Practical Use of RNA Interference: Oral Delivery of Double-stranded RNA in Liposome Carriers for Cockroaches.

RNA interference (RNAi) has been widely applied for uncovering the biological functions of numerous genes, and has been envisaged as a pest control tool operating by disruption of essential gene expression. Although different methods, such as injection, feeding, and soaking, have been reported for successful delivery of double-stranded RNA (dsRNA), the efficiency of RNAi through oral delivery of dsRNA is highly variable among different insect groups. The German cockroach, Blattella germanica, is highly sensitive to the injection of dsRNA, as shown by many studies published previously. The present study describes a method to demonstrate that the dsRNA encapsulated with liposome carriers is sufficient to retard the degradation of dsRNA by midgut juice. Notably, the continuous feeding of dsRNA encapsulated by liposomes significantly reduces the tubulin expression in the midgut, and led to the death of cockroaches. In conclusion, the formulation and utilization of dsRNA lipoplexes, which protect dsRNA against nucleases, could be a practical use of RNAi for insect pest control in the future.

Oral delivery of dsRNA lipoplexes to German cockroach protects dsRNA from degradation and induces RNAi response.

BACKGROUND: In the past years, the concept of RNAi application for insect pest control has been proposed, considering the disruption of vital genes. However, the efficiency of RNAi is variable between different insect groups, especially by oral delivery of dsRNA. The purpose of this study is to assess the possibilities of RNAi as a tool for pest control using oral delivery of the dsRNAs encapsulated by liposome in the German cockroach Blattella germanica, which is highly sensitive to RNAi by injection of dsRNAs. RESULTS: Injecting dsRNA into the abdomen of B. germanica caused dramatic depletion of essential α-tubulin gene and mortality. In contrast, oral delivery of the naked dsRNA resulted in lower RNAi efficiency, accounting for rapid degradation of the dsRNA in the midgut of B. germanica. Notably, we have further demonstrated that continuous ingestion of dsRNA lipoplexes in which dsRNA was encapsulated with a cationic liposome carrier was sufficient to slow down the degradation of dsRNA in the midgut and to increase the mortality of the German cockroach by significantly inhibiting α-tubulin expression in the midgut. CONCLUSION: We provide empirical evidence that the formulation of dsRNA lipoplexes could be a plausible approach for insect pest control based on RNAi. © 2016 Society of Chemical Industry.

Cryptochrome 2 mediates directional magnetoreception in cockroaches.

The ability to perceive geomagnetic fields (GMFs) represents a fascinating biological phenomenon. Studies on transgenic flies have provided evidence that photosensitive Cryptochromes (Cry) are involved in the response to magnetic fields (MFs). However, none of the studies tackled the problem of whether the Cry-dependent magnetosensitivity is coupled to the sole MF presence or to the direction of MF vector. In this study, we used gene silencing and a directional MF to show that mammalian-like Cry2 is necessary for a genuine directional response to periodic rotations of the GMF vector in two insect species. Longer wavelengths of light required higher photon fluxes for a detectable behavioral response, and a sharp detection border was present in the cyan/green spectral region. Both observations are consistent with involvement of the FADox, FAD(•-) and FADH(-) redox forms of flavin. The response was lost upon covering the eyes, demonstrating that the signal is perceived in the eye region. Immunohistochemical staining detected Cry2 in the hemispherical layer of laminal glia cells underneath the retina. Together, these findings identified the eye-localized Cry2 as an indispensable component and a likely photoreceptor of the directional GMF response. Our study is thus a clear step forward in deciphering the in vivo effects of GMF and supports the interaction of underlying mechanism with the visual system.

Characterization and expression analysis of adipokinetic hormone and its receptor in eusocial aphid Pseudoregma bambucicola.

Aphids display an extraordinary phenotypic plasticity ranging from widespread reproductive and wing polyphenisms to the occurrence of sterile or subfertile soldier morphs restricted to eusocial species of the subfamilies Eriosomatinae and Hormaphidinae. Individual morphs are specialized by their behavior, anatomy, and physiology to perform different roles in aphid societies at different stages of the life cycle. The capacity of the insects to cope with environmental stressors is under the control of a group of neuropeptides of the adipokinetic hormone/red pigment-concentrating hormone family (AKH/RPCH) that bind to a specific receptor (AKHR). Here, we describe the molecular characteristics of AKH and AKHR in the eusocial aphid Pseudoregma bambucicola. The sequence of the bioactive AKH decapeptide and the intron position in P. bambucicola AKH preprohormone were found to be identical to those in a phylogenetically distant aphid Dreyfusia spp. (Adelgidae). We detected four transcript variants of AKHR that are translated into three protein isoforms. Further, we analyzed AKH/AKHR expression in different tissues and insects of different castes. In wingless females, a remarkable amount of AKH mRNA was only expressed in the heads. In contrast, AKHR transcript levels increased in the order gut

Termite Assemblage Pattern and Niche Partitioning in a Tropical Forest Ecosystem.

Termites are major plant decomposers in tropical forest ecosystems, but their cryptic nature poses an obstacle for studying their ecological roles in depth. In the current study, we quantified climatic and geographic information of 137 termite collection sites in the Kenting National Park, Taiwan, and described the ecological niches and assemblage patterns of 13 termite species of three families. Three major assemblage patterns are reported. First, the three termite families were found in most landcovering types with similar number of species, which indicated that each family played a unique role in the ecosystem. Second, average numbers of termite species were not different among collection sites, but the total number of termite species found in each landcovering type was different, which indicated that termite niche capacity in each small area was the same but some landcovering types were composed of diverse microhabitats to host more termite species. Third, termite species of every family showed distinct moisture preferences in their habitat choices. In addition to the three assemblage patterns, we found that niche size of the advanced termite family, Termitidae, was larger than that of the primitive termite families, Rhinotermitidae or Kalotermitidae. The broader choices of cellulosic materials as food sources may allow Termitidae to adapt to more diverse environments than exclusive wood feeders. Termite niche quantification could further be used to study termite pest adaption in urban areas, interspecific competition between native and invasive species, and plant decomposition processes.

Expression of stress-related genes in the parthenogenetic forms of the pea aphid, Acyrthosiphon pisum.

Aphids are an economically important group of insects that have an intricate life cycle with seasonal polyphenism. This study aimed to explore the physiological background of aphid migration from unfavorable nutritional conditions to a new, intact host plant. Specifically, the relative expression of stress/metabolism-related genes and changes in metabolic reserves were determined for the winged and wingless forms of female pea aphids, Acyrthosiphon pisum, under two different nutritional conditions. The expression level was determined for the following sets of genes: the adipokinetic hormone (AKH) and its receptor, enzymes involved in carbohydrate and lipid metabolism, detoxifying enzymes, and genes encoding exoskeleton/cuticular proteins and cytoskeleton proteins. In both forms, the transcription of the adipokinetic hormone was upregulated during nutritional stress, whereas its receptor mRNA levels remained unchanged. Similarly, the expression of genes engaged in glycogen and triglyceride degradation was elevated. Glycogen reserves and phospholipids appeared to be used during stress. In comparison, nutrient rich reproductively active females of both forms appeared to use triglycerides. Moreover, we revealed changes in the mRNA level of the detoxifying genes delta-class glutathione S-transferase (GST-δ) and cytochrome P450 monooxygenase (CYP450), as well as the CP gene (which encodes exoskeleton/cuticular proteins) and the cofilin gene (the products of which influence cytoskeleton organization). These results indicate the possible correlation between nutritional stress, energy content, AKH, and the stress-related enzymes of different metabolic pathways in winged and wingless forms of A. pisum.

Circadian regulation of permethrin susceptibility by glutathione S-transferase (BgGSTD1) in the German cockroach (Blattella germanica).

The daily susceptibility rhythm to permethrin and the expression level of the delta class glutathione S-transferase (BgGSTD1) gene were investigated in Blattella germanica. Male cockroaches were exposed to the same concentration of permethrin at different times in a light-dark cycle, and results showed that the highest resistance occurred at night. Furthermore, the circadian rhythmicity of permethrin susceptibility was demonstrated by the highest resistance at subjective night under constant darkness. The mRNA level of the BgGSTD1 gene in the fat body of B. germanica peaked early in the day or subjective day under light-dark or constant dark conditions, whereas enzyme activity of cytosolic GSTs did not reflect the rhythmic pattern as well as BgGSTD1 expression. RNA interference (RNAi) was employed to study the function of BgGSTD1 in the circadian rhythm of permethrin susceptibility in B. germanica. Both BgGSTD1 mRNA level and cytosolic GSTs activity were significantly decreased by dsGSTD1 injection. In addition, survival of B. germanica with silenced BgGSTD1 was significantly decreased at night but not in the day when the cockroaches were exposed to permethrin. Total cytosolic GSTs activity demonstrated that is not the only gene involved in the circadian regulation of the permethrin resistance, although it is one of the major regulators of permethrin resistance.

Super-induction of Dicer-2 expression by alien double-stranded RNAs: an evolutionary ancient response to viral infection?

Dicer-2 is a ribonuclease involved in the insect RNAi pathway. On attempting to knockdown Dicer-2 expression in the insect Blattella germanica by RNAi, we found that treatment with Dicer-2 dsRNA upregulated the targeted mRNA. This unexpected result was also observed after treating with a nucleopolyhedrovirus dsRNA. Experiments with this alien dsRNA showed an all-or-none response with a threshold for inducing Dicer-2 upregulation between 0.4 and 0.04 µg in terms of dsRNA concentration and between 50 and 20 bp in terms of dsRNA length. The response seems specific of dsRNA given that equivalent experiments carried out with dsDNA did not affect Dicer-2 expression. In insects, Dicer-2 is postulated to be a sensor of viral infections and a key antiviral defense element. The upregulation of Dicer-2 expression after dsRNA administration fits well with this sensor role, and the occurrence of this mechanism in B. germanica, a phylogenetically basal insect, suggests that sensing alien RNAs might be an ancestral function of Dicer-2 proteins.

RNA interference unveils functions of the hypertrehalosemic hormone on cyclic fluctuation of hemolymph trehalose and oviposition in the virgin female Blattella germanica.

Hypertrehalosemic hormone (HTH) is a neuropeptide within the adipokinetic hormone (AKH) family that induces a release of trehalose from fat body into hemolymph in a number of insects. In this study, we first showed that female adult German cockroach, Blattella germanica, displayed a cyclic fluctuation of hemolymph trehalose levels correlated to the maturation of oocytes in the reproductive cycle. After cloning the HTH cDNA from the German cockroach (Blage-HTH), expression studies indicated that Blage-HTH mRNA showed the cyclic changes during the first reproductive cycle, where peak values occurred in 8-day-old virgin female cockroaches, which were going to produce oothecae. The functions of Blage-HTH were studied using RNA interference (RNAi) to knockdown its expression. Adult virgin females of B. germanica injected with Blage-HTH dsRNA increased hemolymph trehalose levels in the late period of vitellogenesis more slowly than control. Furthermore, RNAi of Blage-HTH delayed oviposition time and some (10%) individuals did not produce the first ootheca until 15 days after eclosion, whereas the control group produced ootheca before 9 days in all cases.

Functional Characterization of Hypertrehalosemic Hormone Receptor in Relation to Hemolymph Trehalose and to Oxidative Stress in the Cockroach Blattella germanica.

Hypertrehalosemic hormone (HTH) is a peptide hormone that belongs to the adipokinetic hormone/red pigment concentrating hormone (AKH/RPCH) family, which exerts pleiotropic actions related to catabolic reaction and stress response. AKH peptides have been demonstrated to participate in stress response including oxidative stress in several insects. In order to study the signaling pathway of HTH involved in anti-oxidative stress, we have characterized a HIH receptor cDNA in Blattella germanica (Blage-HTHR) in structural and in functional terms using RNA interference (RNAi). Blage-HTHR is expressed in various female adult tissues (brain-CC-CA, ventral nerve cord, midgut, fat body, oviduct), but maximal expression is observed in the fat body. RNAi-mediated knockdown of Blage-HTHR expression results in a significantly lower level of hemolymph trehalose, even though HTH is exogenously administered. Paraquat elicits lethal oxidative stress in B. germanica, and co-injection of paraquat and HTH reduces this detrimental effect and extends the median survival time. Interestingly, the "rescue" effect of HTH on mortality caused by paraquat is diminished in specimens with depleted expression of Blage-HTH and Blage-HTHR. Finally, lipid peroxidation in the hemolymph increases 4 h after paraquat treatment, in comparison with control specimens or with HTH-treated specimens. However, lipid peroxidation induced by paraquat was not "rescued" by HTH in Blage-HTH and Blage-HTHR knockdown specimens. Our results demonstrate that HTH acts as a stress hormone mediating anti-oxidative protection in B. germanica, and that its receptor, Blage-HTHR, is essential for this action.

Proteome mining for novel IgE-binding proteins from the German cockroach (Blattella germanica) and allergen profiling of patients.

Although cockroaches are known to produce allergens that can cause IgE-mediated hypersensitivity reactions, including perennial rhinitis and asthma, the various cockroach allergens have not yet been fully studied. Many proteins from the German cockroach show high IgE reactivity, but have never been comprehensively characterized. To identify these potential allergens, proteins were separated by 2-DE and IgE-binding proteins were analyzed by nanoLC-MS/MS or N-terminal sequencing analysis. Using a combination of proteomic techniques and bioinformatic allergen database analysis, we identified a total of ten new B. germanica IgE-binding proteins. Of these, aldolase, arginine kinase, enolase, Hsp70, triosephosphate isomerase, and vitellogenin have been reported as allergens in species other than B. germanica. Analysis of the Food Allergy Research and Resource Program allergen database indicated that arginine kinase, enolase, and triosephosphate isomerase showed significant potential cross-reactivity with other related allergens. This study revealed that vitellogenin is an important novel B. germanica allergen. Personalized profiling and reactivity of IgE Abs against the panel of IgE-binding proteins varied between cockroach-allergic individuals. These findings make it possible to monitor the individual IgE reactivity profile of each patient and facilitate personalized immunotherapies for German cockroach allergy disorders.

Circadian control of permethrin-resistance in the mosquito Aedes aegypti.

Daily fluctuation of permethrin-resistance was found in adult mosquito Aedes aegypti, the major vector of dengue viruses in Taiwan. We hypothesized there is a relationship between resistance and the circadian clock. To test our hypothesis we correlated changes in the knock-down time (KT(50)) response to permethrin with the expression of the pyrethroid-resistant gene CYP9M9 and the clock gene period (per) during a 12:12h photoperiodic cycle. Rhythmic expression of per peaked at early scotophase of the light-dark cycle and at early subjective night in constant darkness. The values of KT(50) and the expression of CYP9M9 also exhibited circadian rhythms in both susceptible and permethrin-resistant mosquito strains, from which we inferred a link to the circadian clock. The KT(50) was significantly longer in the light than in the dark phase, and the level of CYP9M9 mRNA was maximal in early scotophase, dropped to a minimum in the midnight and then slowly increased through the photophase. Existence of a clock control over mosquito sensitivity to permethrin was further indicated by reduced expression of CYP9M9 and reduced mosquito resistance to permethrin after temporal silencing of the per gene. These data provide the first evidence on the circadian control of insect resistance to permethrin.

Bioenergetic modeling reveals that Chinese green tree vipers select postprandial temperatures in laboratory thermal gradients that maximize net energy intake.

With bioenergetic modeling, we tested the hypothesis that reptiles maximize net energy gain by postprandial thermal selection. Previous studies have shown that Chinese green tree vipers (Trimeresurus s. stejnegeri) have postprandial thermophily (mean preferred temperature T(p) for males =27.8 degrees C) in a linear thigmothermal gradient when seclusion sites and water existed. With some published empirical models of digestion associated factors for this snake, we calculated the average rate (E(net)) and efficiency (K(net)) of net energy gain from possible combinations of meal size, activity level, and feeding frequency at each temperature. The simulations consistently revealed that E(net) maximizes at the T(p) of these snakes. Although the K(net) peaks at a lower temperature than E(net), the value of K(net) remains high (>=0.85 in ratio to maximum) at the peak temperature of E(net). This suggested that the demands of both E(net) and K(net) can be attained by postprandial thermal selection in this snake. In conclusion, the data support our prediction that postprandial thermal selection may maximize net energy gain.

Multiple factors conferring high radioresistance in insect Sf9 cells.

Sf9, a lepidopteran cell line isolated from the fall armyworm, Spodoptera frugiperda, was shown to be significantly more resistant to growth inhibition and apoptosis induction effects of x-ray irradiation than several human cell lines of different origins. The single-cell electrophoresis technique revealed that Sf9 cells showed lower x-ray irradiation-induced DNA damage as well as better efficiency at repairing these damages. In addition, Sf9 cells were lower in both background and x-ray irradiation-induced intracellular oxidative stress, in which the higher intracellular level of reduced glutathione seemed to play a major role. The significance of oxidative stress in determining the radioresistance of Sf9 cells was confirmed by their being more resistant to hydrogen peroxide while equally susceptible to other non-reactive oxygen species of N-nitroso alkylating agents when compared with a human cell line. Although the Sf9 and human cell lines were equally susceptible to the lethal effects of N-nitroso alkylating agents, the components of DNA damage-induced and the repair enzymes involved significantly differ. This phenomenon is also discussed in this report.

Development of the circadian clock in the German cockroach, Blattella germanica.

The cell distribution and immunoreactivity (ir) against period (PER), pigment dispersing factor (PDF) and corazonin (CRZ), were compared between adults and nymphs in the central nervous system of the German cockroach. Although PER-ir cells in the optic lobes (OL) were expressed in the nymphs from the first instar, the links between major clock cells became more elaborated after second/third instar. A circadian rhythm of locomotion was initiated at the fourth/fifth instar. The results suggest that the clock was running from hatching, but the control network needed more time to develop. In addition, the putative downstream regulators, PDF-ir and CRZ-ir, are co-localized in various regions of the brain, indicating potential output routes of the circadian clock. CRZ-ir cells with typical morphology of neurosecretory cells in the dorsolateral protocerebrum send out three neural fibers to reach the ipsilateral corpora cardiaca (CC), the antennal lobe and two hemispheres of the protocerebrum. Based on co-localization with some PER-ir/PDF-ir cells, the CRZ-ir cells have the potential to serve as a bridge between circadian neural signals and endocrine regulation. Based on PDF's role in the regulation of locomotion, our results support the finding that the locomotor circadian rhythm is possibly controlled by a hormonal route.

Pigment dispersing factor: an output regulator of the circadian clock in the German cockroach.

Pigment-dispersing factor (PDF) is a neuropeptide that is synthesized specifically and constantly in the circadian clock cells of many insects. The functions of PDF have not been fully determined, but it might serve as the output and coupling signal of circadian locomotor rhythms. In this experiment, we explore the functions of PDF in the German cockroach with RNA interference technique. Since the 2nd day after pdf double-strand RNA (dsRNA) injection, the amount of pdf mRNA decreased significantly, and this knockdown effect could persist at least 56 days. With immunostaining technique, the clock cells of pdf dsRNA-injected cockroaches could not be stained by anti-PDF antibody. In the behavioral study, pdf dsRNA injection caused rhythmic males to become arrhythmic in light-dark cycles or in constant darkness. In addition, due to the nocturnal nature of the German cockroaches, the locomotor activity increased after lights-off or entering subjective night. However, this activity peak gradually disappeared after pdf dsRNA injection. Based on these 2 lines of evidences, PDF serves as an output regulator of locomotor circadian rhythm in the German cockroach.

Corazonin- and PDF-immunoreactivities in the cephalic ganglia of termites.

Antisera against the pigment-dispersing factor (PDF) and corazonin (Crz) reacted with distinct sets of neurons in the cephalic ganglia of termites. The locations of immunoreactive cells were similar but their numbers differed among the eight species examined: PDF-ir occurred in 0-6 cells in each optic lobe and 1-2 pairs of cells in the subosophageal ganglion (SOG), and Crz-ir in 0-2 pairs of cells in the pars intecerebralis, 3-14 cells in each lateral protocerebrum, and 0-6 pairs of cells in the SOG. Staining patterns were identical in the pseudergates, soldiers, and substitutive reproductives of Prorhinotermes simplex. Workers and soldiers were compared in the remaining 7 species. The only caste divergence was detected in Coptotermes formosanus, in which the soldiers differed from the workers by lack of 4 Crz-ir perikarya in the pars intercerebralis and occasionally also by the absence of 2 Crz-ir perikarya in the SOG. Diurnal changes in PDF-ir and Crz-ir were examined in P. simplex kept under long day (18:6h light:darkness) or short day (10:14 h) photoperiods. No circadian fluctuations in the distribution or the intensity of immunostaining were found in the pseudergates and soldiers that were sacrificed in 4h intervals or in the male and female substitutive reproductives examined in 6h intervals.

Mapping the cellular network of the circadian clock in two cockroach species.

The German cockroach, Blattella germanica, and the double-striped cockroach, B. bisignata, are sibling species with a similar period sequence but a distinctive circadian rhythm in locomotion. The cell distribution of immunoreactivity (ir) against three clock-related proteins, Period (PER), Pigment Dispersing Factor (PDF), and Corazonin (CRZ), was compared between the species. The PER-ir cells tend to form clusters and are sprayed out in the central nervous system. Three major PER-ir cells are located in the optic lobes, which are the sites of the major circadian clock. They are interconnected with PER-ir axon bundles. Interestingly, the potential output signal of the circadian clock, PDF, is co-localized with PER in all three groups of cells. However, only two CRZ-ir cells and their axons are found in the optic lobes and they are not co-localized with PER-ir or PDF-ir cells and axons. Since only one circadian rhythm is expressed in locomotion, the time signals from both major clocks in optic lobes are coupled by connection with PDF-ir axons. A group of 3-4 PER-ir cells in the protocerebrum display typical characteristics of neurosecretary cells. In addition, there are numerous, small PER-ir and PDF-ir co-localized cells in the pars intercerebralis (PI), which have direct connections with the neurohemoorgan, corpora cardiaca, through PER-ir and PDF-ir axons. Based on these findings, the cellular connection shows a circadian control through the endocrine route. For the rest of central nervous system, only a few PER-ir and PDF-ir cells or axons are detected. This finding implies the circadian clock for locomotion is not located in subesophageal ganglion, thoracic or abdominal ganglia, but may use other neural messengers to pass on circadian signals. Since the overall distribution pattern of the clock cells are the same for B. germanica and B. bisignata, the possible explanation for the different expressions of locomotion between the species depends on genes downstream of per, pdf, and crz.

Distribution of corazonin and pigment-dispersing factor in the cephalic ganglia of termites.

Distribution of neurones detectable with antisera to the corazonin (Crz) and the pigment-dispersing factor (PDF) was mapped in the workers or pseudergates of 10 species representing six out of seven termite families. All species contained two triads of Crz-immunoreactive (Crz-ir) neurones in the protocerebrum. Their fibres were linked to the opposite hemisphere, formed a network in the fronto-lateral protocerebrum, and projected to the corpora cardiaca (CC); in most species the fibres also supplied the deuto- and tritocerebrum and the frontal ganglion. Some species possessed additional Crz-ir perikarya in the protocerebrum and the suboesophageal ganglion (SOG). The PDF-ir somata were primarily located in the optic lobe (OL) and SOG. OL harboured a group (3 groups in Coptotermes) of 2-6 PDF-ir cells with processes extending to the medulla, connecting to the contralateral OL, forming 1-2 networks in the protocerebrum, and in most species running also to CC. Such a PDF-ir system associated with the OL was missing in Reticulitermes. Except for Mastotermes, the termites contained 1-2 PDF-ir cell pairs in the SOG and two species had additional perikarya in the protocerebrum. The results are consistent with the view of a monophyletic termite origin and demonstrate how the Crz-ir and PDF-ir systems diversified in the course of termite phylogeny.