An OBP gene highly expressed in non-chemosensory tissues affects the phototaxis and reproduction of Spodoptera frugiperda.

Insect odorant binding proteins (OBPs) were initially regarded as carriers of the odorants involved in chemosensation. However, it had been observed that a growing number of OBP genes exhibited broad expression patterns beyond chemosensory tissues. Here, an OBP gene (OBP31) was found to be highly expressed in the larval ventral nerve cord, adult brain and male reproductive organ of Spodoptera frugiperda. An OBP31 knockout strain (OBP31-/- ) was generated by CRISPR/Cas9 mutagenesis. For OBP31-/- , the larvae needed longer time to pupate, but there was no difference in the pupal weight between OBP31-/- and wild type (WT). OBP31-/- larvae showed stronger phototaxis than the WT larvae, indicating the importance of OBP31 in light perception. For mating rhythm of adults, OBP31-/- moths displayed an earlier second mating peak. In the cross-pairing of OBP31-/- and WT moths, the mating duration was longer, and hatchability was lower in OBP31-/- group and OBP31+/- ♂ group than that in the WT group. These results suggested that OBP31 played a vital role in larval light perception and male reproductive process and could provide valuable insights into understanding the biological functions of OBPs that were not specific in chemosensory tissues.

A nonsensory odorant-binding protein plays an important role in the larval development and adult mating of Spodoptera frugiperda.

Odorant-binding proteins (OBPs) play key roles in the perception of semiochemicals in insects. Several OBPs in insect olfactory systems have been functionally characterized, and they provide excellent targets for pest control. The functions of some OBPs that are highly expressed in the nonsensory organs of insects remain unclear. Here, the physiological function of an OBP (OBP27) that was highly expressed in the nonsensory organs of Spodoptera frugiperda was studied. OBP27 was nested within the Plus-C cluster according to phylogenetic analysis. The transcription of OBP27 steadily increased throughout the development of S. frugiperda, and transcripts of this gene were abundant in the fat body and male reproductive organs. An OBP27 knockout strain with an early frameshift mutation was obtained using the clustered regularly interspaced palindromic repeats (CRISPR) / CRISPR-associated protein 9 (Cas9) system. The development time of OBP27-/- larvae was significantly longer than that of other larvae. Both male and female OBP27-/- pupae weighed significantly less than wild-type (WT) pupae. In crosses of OBP27-/- males or females, the mating rate was lower and the mating duration was longer for OBP27-/- male-WT female pairs than for WT-WT pairs. By contrast, the mating rate, hatching rate, and number of eggs of OBP27-/- female-WT male pairs and WT-WT pairs were similar. These findings indicate that OBP27 plays an important role in the larval development and mating process in male adults. Generally, our findings provide new insights into the physiological roles of nonsensory OBPs.

CircHIPK3 promotes bone microvascular endothelial cell proliferation, migration and angiogenesis by targeting miR-7 and KLF4/VEGF signaling in steroid-induced osteonecrosis of the femoral head.

BACKGROUND: Circular RNA homeodomain interacting protein kinase 3 (circHIPK3) has been implicated in facilitating angiogenesis in various conditions. However, its role in steroid-induced osteonecrosis of the femoral head (ONFH) remains unclear. OBJECTIVES: To investigate whether circHIPK3 promotes bone microvascular activity and angiogenesis by targeting miR-7 and Krüppel-like factor 4 (KLF4)/vascular endothelial growth factor (VEGF) signaling in ONFH. MATERIAL AND METHODS: Fifty patients with steroid-induced ONFH undergoing hip-preserving surgery or total hip arthroplasty were included in this study. The expression of circHIPK3, miR-7 and KLF4 was evaluated using reverse transcription polymerase chain reaction (RT-PCR) in necrotic and healthy samples of the femoral head. Bone microvascular endothelial cells (BMECs) were extracted and cultured with 0.1 mg/mL hydrocortisone to create a hormonally deficient cell model. These BMECs were then transfected with either circHIPK3 overexpressing or silencing plasmids with or without miR-7 mimics. The MTT assays were used to detect cell proliferation. Scratch assays were used to assess the migration ability of the BMECs. The tube formation was carried out using an in vitro Matrigel angiogenesis assay. Annexin V-FITC/PI and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assays were used to assess the degree of apoptosis. Western blot assays were carried out to discern KLF4 and VEGF expression. The interactions of circHIPK3, miR-7 and KLF4 were confirmed using luciferase, RNA-binding protein immunoprecipitation (RIP), RNA pull-down, and fluorescence in situ hybridization (FISH) assays. RESULTS: The circHIPK3 and KLF4 expression was decreased, whereas miR-7 expression was increased in necrotic tissues compared to non-necrotic samples. Both circHIPK3 and KLF4 expression correlated negatively with miR-7. The overexpression of circHIPK3 promoted the proliferative, migratory and angiogenic capabilities of the BMECs, while adding an miR-7 mimic reversed these effects. At the same time, the overexpression of circHIPK3 reduced the apoptosis rate of the BMECs and increased KLF4 and VEGF protein expression, but adding an miR-7 mimic reversed these effects. The FISH, RNA pull-down, RIP, and luciferase assays revealed an interaction between circHIPK3, miR-7 and KLF4. CONCLUSIONS: The circHIPK3 promotes BMEC proliferation, migration and angiogenesis by targeting miR-7 and KLF4/VEGF signaling.

Deciphering the active constituents of Dabushen decoction of ameliorating osteoarthritis via PPARγ preservation by targeting DNMT1.

Osteoarthritis (OA) is a multifactorial and chronic degenerative joint disease. Due to the adverse effects of currently used drugs, a safer and more effective therapy for treating OA is needed. Peroxisome proliferator-activated receptor-γ (PPARγ) is a key protein protecting cartilage. DNMT1-mediated hypermethylation of PPARγ promoter leads to its suppression. Therefore, DNMT1 might be an effective target for exerting cartilage protective effects by regulating the epigenetic expression of PPARγ. Dabushen decoction (DD) is a representative prescription of Dunhuang ancient medical prescription, which has a potential therapeutic effect on OA. So far, the research of the efficacy and material basis of DD in the treatment of OA remains unclear. In this study, Micro-CT, HE staining, S-O staining, and immunohistochemistry analysis were used to demonstrate that DD increased the expression of PPARγ and collagen synthesis in an OA rat model. Next, the structure of DNMT1 was used to screen the active constituents of DD by molecular docking method for treatment OA. Seven potential active constituents, including isoliquiritigenin, emodin, taxifolin, catalpol, alisol A, zingerone, and schisandrin C were hited. The protective effect of the potential active constituents to chondrocytes were evaluated by protein capillary electrophoresis, immunofluorescence assays, and ex vivo culture of rat knee cartilage. The five constituents, such as alisol A, emodin, taxifolin, isoliquiritigenin, and schisandrin C could promote the expression of PPARγ and ameliorate IL-1ß-induced downregulation of collagen II and the production of MMP-13. Alisol A and Emodin could effectively mitigate cartilage damage. At last, molecular dynamics simulations with MM-GBSA method was applied to investigate the interaction pattern of the active constituents and DNMT1 complexes. The five constituents, such as alisol A, emodin, taxifolin, isoliquiritigenin, and schisandrin C achieved a stable binding pattern with DNMT1, in which alisol A has a relatively high binding free energy. In conclusion, this study elucidates that the active constituents of DD (alisol A, emodin, taxifolin, isoliquiritigenin, and schisandrin C) could ameliorate osteoarthritis via PPARγ preservation by targeting DNMT1.These findings facilitated clinical use of DD and provided a valuable strategy for developing natural epigenetic modulators from Chinese herbal formula.

Effects of temperature on fitness costs in chlorpyrifos-resistant brown planthopper, Nilaparvata lugens (Hemiptera: Delphacidae).

Insecticide resistance is inevitable if an insecticide is widely used to control insect pests. Fortunately, the resistance-associated fitness costs often give chances to manage resistances. In most cases, the fitness cost in resistant insects is often evaluated under laboratory conditions for insect development, which limits its practical application in pest control in the field. In a laboratory population R9 with 253-fold resistance to chlorpyrifos after nine-generation selection with chlorpyrifos, the relative fitness was only 0.206 under laboratory conditions (25°C, humidity 70%-80% and 16 h light/8 h dark photoperiod), when compared to S9, a susceptible counterpart (resistance ratio = 2.25-fold) from the same origin as R9 but without any selection with insecticides. Temperatures varied the resistance-associated fitness costs, with enhanced costs at high temperatures and reduced costs at low temperatures, such as 0.174 at 32°C and 0.527 at 18°C. The copulation rate and fecundity were two key factors for the reduced costs at low temperatures. Another finding was that R9 individuals needed much more time to recover from heat shock than that of S9, but R9 and S9 individuals were similarly sensitive to cold shock. The low fitness cost at low temperatures would increase the overwintering population, which might further increase risks of rapid development and widespread distribution of chlorpyrifos resistance in Nilaparvata lugens.

Induction of P450 genes in Nilaparvata lugens and Sogatella furcifera by two neonicotinoid insecticides.

Nilaparvata lugens and Sogatella furcifera are two primary planthoppers on rice throughout Asian countries and areas. Neonicotinoid insecticides, such as imidacloprid (IMI), have been extensively used to control rice planthoppers and IMI resistance consequently occurred with an important mechanism from the over-expression of P450 genes. The induction of P450 genes by IMI may increase the ability to metabolize this insecticide in planthoppers and increase the resistance risk. In this study, the induction of P450 genes was compared in S. furcifera treated with IMI and nitromethyleneimidazole (NMI), in two planthopper species by IMI lethal dose that kills 85% of the population (LD85 ), and in N. lugens among three IMI doses (LD15 , LD50 and LD85 ). When IMI and NMI at the LD85 dose were applied to S. furcifera, the expression changes in most P450 genes were similar, including the up-regulation of nine genes and down-regulation of three genes. In terms of the expression changes in 12 homologous P450 genes between N. lugens and S. furcifera treated with IMI at the LD85 dose, 10 genes had very similar patterns, such as up-regulation in seven genes, down-regulation in one gene and no significant changes in two genes. When three different IMI doses were applied to N. lugens, the changes in P450 gene expression were much different, such as up-regulation in four genes at all doses and dose-dependent regulation of the other nine genes. For example, CYP6AY1 could be induced by all IMI doses, while CYP6ER1 was only up-regulated by the LD50 dose, although both genes were reported important in IMI resistance. In conclusion, P450 genes in two planthopper species showed similar regulation patterns in responding to IMI, and the two neonicotinoid insecticides had similar effects on P450 gene expression, although the regulation was often dose-dependent.

New insight into foregut functions of xenobiotic detoxification in the cockroach Periplaneta americana.

The physiological functions of insect foregut, especially in xenobiotic detoxification, are scarcely reported because of unimportance in appearance and insufficient molecular information. The cockroach Periplaneta americana, an entomological model organism, provides perfect material to study physiological functions of foregut tissue due to its architectural feature. Through Illumina sequencing of foregut tissue from P. americana individuals (control) or insects treated with cycloxaprid, as a novel neonicotinoid insecticide, 54 193 166 clean reads were obtained and further assembled into 53 853 unigenes with an average length of 366 bp. Furthermore, the number of unigenes involved in xenobiotic detoxification was analyzed, mainly including 70 cytochrome P450s, 12 glutathione S-transferases (GSTs), seven carboxylesterases (CarEs) and seven adenosine triphosphate-binding cassette (ABC) transporters. Compared to control, the expression of 22 xenobiotic detoxification unigenes was up-regulated after cycloxaprid application, mainly containing 18 P450s, one GST, two CarEs and one ABC adenosine triphosphate transporter, indicating that the oxidation-reduction was the major reactive process to cycloxaprid application. Through quantitative real-time polymerase chain reaction analysis, the expression of selected unigenes (six P450s, one GST and one CarE) was up-regulated at least two-fold following cycloxaprid treatment, and was generally in agreement with transcriptome data. Compared to the previous midgut transcriptome of P. americana, it looks like the expressive abundance of the xenobiotic detoxification unigenes might be important factors to the detoxifying functional differences between foregut and midgut. In conclusion, insect foregut would also play important roles in the physiological processes related to xenobiotic detoxification.

The insecticidal activity and action mode of an imidacloprid analogue, 1-(3-pyridylmethyl)-2-nitroimino-imidazolidine.

Neonicotinoids, such as imidacloprid, are key insecticides extensively used for control of Nilaparvata lugens. However, imidacloprid resistance has been reported in many Asian countries in recent years. To understand the roles of the chlorine atom of pyridyl group on insecticidal activity and resistance, the atom was removed to generate an imidacloprid analogue DC-Imi (DesChlorine Imidacloprid). DC-Imi showed significantly higher toxicity than imidacloprid in the susceptible strain of N. lugens, but had medium level cross-resistance in an imidacloprid-resistant strain. In Xenopus oocyte expressed nicotinic acetylcholine receptors (nAChRs) Nlα1/rß2, the inward currents evoked by DC-Imi were detected and could be blocked by typical nAChRs antagonist dihydro-ß-erythroidine (DHßE), which demonstrated that DC-Imi acted as an agonist on insect nAChRs. The efficacy of DC-Imi on Nlα1/rß2 was 1.8-fold higher than that of imidacloprid. In addition, the influence of an imidacloprid resistance associated mutation (Y151S) on agonist potencies was evaluated. Compared with the wild-type receptor, the mutation reduced maximal inward current of DC-Imi to 55.6% and increased half maximal effective concentration (EC50 ) to 3.53-fold. Compared with imidacloprid (increasing EC50 to 2.38-fold of wild-type receptor), Y151S mutation decreased DC-Imi potency more significantly. The results indicated that the selective and possibly high toxicities could be achieved through the modification of 6-chloro-3-pyridyl group in imidacloprid and other neonicotinoids.