies exhibit a high crystal quality and reproducibility, this method is considered a reliable strategy for SRCRD protein expression and purification. To further confirm the structural integrity of the refolded SRCRD protein, the purified protein was subjected to crystallization using sitting-drop vapor diffusion method. The obtained crystals of SRCRD diffracted X-rays to a resolution of 1.47 Å. The solved crystal structure appeared to be highly conserved, with four disulfide bonds appropriately formed. The surface charge distribution of homologous SRCRD proteins indicates that the negatively charged region at the surface is associated with their calcium-dependent ligand recognition. These results suggest that a high-quality SRCRD protein expressed by E. coli SHuffle T7 can be successfully folded and purified, providing new options for the expression of members of the scavenger receptor superfamily.