Red Flour Beetle Tribolium Castaneum Research Articles

Attachment of the blastoderm to the vitelline envelope affects gastrulation of insects.

During gastrulation, physical forces reshape the simple embryonic tissue to form the complex body plans of multicellular organisms1. These forces often cause large-scale asymmetric movements of the embryonic tissue2,3. In many embryos, the gastrulating tissue is surrounded by a rigid protective shell4. Although it is well-recognized that gastrulation movements depend on forces that are generated by tissue-intrinsic contractility5,6, it is not known whether interactions between the tissue and the protective shell provide additional forces that affect gastrulation. Here we show that a particular part of the blastoderm tissue of the red flour beetle (Tribolium castaneum) tightly adheres in a temporally coordinated manner to the vitelline envelope that surrounds the embryo. This attachment generates an additional force that counteracts tissue-intrinsic contractile forces to create asymmetric tissue movements. This localized attachment depends on an αPS2 integrin (inflated), and the knockdown of this integrin leads to a gastrulation phenotype that is consistent with complete loss of attachment. Furthermore, analysis of another integrin (the αPS3 integrin, scab) in the fruit fly (Drosophila melanogaster) suggests that gastrulation in this organism also relies on adhesion between the blastoderm and the vitelline envelope. Our findings reveal a conserved mechanism through which the spatiotemporal pattern of tissue adhesion to the vitelline envelope provides controllable, counteracting forces that shape gastrulation movements in insects.

Digital gene expression profiling in larvae of Tribolium castaneum at different periods post UV-B exposure

UV-B radiation is an important environmental factor. Exposure to excess UV-B radiation can cause serious effects on the development, survival, and reproduction of different organisms. Plants and animals have developed many different strategies to cope with UV-B-induced damage, but the physiological response of insects to UV-B remains unclear. In the present study, the red flour beetle Tribolium castaneum (Herbst) was used to assess the stress response of UV-B. The underlying molecular mechanisms were explored using RNA sequencing. We investigated the transcriptomic profile of T. castaneum larvae at 4 and 24 h after treatment with UV-B radiation via digital gene expression analysis. The 310 and 996 differentially expressed genes were detected at 4 and 24 h, respectively. Then the biological functions and associated metabolic processes of these genes were determined by Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathway analysis. The reliability of the data was verified using qRT-PCR. The results indicated that several differentially expressed genes are involved in antioxidation, DNA repair, protein folding, carbon flux diversion, and the extracellular matrix to protect against UV-B-induced damage. This study will increase our understanding of the molecular mechanism underlying insect response to UV-B radiation.

Roles of Polycomb group proteins Enhancer of zeste (E(z)) and Polycomb (Pc) during metamorphosis and larval leg regeneration in the flour beetle Tribolium castaneum

Many organisms both undergo dramatic morphological changes during post-embryonic development and also regenerate lost structures, but the roles of epigenetic regulators in such processes are only beginning to be understood. In the present study, the functions of two histone modifiers were examined during metamorphosis and larval limb regeneration in the red flour beetle Tribolium castaneum. Polycomb (Pc), a member of Polycomb repressive complex 1 (PRC1), and Enhancer of zeste (E(z)), a member of Polycomb repressive complex 2 (PRC2), were silenced in larvae using RNA interference. In the absence of Pc, the head appendages of adults transformed into a leg-like morphology, and the legs and wings assumed a metathoracic identity, indicating that Pc acts to specify proper segmental identity. Similarly, silencing of E(z) led to homeotic transformation of legs and wings. Additional defects were also observed in limb patterning as well as eye morphogenesis, indicating that PcG proteins play critical roles in imaginal precursor cells. In addition, larval legs and antennae failed to re-differentiate when either Pc or E(z) was knocked down, indicating that histone modification is necessary for proper blastema growth and differentiation. These findings indicate that PcG proteins play extensive roles in postembryonic plasticity of imaginal precursor cells.

Transgenerational Developmental Effects of Immune Priming in the Red Flour Beetle Tribolium castaneum.

Immune priming, the increased chance to survive a secondary encounter with a pathogen, has been described for many invertebrate species, which lack the classical adaptive immune system of vertebrates. Priming can be specific even for closely related bacterial strains, last up to the entire lifespan of an individual, and in some species, it can also be transferred to the offspring and is then called transgenerational immune priming (TGIP). In the red flour beetle Tribolium castaneum, a pest of stored grains, TGIP has even been shown to be transferred paternally after injection of adult beetles with heat-killed Bacillus thuringiensis. Here we studied whether TGIP in T. castaneum is also transferred to the second filial generation, whether it can also occur after oral and injection priming of larvae and whether it has effects on offspring development. We found that paternal priming with B. thuringiensis does not only protect the first but also the second offspring generation. Also, fitness costs of the immune priming became apparent, when the first filial generation produced fewer offspring. Furthermore, we used two different routes of exposure to prime larvae, either by injecting them with heat-killed bacteria or orally feeding them B. thuringiensis spore culture supernatant. Neither of the parental larval priming methods led to any direct benefits regarding offspring resistance. However, the injections slowed down development of the injected individuals, while oral priming with both a pathogenic and a non-pathogenic strain of B. thuringiensis delayed offspring development. The long-lasting transgenerational nature of immune priming and its impact on offspring development indicate that potentially underlying epigenetic modifications might be stable over several generations. Therefore, this form of phenotypic plasticity might impact pest control and should be considered when using products of bacterial origin against insects.

Separation between maternal and paternal effects on offspring following exposure of adult red flour beetles to two stressors

1. The contribution of non‐genetic maternal effects to offspring performance is well established and the evidence for paternal effects has also been increasing recently. Studies determining the relative contributions of the two parents to offspring success are, however, still rare.2. In this study, two stressors were applied to adult red flour beetles (Tribolium castaneum) – starvation and cold stress – and a full‐factorial design was used to distinguish between maternal and paternal reproductive decisions and their effects on the offspring.3. Starvation had a stronger negative effect than cold stress on both males and females, and the likelihood of starved females producing offspring was very low. Furthermore, starved fathers led to lower offspring mass at the larval stage, probably leading to impaired starvation tolerance of the offspring.4. Cold‐stressed fathers were less likely than unstressed fathers to reproduce, whereas cold‐stressed mothers demonstrated a similar effect by producing fewer offspring.5. Applying stress probably led to energy saving that came at the expense of reproduction intensity. It is suggested that the smaller offspring mass is a negative consequence of the parental exposure to stress.6. The differences between the consequences of the two stressors applied and between the relative contribution of each parent could perhaps be explained by the distinct physiological responses of each sex to each of the stressors.

Tribolium castaneum as a whole-animal screening system for the detection and characterization of neuroprotective substances.

Parkinson's disease (PD) is a movement disorder caused by the progressive loss of dopaminergic neurons. Natural antioxidants and plant extracts with neuroprotective properties offer a promising new therapeutic approach for PD patients, but a suitable large-scale screening system is required for their discovery and preclinical analysis. Here we used the red flour beetle (Tribolium castaneum ) as a whole-animal screening system for the detection and characterization of neuroprotective substances. Paraquat was added to the diet of adult beetles to induce PD-like symptoms, which were quantified using a novel positive geotaxis behavioral assay. These paraquat-induced behavioral changes were reduced in beetles fed on diets supplemented with l-dihydroxyphenylalanine, ascorbic acid, curcumin, hempseed flour, or the Chinese herb gou-teng. T. castaneum is, therefore, a valuable model for the screening of neuroprotective substances in chemical libraries and plant extracts and could be developed as a model for the preclinical testing of therapeutic candidates for the treatment of neurodegenerative diseases, such as PD.

Response of Tribolium castaneum to dietary mannitol, with remarks on its possible nutritive effects.

Mannitol, one of the sugar alcohols, is often used as a low-calorific carbohydrate by animals. In some insects, mannitol acts as a cryoprotectant to endure coldness, but also become a poisonous agent. Adults of the red flour beetle Tribolium castaneum were shown to recognize mannitol as a factor stimulating their feeding behavior, but it remains unclear whether T. castaneum can utilize mannitol as a source of nutrition, because the enzymes needed to metabolize mannitol are unknown in this species. This study shows that T. castaneum utilizes mannitol as a nutrient in a dietary assay based on a sole carbon source added to artificial gypsum diet. The amount of mannitol excreted was less than that ingested, suggesting that it is absorbed in the insect body. The hemolymph of T. castaneum contained no mannitol but contained trehalose, a known blood sugar in insects, even after being fed mannitol. This study also revealed that dietary mannitol was metabolized to triglyceride, the main component of the fat body, forming lipid droplets. It was found that metabolites of a mannitol-supplemented diet extend the lifespan of T. castaneum, compared with those obtained by metabolizing a mannitol-free diet. Given that the insects presented transcriptional changes upon being fed carbohydrates, it might be possible to identify specific genes related to mannitol-specific metabolism by their upregulation upon mannitol intake in T. castaneum. The present study investigated mannitol-responsive gene expression using RNA-Seq. Twenty-eight genes, including those encoding trehalose-6-phosphate synthase and fatty acid synthase, were differentially expressed between beetles that were fed or not fed mannitol. The identification of upregulated genes provides us with important insights into the molecular events following mannitol intake.

Dnmt1 has an essential function despite the absence of CpG DNA methylation in the red flour beetle Tribolium castaneum

Epigenetic mechanisms, such as CpG DNA methylation enable phenotypic plasticity and rapid adaptation to changing environments. CpG DNA methylation is established by DNA methyltransferases (DNMTs), which are well conserved across vertebrates and invertebrates. There are insects with functional DNA methylation despite lacking a complete set of Dnmts. But at least one of the enzymes, DNMT1, appears to be required to maintain an active DNA methylation system. The red flour beetle, Tribolium castaneum, lacks Dnmt3 but possesses Dnmt1 and it has been controversial whether it has a functional DNA methylation system. Using whole genome bisulfite sequencing, we did not find any defined patterns of CpG DNA methylation in embryos. Nevertheless, we found Dnmt1 expressed throughout the entire life cycle of the beetle, with mRNA transcripts significantly more abundant in eggs and ovaries. A maternal knockdown of Dnmt1 caused a developmental arrest in offspring embryos. We show that Dnmt1 plays an essential role in T. castaneum embryos and that its downregulation leads to an early developmental arrest. This function appears to be unrelated to DNA methylation, since we did not find any evidence for this modification. This strongly suggests an alternative role of this protein.

Insecticidal efficacy of abamectin against red flour beetle Tribolium castaneum (Herbst) (Coleoptera: Tenebrionidae): influence of dose, exposure interval, relative humidity and temperature: Poster

Insecticidal efficacy of abamectin against red flour beetle Tribolium castaneum (Herbst) (Coleoptera: Tenebrionidae): influence of dose, exposure interval, relative humidity and temperature: Poster

On-farm comparison of different postharvest storage technologies for effectiveness in pest management in a maize farming system of Tanzania central corridor.

Seven methods for storing maize were compared with traditional practice of storing maize in polypropylene bags. Twenty farmers managed the experiment under their prevailing conditions for 30 weeks. Stored grain was assessed for damage every six weeks. The dominant storage insect pests identified were the Maize weevil (Sitophilus zeamais) and the Red flour beetle (Tribolium castaneum). There was no significant difference (F = 87.09; P < 0.0001) in insect control and grain damage between hermetic storage and fumigation with insecticides. However, the insecticide treated polypropylene yarn (ZeroFly®) did not control insect infestation of grain for the experimental period under farmers’ management. Grain damage was significantly lower in hermetic storage and fumigated grain than ZeroFly® and polypropylene bags without fumigation. No significant difference in grain damage was found between airtight treatment alone and when combined with the use of insecticides. During storage, S. zeamais was predominant and could be of more economic importance than T. castaneum as far as maize damage is concerned. Even though ZeroFly®, and polypropylene bags without grain treatment did not control storage pests, farmers still prefered this cheap technology. Hermetic storage techniques can be recommended to farmers without the use of insecticides provided they are inexpensive, and the proper application of technologies is ensured.

Bio-nanosilver synthesized by the entomopathogenic nematode-symbiotic bacterium as bio-insecticide for the red flour beetle ( Tribolium castaneum ):

Biological control can be another important way to manage post-harvest insect pests. Some organisms that showed biological control activity against some soil pests are insect-parasitic nematodes. There are two different species of nematodes, steinernematids and heterorhabditids, who carry within their bodies insect-pathogenic bacteria. Xenorhabdus spp are bacteria which infest steinernematids and Photorhabdus spp. bacteria infect heterorhabditids. The study aimed to develop pesticide alternatives by synthesizing silver bio-nanoparticles (AgNPs) using Xenorhabdus indica bacterial filtrate. The nanoparticles synthesized by the bacterial strains were purified and its cytotoxicity and bioactivity was examined against the larvae of the Tribolium castaneum. AgNPs were characterized by Scanning Electron Microscopy and X-Ray diffraction analysis, and the results revealed that the obtained nanoparticles are nanosilver with sizes ranging from 30 to 70 nm, with spherical shape and nonsmoothed surface. Insect larvae were initially exposed to descending concentrations (100, 50, 25, 10 and 5 µg/ml) of the biosynthesized nanosilver for 48 hours. Results of the bioassay showed that mortality of treated larvae was concentration-dependent with LC50 of 25 µg/ml. Higher mortality percentage (89%) was observed with the concentration 100 ug/ml and the lower one was obtained by the concentration 5 µg/ml (60%). Subsequently, data of the present study suggest these bio-AgNPs-bacterial filtrate complexes could be used as potentially effective eco-friend bio-control candidates. However, testing other types of bio-synthesized nanomaterials, and its vital effect as bio-insecticide for storage insect species are still under investigation.

Phytochemical-based nano emulsions for stored grain protection:

Stored grain losses caused by pest insects contribute significantly to the global food crisis. Currently, there are two main chemical control methods against stored product insect pests: fumigation with very toxic gases and grain protection by residual contact insecticides. Today, the global tendency is to prevent/reduce the wide use of insecticides, which have high toxicity to humans and harm the environment. Therefore, there is an urgent need to develop alternative eco-friendly approaches for stored insect pest control. Essential oils from Micromeria fruticosa and Mentha rotundifolia (Fam. Labiatae) and their main constituent pulegone which previously were shown by us as very active against stored product insect pests, were encapsulated into coarse and nano emulsions. The insecticidal activity of the developed formulations against primary internal insect rice weevil (Sitophilus oryzae L.) and secondary external pest red flour beetle (Tribolium castaneum Herbst) was evaluated in laboratory and pilot experiments. It was found that the phytochemical-based nano emulsions offered significant advantages and provided powerful and prolonged biological activity compare with the coarse formulations. The developed nano emulsions could serve as a natural, effective, low-toxify for human, and environmentally preferred method for protection stored grain and dry food products from pest insects.

The responses of Tribolium castaneum to wheat germ oil and fungal produced volatiles: Presentation

The red flour beetle Tribolium castaneum Herbst (Coleoptera: Tenebrionidae) is a significant pest affecting a wide variety of different stored products around the Globe. Despite its economic impact, there is evidence that the lures currently used in traps to monitor for this species are largely ineffective. Based on the evolutionary history of T. castaneum, and the ecological niche it occupies, the volatiles of wheat germ oil and volatiles produced by grain-associated fungi have the potential to act as attractants for this species. We used electroantennography (EAG) to measure the electrophysiological response elicited by sixty-eight volatile compounds found in wheat germ oil and/or grain-associated fungi in two T. castaneum strains; an established lab population (CTC12 strain) and a recently caught wild population. Many volatile compounds from both sources elicited strong antennal depolarisations, and the responses of both strains were highly correlated. We then tested whether the compounds that triggered the strongest antennal depolarisations also elicited behavioural responses by using Y-tube olfactometer bioassays and identified several compounds attractive to both strains. The discovery of novel compounds that elicit strong EAG signals and behavioural responses could prove useful in the design of improved lures for T. castaneum and other stored product pests. Our future research will identify how effective these attractive volatiles might be when used in combination, and when used under conditions that more closely replicate a stored product environment.

Costs of walking: differences in egg size and starvation resistance of females between strains of the red flour beetle (Tribolium castaneum) artificially selected for walking ability.

In many animals, movement often affects fitness components such as foraging or migration. On the other hand, individuals with higher mobility also often have fitness costs. This trade-off between movement and other traits may explain the maintenance of variation in moving ability in a population. However, few studies have focused on movement by walking, although many previous studies of insects have investigated the evolution of variations in mobility with wing polymorphism. In this study, we focused on the walking ability of the red flour beetle (Tribolium castaneum) and investigated whether females with higher than lower walking ability have fitness costs. The present results showed that females with genetically higher walking ability produced smaller eggs and had lower starvation resistance than females with lower walking ability. These results suggest that higher walking ability is costly for females, and this fitness cost may explain maintenance of variations of walking ability in a population.

Red flour beetle (Tribolium castaneum): From population genetics to functional genomics.

Tribolium castaneum is a small and low maintenance beetle that has emerged as a most suitable insect model for studying developmental biology and functional genetic analysis. Diverse population genetic studies have been conducted using Tribolium as the principal model to establish basic facts and principles of inbreeding experiments and response to the selection and other quantitative genetics fundamentals. The advanced molecular genetic studies presently focused on the use of Tribolium as a typical invertebrate model for higher diploid eukaryotes. After a whole genome sequencing of Tribolium, many areas of functional genomics were unraveled, which enabled the use of it in many technical approaches of genomics. The present text reviews the use of Tribolium in techniques such as RNAi, transgenic studies, immune priming, immunohistochemistry, in situ hybridization, gene sequencing for characterization of microRNAs, and gene editing using engineered endonuclease. In contrast to Drosophila, the T. castaneum holds a robust systemic RNAi response, which makes it an excellent model for comparative functional genetic studies.

The induction of larval resource preference in heterogeneous habitats

1. Animals often have to choose between multiple food sources in their habitat, and these potentially complex decisions have a large impact on their fitness. Among other factors, previous experience with an alternative resource can significantly increase subsequent preference for the resource (‘induction of preference’).2. Induction of resource preference is particularly relevant in spatially or temporally heterogeneous habitats. Although most mobile species – especially generalists – probably frequently encounter habitat heterogeneity, the impact of preference induction on individual behaviour and fitness in heterogeneous habitats is poorly understood. This study analysed larval preference induction in wheat‐adapted generalist red flour beetles, Tribolium castaneum (Coleoptera: Tenebrionidae), under three types of habitat heterogeneity.3. First, the induction of preference for novel cereal flours under temporal heterogeneity was analysed, exposing larvae to new resources during development. Larvae preferred a new resource if they experienced it recently, but the magnitude of induction varied across resources.4. Specific induction for a homogenous mix of wheat and a novel resource was also observed, with larvae preferring the mix over either pure resource.5. To analyse induction under spatial heterogeneity, beetle eggs were placed in one of two alternative resource patches and the preference of emerged larvae was tested. Hatching into a novel resource did not always induce preference.6. Finally, it was found that induction of preference for new resources could be maladaptive for larval development. In sum, this work demonstrates that experience‐based plasticity of larval resource choice may strongly impact larval preference and fitness in heterogeneous habitats.

Limited variations in susceptibility to an insecticidal double-stranded RNA (dsvATPaseE) among a laboratory strain and seven genetically differentiated field populations of Tribolium castaneum

There has been a considerable growth in interest to use RNA interference (RNAi) as a novel insect pest management strategy in the past 10 years. However, there has been virtually no information on insect population variations in response to double-stranded RNA (dsRNA) molecules. The objective of this study was to generate baseline susceptibilities of the red flour beetle (Tribolium castaneum) to an insecticidal dsRNA targeting vacuolar H+-ATPase subunit E gene (dsvATPaseE), and correlate the susceptibility data with sequence and expression variations of the target gene (vATPaseE), expression variations of the RNAi core genes, and overall genetic differences among a laboratory strain and seven geographical field populations of T. castaneum collected in China. Our results showed limited variations in the LD50 values of dsvATPaseE, which ranged from 0.10 to 0.29 ng/larva among the laboratory strain and the seven field populations. Considering the overlapping of the 95% confidence intervals of their LD50 values, there were no significant differences among the laboratory strain and field populations. We also found limited sequence polymorphisms and low frequencies of the polymorphisms of vATPaseE, and limited variations (<2-fold) of the endogenous expression of vATPaseE among the laboratory strain and field populations. However, we found considerable genetic variations among the individuals within each field population for most of eight loci and moderate to large genetic variations among the field populations. These results demonstrated that although the genetic variabilities were considerable among these field populations, the efficiency of RNAi targeting vATPaseE was highly consistent in T. castaneum. Our study provides work frames of resistance risk assessment for RNAi-based insect pest management programs.

Hyperactive piggyBac transposase improves transformation efficiency in diverse insect species

Even in times of advanced site-specific genome editing tools, the improvement of DNA transposases is still on high demand in the field of transgenesis: especially in emerging model systems where evaluated integrase landing sites have not yet been created and more importantly in non-model organisms such as agricultural pests and disease vectors, in which reliable sequence information and genome annotations are still pending. In fact, random insertional mutagenesis is essential to identify new genomic locations that are not influenced by position effects and thus can serve as future stable transgene integration sites. In this respect, a hyperactive version of the most widely used piggyBac transposase (PBase) has been engineered. The hyperactive version (hyPBase) is currently available with the original insect codon-based coding sequence (ihyPBase) as well as in a mammalian codon-optimized (mhyPBase) version. Both facilitate significantly higher rates of transposition when expressed in mammalian in vitro and in vivo systems compared to the classical PBase at similar protein levels. Here we demonstrate that the usage of helper plasmids encoding the hyPBase - irrespective of the codon-usage - also strikingly increases the rate of successful germline transformation in the Mediterranean fruit fly (Medfly) Ceratitis capitata, the red flour beetle Tribolium castaneum, and the vinegar fly Drosophila melanogaster. hyPBase-encoding helpers are therefore highly suitable for the generation of transgenic strains of diverse insect orders. Depending on the species, we achieved up to 15-fold higher germline transformation rates compared to PBase and generated hard to obtain transgenic T. castaneum strains that express constructs affecting fitness and viability. Moreover, previously reported high sterility rates supposedly caused by hyPBase (iPB7), encoded by ihyPBase, could not be confirmed by our study. Therefore, we value hyPBase as an effective genetic engineering tool that we highly recommend for insect transgenesis.

Responses to relaxed and reverse selection in strains artificially selected for duration of death-feigning behavior in the red flour beetle, Tribolium castaneum

Divergent lines selected artificially for many generations make it possible to answer two questions: (1) whether genetic variation still exists within the selected population; and (2) whether the selection itself is costly for the selected strain. In previous studies, the red flour beetle Tribolium castaneum was divergently selected artificially for duration of death-feigning, and strains selected for longer (L-strain) and shorter (S-strain) durations of death-feigning have been established (Miyatake et al. 2004, 2008). Because the selection experiments have been conducted for more than 27 generations, genetic variation may be eroded. Furthermore, because another previous study reported physiological costs to L-strains, the L-strains selected artificially for longer duration of death-feigning may have suffered more costs than the S-strains. In the present study, therefore, we relaxed the selection pressure after the 27th or 30th generation of S- and L-strains. We also carried out reverse selection during the most recent eight generations of S- and L-strains. The results showed that each strain clearly responded to relaxation of selection and reverse selection, suggesting that (1) additive genetic variation still existed in both strains after long-term selection, and (2) selection for shorter and longer duration of death-feigning was costly. These results suggest that anti-predator behavior is controlled by many loci, and longer or shorter duration of death-feigning is costly in a laboratory without predators.

A Systematic Study of RNAi Effects and dsRNA Stability in Tribolium castaneum and Acyrthosiphon pisum, Following Injection and Ingestion of Analogous dsRNAs.

RNA interference (RNAi) effects in insects are highly variable and may be largely dependent upon the stability of introduced double-stranded RNAs to digestion by nucleases. Here, we report a systematic comparison of RNAi effects in susceptible red flour beetle (Tribolium castaneum) and recalcitrant pea aphid (Acyrthosiphon pisum) following delivery of dsRNAs of identical length targeting expression of V-type ATPase subunit E (VTE) and inhibitor of apoptosis (IAP) genes. Injection and ingestion of VTE and IAP dsRNAs resulted in up to 100% mortality of T. castaneum larvae and sustained suppression (>80%) of transcript levels. In A. pisum, injection of VTE but not IAP dsRNA resulted in up to 65% mortality and transient suppression (ca. 40%) of VTE transcript levels. Feeding aphids on VTE dsRNA reduced growth and fecundity although no evidence for gene suppression was obtained. Rapid degradation of dsRNAs by aphid salivary, haemolymph and gut nucleases contrasted with stability in T. castaneum larvae where it appears that exo-nuclease activity is responsible for relatively slow digestion of dsRNAs. This is the first study to directly compare RNAi effects and dsRNA stability in receptive and refractory insect species and provides further evidence that dsRNA susceptibility to nucleases is a key factor in determining RNAi efficiency.