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.     

Amblyomma americanum tick saliva insulin‐like growth factor binding protein‐related protein 1 binds insulin but not insulin‐like growth factors

Silencing Amblyomma americanum insulin‐like growth factor binding protein‐related protein 1 (AamIGFBP‐rP1) mRNA prevented ticks from feeding to repletion. In this study, we used recombinant (r)AamIGFBP‐rP1 in a series of assays to obtain further insight into the role(s) of this protein in tick feeding regulation. Our results suggest that AamIGFBP‐1 is an antigenic protein that is apparently exclusively expressed in salivary glands. We found that both males and females secrete AamIGFBP‐rP1 into the host during feeding and confirmed that female ticks secrete this protein from within 24–48 h after attachment. Our data suggest that native AamIGFBP‐rP1 is a functional insulin binding protein in that both yeast‐ and insect cell‐expressed rAamIGFBP‐rP1 bound insulin, but not insulin‐like growth factors. When subjected to anti‐blood clotting and platelet aggregation assays, rAamIGFBP‐rP1 did not have any effect. Unlike human IGFBP‐rP1, which is controlled by trypsinization, rAamIGFBP‐rP1 is resistant to digestion, suggesting that the tick protein may not be under mammalian host control at the tick feeding site. The majority of tick‐borne pathogens are transmitted 48 h after the tick has attached. Thus, the demonstrated antigenicity and secretion into the host within 24–48 h of the tick starting to feed makes AamIGFBP‐rP1 an attractive target for antitick vaccine development.     

Interactions of two odorant‐binding proteins influence insect chemoreception

It is well known that the odorant‐binding proteins (OBPs) play crucial roles in insect olfactory detection. To explore if interactions of OBPs affect olfactory coding in the rice leaffolder Cnaphalocrocis medinalis ligand‐binding experiments, molecular docking, RNA interference and electrophysiological recording were performed. The binding activity of two C. medinalis OBPs (CmedOBPs) to rice plant volatiles showed wide flexibility depending on the structure of ligands and interactions of CmedOBPs involved. The binding sites of CmedOBP2 and CmedOBP3 to rice plant volatiles were well predicted by three‐dimensional structure modelling and molecular docking experiments. In addition, the interactions of these two CmedOBPs in the perception of rice volatiles were demonstrated by RNA interference experiments. When a single double‐stranded RNA (dsRNA)‐CmedOBP2 was injected, the expression of CmedOBP2 was significantly reduced and the expression of CmedOBP3 was significantly increased, and vice versa. When both dsRNA‐CmedOBP2 and 3 were injected together, greater reduction of electroantennogram responses to rice plant volatiles was induced than that seen with individual injection of either dsRNA‐CmedOBP2 or dsRNA‐CmedOBP3. These results clearly indicate that the interactions of CmedOBP2 and CmedOBP3 have significant effects on C. medinalis during the detection of host plant volatiles.     

Suppressive effects of neutrophil by Salp16‐like salivary gland proteins from Ixodes persulcatus Schulze tick

Salp16, a 16‐kDa tick salivary gland protein, is known to be the molecule involved in the transmission of Anaplasma phagocytophilum, an obligate intracellular pathogen causing zoonotic anaplasmosis, from its mammalian hosts to Ixodes scapularis. Recently, the presence of A. phagocytophilum was documented in Japan and Ixodes persulcatus was identified as one of its vectors. The purpose of this study was to identify Salp16 genes in I. persulcatus and characterize their function. Two cDNA clones encoding the Salp16‐like sequences were obtained from the salivary glands of fed female I. persulcatus ticks and designated Salp16 Iper1 and Iper2. Gene expression analyses showed that the Salp16 Iper genes were expressed specifically in the salivary glands and were up‐regulated by blood feeding. These proteins attenuated the oxidative burst of activated bovine neutrophils and inhibited their migration induced by the chemoattractant interleukin‐8 (IL‐8). These results demonstrate that Salp16 Iper proteins contribute to the establishment of blood feeding as an immunosuppressant of neutrophil, an essential factor in innate host immunity. Further examination of the role of Salp16 Iper in the transmission of pathogens, including A. phagocytophilum, will increase our understanding of the tick–host–pathogen interface.     

RNA interference in ticks: a study using histamine binding protein dsRNA in the female tick Amblyomma americanum

RNA interference (RNAi), a gene silencing process, has been recently exploited to determine gene function by degrading specific mRNAs in several eukaryotic organisms. We constructed a double stranded RNA (dsRNA) from a previously cloned putative Amblyomma americanum histamine binding protein (HBP) to test the significance of using this methodology in the assessment of the function and importance of gene products in ectoparasitic ticks. The female salivary glands incubated in vitro with HBP dsRNA had a significantly lower histamine binding ability. In addition, the injection of HBP dsRNA into the unfed females led both to a reduced histamine binding ability in the isolated salivary glands and to an aberrant tick feeding pattern or host response. Molecular data demonstrated less expression of the HBP mRNA in the RNAi group. Taken together, these results suggest that RNAi might be an important tool for assessing the significance of tick salivary gland secreted proteins modulating responses at the tick-host interface.     

The involvement of clathrin‐mediated endocytosis and two Sid‐1‐like transmembrane proteins in double‐stranded RNA uptake in the Colorado potato beetle midgut

RNA interference (RNAi) is a powerful tool in entomology and shows promise as a crop protection strategy, but variability in its efficiency across different insect species limits its applicability. For oral uptake of the double‐stranded RNA (dsRNA), the RNAi trigger, two different mechanisms are known: systemic RNA interference deficient‐1 (Sid‐1) transmembrane channel‐mediated uptake and clathrin‐mediated endocytosis. So far, a wide range of experiments has been conducted, confirming the involvement of one of the pathways in dsRNA uptake, but never both pathways in the same species. We investigated the role of both pathways in dsRNA uptake in the Colorado potato beetle, Leptinotarsa decemlineata, known to have an efficient RNAi response. Through RNAi‐of‐RNAi experiments, we demonstrated the contribution of two different sid‐1‐like (sil) genes, silA and silC, and clathrin heavy chain and the 16kDa subunit of the vacuolar H⁺ ATPase (vha16), elements of the endocytic pathway, to the RNAi response. Furthermore, the sid‐1‐like genes were examined through phylogenetic and hydrophobicity analysis. This article reports for the first time on the involvement of two pathways in dsRNA uptake in an insect species and stresses the importance of evaluating both pathways through a well‐devised reporter system in any future experiments on cellular dsRNA uptake.     

Differential responses of migratory locusts to systemic RNA interference via double‐stranded RNA injection and feeding

The migratory locust, Locusta migratoria, is one of the most destructive agricultural pests and has been widely used as a model system for insect physiology, neurobiology and behavioural research. In the present study, we investigated the effects of RNA interference (RNAi) using two delivery methods for double‐stranded RNA (dsRNA) molecules, namely, injection and feeding, to develop a potential new pest control strategy. Our results showed that locusts have a sensitive and systemic response to the injection of dsRNAs in a dose‐dependent manner, but do not respond to the feeding of dsRNAs. Further experiments suggested that the ineffectiveness of dsRNA feeding was attributable to the rapid degradation of dsRNA, which was probably induced by nuclease enzymes in the locust midgut. Moreover, we identified almost all the homologous genes involved in the endocytosis‐mediated dsRNA uptake from the locust genome, which provided possible clues regarding the dsRNA uptake mechanisms from the intestine to the midgut epithelium. These findings reveal the differential response models of fourth instar locust nymphs to dsRNA delivery methods, contribute to the current understanding of insect RNAi mechanisms and provide important information for the further application of RNAi as a genetic tool and pest control strategy.     

Nuclear factor erythroid‐derived 2–related factor 2 activates glutathione S‐transferase expression in the midgut of Spodoptera litura (Lepidoptera: Noctuidae) in response to phytochemicals and insecticides

Detoxication enzymes play an important role in insect resistance to xenobiotics such as insecticides and phytochemicals. We studied the pathway for activating the expression of glutathione S‐transferases (GSTs) in response to selected xenobiotics. An assay of the promoter activity of GST epsilon 1 (Slgste1) of Spodoptera litura led to the discovery of a cis‐regulating element. An antioxidant response element was activated in response to indole‐3‐carbinol (I3C) and chlorpyrifos (CPF) and was able to bind with the xenobiotic sensor protein nuclear factor erythroid‐derived 2–related factor 2 (SlNrf2). SlNrf2 and Slgste1 were responsive to reactive oxygen species induced by I3C and CPF in a S. litura cell line, as well as in S. litura midguts. SlNrf2 RNA interference (RNAi) reduced the message RNA levels of Slgste1 and the peroxidase activity of GSTs in response to I3C, xanthotoxin, CPF and deltamethrin. SlNrf2 RNAi and inhibitor treatment of GST activity decreased the viability of I3C‐treated cells. These results indicate that SlNrf2 activates the expression of GSTs in response to oxidative stresses caused by exposure to xenobiotics.     

Live imaging reveals a biphasic mode of dissemination of Borrelia burgdorferi within ticks

Lyme disease is caused by transmission of the spirochete Borrelia burgdorferi from ticks to humans. Although much is known about B. burgdorferi replication, the routes and mechanisms by which it disseminates within the tick remain unclear. To better understand this process, we imaged live, infectious B. burgdorferi expressing a stably integrated, constitutively expressed GFP reporter. Using isolated tick midguts and salivary glands, we observed B. burgdorferi progress through the feeding tick via what we believe to be a novel, biphasic mode of dissemination. In the first phase, replicating spirochetes, positioned at varying depths throughout the midgut at the onset of feeding, formed networks of nonmotile organisms that advanced toward the basolateral surface of the epithelium while adhering to differentiating, hypertrophying, and detaching epithelial cells. In the second phase of dissemination, the nonmotile spirochetes transitioned into motile organisms that penetrated the basement membrane and entered the hemocoel, then migrated to and entered the salivary glands. We designated the first phase of dissemination “adherence-mediated migration” and provided evidence that it involves the inhibition of spirochete motility by one or more diffusible factors elaborated by the feeding tick midgut. Our studies, which we believe are the first to relate the transmission dynamics of spirochetes to the complex morphological and developmental changes that the midgut and salivary glands undergo during engorgement, challenge the conventional viewpoint that dissemination of Lyme disease–causing spirochetes within ticks is exclusively motility driven.     

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.     

RNA interference and functional characterization of a tergal gland alpha amylase in the German cockroach,Blattella germanica L.

German cockroach males possess tergal glands that secrete a combination of oligosaccharides, lipids and proteins. Four major proteins occur in the secretion, with one being the 63 kDa alpha‐amylase Blattella germanica Tergal Gland protein‐1 (BGTG‐1). Denaturing and starch gel electrophoresis coupled with peptide sequencing verified amylase activity for the BGTG‐1 protein. BGTG‐1 gene expression profiles were determined by using quantitative real‐time PCR to compare messenger RNA abundance among isolated tissues of males, females and gravid females. Differences in BGTG‐1 gene expression occurred among male tissues, with tergal gland tissue showing the highest expression. Tissues of nongravid and gravid females had significantly lower expression in comparison with male tergal glands (gravid females lowest). RNA interference (RNAi) was used to silence BGTG‐1 gene expression by injecting BGTG‐1 homologous double‐stranded RNA (dsRNA) into male cockroaches. Groups injected with BGTG‐1 dsRNA showed ～90% lower BGTG‐1 gene and protein expression compared to controls, which correlated with lower amylase activity in colorimetric assays. However, behavioural assays comparing precopulatory behaviour and mating success between RNAi and control males did not reveal differences. These results connect amylase gene expression and activity in tergal gland tissue but suggest other factors, such as other tergal gland components, may contribute more strongly to mating success.