Feeding‐based RNA interference of a trehalose phosphate synthase gene in the brown planthopper,Nilaparvata lugens

The brown planthopper, Nilaparvata lugens, is the most devastating rice insect pest to have given rise to an outbreak in recent years. RNA interference (RNAi) is a technological breakthrough that has been developed as a powerful tool for studying gene function and for the highly targeted control of insect pests. Here, we examined the effects of using a feeding‐based RNAi technique to target the gene trehalose phosphate synthase (TPS) in N. lugens. The full‐length cDNA of N. lugens TPS (NlTPS) is 3235 bp and has an open reading frame of 2424 bp, encoding a protein of 807 amino acids. NlTPS was expressed in the fat body, midgut and ovary. Quantitative real‐time PCR (qRT‐PCR) analysis revealed that NlTPS mRNA is expressed continuously with little change during the life of the insect. Efficient silencing of the TPS gene through double‐stranded RNA (dsRNA) feeding led to rapid and significant reduction levels of TPS mRNA and enzymatic activity. Additionally, the development of N. lugens larvae that had been fed with the dsRNA was disturbed, resulting in lethality, and the cumulative survival rates dropped to 75.56, 64.44, 55.56 and 40.00% after continuous ingestion of 0.5 µg/µl dsRNA for 2, 4, 7 and 10 days, respectively. These values were significantly lower than those of the insects in the control group, suggesting that NlTPS dsRNA may be useful as a means of insect pest control.     

Pharmacological characterization of cis-nitromethylene neonicotinoids in relation to imidacloprid binding sites in the brown planthopper, Nilaparvata lugens

Neonicotinoid insecticides, such as imidacloprid, are selective agonists of the insect nicotinic acetylcholine receptors (nAChRs) and extensively used in areas of crop protection and animal health to control a variety of insect pest species. Here we describe that two cis‐nitromethylene neonicotinoids (IPPA152002 and IPPA152004), recently synthesized in our laboratory, discriminated between the high and low affinity imidacloprid binding sites in the brown planthopper, Nilaparvata lugens, a major insect pest of rice crops in many parts of Asia. [³H]imidacloprid has two binding sites with different affinities (Kd value of 0.0035 ± 0.0006 nM for the high‐affinity site and 1.47 ± 0.22 nM for the low‐affinity site). Although the cis‐nitromethylene neonicotinoids showed low displacement ability (Ki values of 0.15 ± 0.03 µM and 0.42 ± 0.07 µM for IPPA152002 and IPPA152004, respectively) against [³H]imidacloprid binding, low concentrations (0.01 µM) of IPPA152002 completely inhibited [³H]imidacloprid binding at its high‐affinity site. In Xenopus oocytes co‐injected with cRNA encoding Nlα1 and rat β2 subunits, obvious inward currents were detected in response to applications of IPPA152002 and IPPA152004, although the agonist potency is reduced to that of imidacloprid. The previously identified Y151S mutation in Nlα1 showed significant effects on the agonist potency of IPPA152002 and IPPA152004, such as a 75.8% and 70.6% reduction in Imax, and a 2.4‐ and 2.1‐fold increase in EC₅₀. This data clearly shows that the two newly described cis‐nitromethylene neonicotinoids act on insect nAChRs and like imidacloprid, discriminated between high and low affinity binding sites in N. lugens native nAChRs. These compounds may be useful tools to further elucidate the pharmacology and nature of neonicotinoid binding sites.     

dsRNA uptake and persistence account for tissue‐dependent susceptibility to RNA interference in the migratory locust,Locusta migratoria

RNA interference (RNAi) by introducing double‐stranded RNA (dsRNA) is a powerful approach to the analysis of gene function in insects; however, RNAi responses vary dramatically in different insect species and tissues, and the underlying mechanisms remain poorly understood. The migratory locust, a destructive insect pest and a hemimetabolic insect with panoistic ovaries, is considered to be a highly susceptible species to RNAi via dsRNA injection, but its ovary appears to be completely insensitive. In the present study, we showed that dsRNA persisted only briefly in locust haemolymph. The ovariole sheath was permeable to dsRNA, but injected dsRNA was not present in the follicle cells and oocytes. The lack of dsRNA uptake into the follicle cells and oocytes is likely to be the primary factor that contributes to the ineffective RNAi response in locust ovaries. These observations provide insights into tissue‐dependent variability of RNAi and help in achieving successful gene silencing in insensitive tissues.     

Transgene‐mediated suppression of the RNA interference pathway in Aedes aegypti interferes with gene silencing and enhances Sindbis virus and dengue virus type 2 replication

RNA interference (RNAi) is the major innate antiviral pathway in Aedes aegypti that responds to replicating arboviruses such as dengue virus (DENV) and Sindbis virus (SINV). On the one hand, the mosquito's RNAi machinery is capable of completely eliminating DENV2 from Ae. aegypti. On the other, transient silencing of key genes of the RNAi pathway increases replication of SINV and DENV2, allowing the viruses to temporally overcome dose‐dependent midgut infection and midgut escape barriers (MEB) more efficiently. Here we expressed Flock house virus B2 (FHV‐B2) from the poly‐ubiquitin (PUb) promoter in Ae. aegypti using the ΦC31 site‐directed recombination system to investigate the impact of transgene‐mediated RNAi pathway suppression on infections with SINV‐TR339eGFP and DENV2‐QR94, the latter of which has been shown to be confronted with a strong MEB in Ae. aegypti. FHV‐B2 was constitutively expressed in midguts of sugar‐ and blood‐fed mosquitoes of transgenic line PUbB2 P61. B2 over‐expression suppressed RNA silencing of carboxypeptidase A‐1 (AeCPA‐1) in midgut tissue of PUbB2 P61 mosquitoes. Following oral challenge with SINV‐TR339eGFP or DENV2‐QR94, mean titres in midguts of PUbB2 P61 females were significantly higher at 7 days post‐bloodmeal (pbm) than in those of nontransgenic control mosquitoes. At 14 days pbm, infection rates of carcasses were significantly increased in PubB2 P61 mosquitoes infected with SINV‐TR339eGFP. Following infection with DENV2‐QR94, midgut infection rates were significantly increased in the B2‐expressing mosquitoes at 14 days pbm. However, B2 expression in PUbB2 P61 did not increase the DENV2‐QR94 dissemination rate, indicating that the infection phenotype was not primarily controlled by RNAi.     

RNA interference‐mediated depletion of N‐ethylmaleimide Sensitive Fusion Protein and Synaptosomal Associated Protein of 25 kDa results in the inhibition of blood feeding of the Gulf Coast tick,Amblyomma maculatum

The signalling pathways in tick salivary glands that control ‘sialo‐secretome’ secretion at the tick?host interface remain elusive; however, this complex process is essential for successful feeding and manipulation of the host haemostatic response. Exocytosis of the sialo‐secretome in the salivary glands requires a core of soluble N‐ethylmaleimide‐sensitive fusion (NSF) attachment proteins (SNAPs) and receptor proteins (SNAREs). SNAREs have been identified as the key components in regulating the sialo‐secretome in the salivary gland cells. In this study, we utilized RNA interference to investigate the functional role of two Amblyomma maculatum SNARE complex proteins, AmNSF and AmSNAP‐25, in the tick salivary glands during extended blood feeding on the vertebrate host. Knock‐down of AmNSF and AmSNAP‐25 resulted in death, impaired feeding on the host, lack of engorgement and inhibited oviposition in ticks. Depletion also led to important morphological changes in the collapse of the Golgi apparatus in the salivary gland cells. Our results imply a functional significance of AmNSF and AMSNAP‐25 in prolonged tick feeding, and survival on the host. Further characterization of the factors that regulate exocytosis may lead to novel approaches to prevent tick‐borne diseases.     

Division of labour in honey bees: age‐ and task‐related changes in the expression of octopamine receptor genes

The honey bee (Apis mellifera L.) has developed into an important ethological model organism for social behaviour and behavioural plasticity. Bees perform a complex age‐dependent division of labour with the most pronounced behavioural differences occurring between in‐hive bees and foragers. Whereas nurse bees, for example, stay inside the hive and provide the larvae with food, foragers leave the hive to collect pollen and nectar for the entire colony. The biogenic amine octopamine appears to play a major role in division of labour but the molecular mechanisms involved are unknown. We here investigated the role of two characterized octopamine receptors in honey bee division of labour. AmOctαR1 codes for a Ca²⁺‐linked octopamine receptor. AmOctβR3/4 codes for a cyclic adenosine monophosphate‐coupled octopamine receptor. Messenger RNA expression of AmOctαR1 in different brain neuropils correlates with social task, whereas expression of AmOctβR3/4 changes with age rather than with social role per se. Our results for the first time link the regulatory role of octopamine in division of labour to specific receptors and brain regions. They are an important step forward in our understanding of complex behavioural organization in social groups.     

Regulatory mechanisms of C4b‐binding protein (C4BP)α and β expression in rat hepatocytes by lipopolysaccharide and interleukin‐6

Summary. Background: C4b‐binding protein (C4BP), a multimeric protein structurally composed of α chains (C4BPα) and a β chain (C4BPβ), regulates the anticoagulant activity of protein S (PS). Patients with sepsis have increased levels of plasma C4BP, which appears to be induced by interleukin (IL)‐6. However, it is not fully understood how lipopolysaccharide (LPS) and IL‐6 affect the plasma C4BP antigen level and C4BPα and C4BPβ expression in hepatocytes. Objectives: To assess the effect of LPS and IL‐6 on plasma C4BP, PS–C4BP complex levels, PS activity, and C4BP expression by rat liver in vivo and on C4BP expression by isolated rat hepatocytes in vitro. Results: Plasma C4BP antigen level transiently decreased from 2 to 12 h after LPS (2 mg kg^?1) injection, and then it abruptly increased up to 24 h after LPS injection. Plasma C4BP antigen level increased until 8 h after IL‐6 (10 μg kg^?1) injection, and then gradually decreased up to 24 h after IL‐6 injection. LPS significantly decreased the protein and mRNA expression of both C4BPα and C4BPβ in rat hepatocytes, and this effect was inhibited by NFκB and MEK/ERK inhibitors. IL‐6 mediated increase in C4BPβ expression in rat hepatocytes, which leads to increased plasma PS–C4BP complex level and to decreased plasma PS activity, was inhibited by inhibition of STAT‐3. Conclusion: LPS decreases both C4BPα and C4BPβ expression via the NFκB and MEK/ERK pathways, whereas IL‐6 specifically increases C4BPβ expression via the STAT‐3 pathway, causing an increase in plasma PS–C4BP complex, and thus decreasing the anticoagulant activity of PS.