RNA interference to combat the Asian tiger mosquito in Europe: A pathway from design of an innovative vector control tool to its application.

The Asian tiger mosquito Aedes albopictus is currently spreading across Europe, facilitated by climate change and global transportation. It is a vector of arboviruses causing human diseases such as chikungunya, dengue hemorrhagic fever and Zika fever. For the majority of these diseases, no vaccines or therapeutics are available. Options for the control of Ae. albopictus are limited by European regulations introduced to protect biodiversity by restricting or phasing out the use of pesticides, genetically modified organisms (GMOs) or products of genome editing. Alternative solutions are thus urgently needed to avoid a future scenario in which Europe faces a choice between prioritizing human health or biodiversity when it comes to Aedes-vectored pathogens. To ensure regulatory compliance and public acceptance, these solutions should preferably not be based on chemicals or GMOs and must be cost-efficient and specific. The present review aims to synthesize available evidence on RNAi-based mosquito vector control and its potential for application in the European Union. The recent literature has identified some potential target sites in Ae. albopictus and formulations for delivery. However, we found little information concerning non-target effects on the environment or human health, on social aspects, regulatory frameworks, or on management perspectives. We propose optimal designs for RNAi-based vector control tools against Ae. albopictus (target product profiles), discuss their efficacy and reflect on potential risks to environmental health and the importance of societal aspects. The roadmap from design to application will provide readers with a comprehensive perspective on the application of emerging RNAi-based vector control tools for the suppression of Ae. albopictus populations with special focus on Europe.

Elucidation of the MicroRNA Transcriptome in Western Corn Rootworm Reveals Its Dynamic and Evolutionary Complexity.

Diabrotica virgifera virgifera (western corn rootworm, WCR) is one of the most destructive agricultural insect pests in North America. It is highly adaptive to environmental stimuli and crop protection technologies. However, little is known about the underlying genetic basis of WCR behavior and adaptation. More specifically, the involvement of small RNAs (sRNAs), especially microRNAs (miRNAs), a class of endogenous small non-coding RNAs that regulate various biological processes, has not been examined, and the datasets of putative sRNA sequences have not previously been generated for WCR. To achieve a comprehensive collection of sRNA transcriptomes in WCR, we constructed, sequenced, and analyzed sRNA libraries from different life stages of WCR and northern corn rootworm (NCR), and identified 101 conserved precursor miRNAs (pre-miRNAs) in WCR and other Arthropoda. We also identified 277 corn rootworm specific pre-miRNAs. Systematic analyses of sRNA populations in WCR revealed that its sRNA transcriptome, which includes PIWI-interacting RNAs (piRNAs) and miRNAs, undergoes a dynamic change throughout insect development. Phylogenetic analysis of miRNA datasets from model species reveals that a large pool of species-specific miRNAs exists in corn rootworm; these are potentially evolutionarily transient. Comparisons of WCR miRNA clusters to other insect species highlight conserved miRNA-regulated processes that are common to insects. Parallel Analysis of RNA Ends (PARE) also uncovered potential miRNA-guided cleavage sites in WCR. Overall, this study provides a new resource for studying the sRNA transcriptome and miRNA-mediated gene regulation in WCR and other Coleopteran insects.

RNAi targeting of rootworm Troponin I transcripts confers root protection in maize.

Western corn rootworm, Diabrotica virgifera virgifera, is the major agronomically important pest of maize in the US Corn Belt. To augment the repertoire of the available dsRNA-based traits that control rootworm, we explored a potentially haplolethal gene target, wings up A (wupA), which encodes Troponin I. Troponin I, a component of the Troponin-Tropomyosin complex, is an inhibitory protein involved in muscle contraction. In situ hybridization showed that feeding on wupA-targeted dsRNAs caused systemic transcript knockdown in D. v. virgifera larvae. The knockdown of wupA transcript, and by extension Troponin I protein, led to deterioration of the striated banding pattern in larval body muscle and decreased muscle integrity. Additionally, the loss of function of the circular muscles surrounding the alimentary system led to significant accumulation of food material in the hind gut, which is consistent with a loss of peristaltic motion of the alimentary canal. In this study, we demonstrate that wupA dsRNA is lethal in D. v. virgifera larvae when fed via artificial diet, with growth inhibition of up to 50% within two days of application. Further, wupA hairpins can be stably expressed and detected in maize. Maize expressing wupA hairpins exhibit robust root protection in greenhouse bioassays, with several maize transgene integration events showing root protection equivalent to commercial insecticidal protein-expressing maize.

Gene silencing in Tribolium castaneum as a tool for the targeted identification of candidate RNAi targets in crop pests.

RNAi shows potential as an agricultural technology for insect control, yet, a relatively low number of robust lethal RNAi targets have been demonstrated to control insects of agricultural interest. In the current study, a selection of lethal RNAi target genes from the iBeetle (Tribolium castaneum) screen were used to demonstrate efficacy of orthologous targets in the economically important coleopteran pests Diabrotica virgifera virgifera and Meligethes aeneus. Transcript orthologs of 50 selected genes were analyzed in D. v. virgifera diet-based RNAi bioassays; 21 of these RNAi targets showed mortality and 36 showed growth inhibition. Low dose injection- and diet-based dsRNA assays in T. castaneum and D. v. virgifera, respectively, enabled the identification of the four highly potent RNAi target genes: Rop, dre4, ncm, and RpII140. Maize was genetically engineered to express dsRNA directed against these prioritized candidate target genes. T0 plants expressing Rop, dre4, or RpII140 RNA hairpins showed protection from D. v. virgifera larval feeding damage. dsRNA targeting Rop, dre4, ncm, and RpII140 in M. aeneus also caused high levels of mortality both by injection and feeding. In summary, high throughput systems for model organisms can be successfully used to identify potent RNA targets for difficult-to-work with agricultural insect pests.

The model beetle Tribolium castaneum can be used as an early warning system for transgenerational epigenetic side effects caused by pharmaceuticals.

Pharmaceuticals are not currently tested for transgenerational and epigenetic side effects. The use of vertebrates as preclinical research models is limited by their long generation times, low numbers of progeny and ethical concerns. In contrast, invertebrates such as insects breed rapidly, produce many offspring and are more ethically acceptable, allowing them to be used for high-throughput screening. Here, we established Tribolium castaneum as a model to screen for the effect of drugs on complex fitness parameters and the expression of epigenetic regulatory genes. We tested diets supplemented with the psychoactive drug valproic acid (VPA), which is a histone deacetylase inhibitor, or the antioxidant curcumin, which is a histone acetyltransferase inhibitor. We found that VPA delayed development, reduced longevity, and increased female body weight compared to a control diet. Fertility and fecundity declined and the expression of epigenetic regulatory genes was induced in the untreated F1 generation. In contrast, curcumin did not affect development or body weight, but it increased longevity, caused a significant reduction in fertility, and induced the expression of epigenetic regulatory genes mostly in the treated F0 generation. VPA and curcumin administered to vertebrate models have similar effects to those we observed in T. castaneum, confirming that this beetle is potentially useful as an alternative model to screen for the epigenetic effect of drugs. T. castaneum also provides a valuable early warning system for transgenerational epigenetic risk factors that are difficult to detect in mammals.

Translocation of bacteria from the gut to the eggs triggers maternal transgenerational immune priming in Tribolium castaneum.

Invertebrates can be primed to enhance their protection against pathogens they have encountered before. This enhanced immunity can be passed maternally or paternally to the offspring and is known as transgenerational immune priming. We challenged larvae of the red flour beetle Tribolium castaneum by feeding them on diets supplemented with Escherichia coli, Micrococcus luteus or Pseudomonas entomophila, thus mimicking natural exposure to pathogens. The oral uptake of bacteria induced immunity-related genes in the offspring, but did not affect the methylation status of the egg DNA. However, we observed the translocation of bacteria or bacterial fragments from the gut to the developing eggs via the female reproductive system. Such translocating microbial elicitors are postulated to trigger bacterial strain-specific immune responses in the offspring and provide an alternative mechanistic explanation for maternal transgenerational immune priming in coleopteran insects.

Tribolium castaneum defensins are primarily active against Gram-positive bacteria.

The red flour beetle Tribolium castaneum is a destructive insect pest of stored food and feed products, and a model organism for development, evolutionary biology and immunity. The insect innate immune system includes antimicrobial peptides (AMPs) with a wide spectrum of targets including viruses, bacteria, fungi and parasites. Defensins are an evolutionarily-conserved class of AMPs and a potential new source of antimicrobial agents. In this context, we report the antimicrobial activity, phylogenetic and structural properties of three T. castaneum defensins (Def1, Def2 and Def3) and their relevance in the immunity of T. castaneum against bacterial pathogens. All three recombinant defensins showed bactericidal activity against Micrococcus luteus and Bacillus thuringiensis serovar tolworthi, but only Def1 and Def2 showed a bacteriostatic effect against Staphylococcus epidermidis. None of the defensins showed activity against the Gram-negative bacteria Escherichia coli and Pseudomonas entomophila or against the yeast Saccharomyces cerevisiae. All three defensins were transcriptionally upregulated following a bacterial challenge, suggesting a key role in the immunity of T. castaneum against bacterial pathogens. Phylogenetic analysis showed that defensins from T. castaneum, mealworms, Udo longhorn beetle and houseflies cluster within a well-defined clade of insect defensins. We conclude that T. castaneum defensins are primarily active against Gram-positive bacteria and that other AMPs may play a more prominent role against Gram-negative species.

Tribolium castaneum as a model for high-throughput RNAi screening.

Coleopteran insects are a highly diverse and successful order, and many beetle species are significant agricultural pests. New biorational strategies for managing populations of beetles and other insect species are needed as pests develop resistance to chemical insecticides and Bt toxins. There is now an opportunity to use genome sequence data to identify genes that are essential for insect growth, development, or survival as new targets for designing control technology. This goal requires a method for high-throughput in vivo screening of thousands of genes to identify candidate genes that, when their expression is disrupted, have a phenotype that may be useful in insect pest control. Tribolium castaneum, the red flour beetle, is a model organism that offers considerable advantages for such screening, including ease of rearing in large numbers, a sequenced genome, and a strong, systemic RNAi response for specific depletion of gene transcripts. The RNAi effect in T. castaneum can be elicited in any tissue and any stage by the injection of dsRNA into the hemocoel, and injection of dsRNA into adult females can even be used to identify phenotypes in offspring. A pilot RNAi screen (iBeetle) is underway. Several T. castaneum genes with promising RNAi phenotypes for further development as mechanisms for plant protection have been identified. These include heat shock protein 90, chitin synthase, the segmentation gene hairy, and a matrix metalloprotease. Candidate genes identified in T. castaneum screens can then be tested in agricultural pest species (in which screening is not feasible), to evaluate their effectiveness for use in potential plant-based RNAi control strategies. Delivery of dsRNA expressed by genetically modified crops to the midgut of phytophagous insects is under investigation as a new tool for very specific protection of plants from insect pest species. The T. castaneum screening platform offers a system for discovery of candidate genes with high potential benefit.

Longevity in the red flour beetle Tribolium castaneum is enhanced by broccoli and depends on nrf-2, jnk-1 and foxo-1 homologous genes.

Diet is generally believed to affect the aging process. The effects of complex foods on life span can be investigated using simple models that produce rapid results and allow the identification of food-gene interactions. Here, we show that 1 % lyophilized broccoli, added to flour as a dietary source, significantly increases the life span of the red flour beetle (Tribolium castaneum) under physiological conditions (32 °C) and under heat stress (42 °C). The beneficial effects of broccoli could also be reproduced by supplementing flour with the isothiocyanate sulforaphane at concentrations found in the broccoli-supplemented diet. We identified stress-resistant genes responsible for these effects on longevity by microinjecting pupae with double-stranded RNA to induce RNA interference (RNAi). The knockdown of transcripts encoding homologs of Nrf-2, Jnk-1 and Foxo-1 reduced the life span of beetles and abrogated the beneficial effects of broccoli, whereas the knockdown of Sirt-1 and Sirt-3 had no impact in either scenario. In conclusion, T. castaneum is a suitable model organism to investigate food-gene interactions that affect stress resistance and longevity, and RNAi can be used to identify functionally relevant genes. As a proof of principle, we have shown here that broccoli increases the longevity of beetles and mediates its effect through signaling pathways that include key stress-resistant factors such as Nrf-2, Jnk-1 and Foxo-1.

Gender- and stressor-specific microRNA expression in Tribolium castaneum.

MicroRNAs (miRNAs) are small non-coding RNAs mediating post-transcriptional regulation of gene expression in eukaryotes. Addressing their role in regulation of physiological adaptations to environmental stress in insects, we selected the red flour beetle Tribolium castaneum as a model. Beetles were fed with the bacterial entomopathogen Pseudomonas entomophila (to mimic natural infection), injected with peptidoglycan (experimental setting of strong immune responses) or subjected to either mild heat shock or starvation. Differential expression of selected immunity- and stress-related genes was quantified using real-time PCR, and expression and induction of 455 mature arthropod miRNAs were determined using proprietary microarrays. We found that Tribolium exhibits both gender- and stressor-specific adjustment of immune gene and miRNA expression. Strikingly, we discovered that the number of stressor-induced miRNAs in females is remarkably higher than in males. This observation could support the hypothesis called Bateman's principle in immunity that predicts gender-specific immune responses because females gain fitness through increased longevity, whereas males gain fitness by increasing mating rates. Our results suggest that Tribolium males and females display differential regulatory elements, both pre- and post-transcriptional, likely resulting from different investment strategies in life-history traits.

Post-embryonic functions of HSP90 in Tribolium castaneum include the regulation of compound eye development.

Heat shock protein 90 (HSP90) belongs to a family of conserved chaperons with multiple roles in stress adaptation and development, including spermatogenesis, oogenesis and embryogenesis in insects. In the red flour beetle, Tribolium castaneum, we found that HSP90 is transiently upregulated during larval development, in prepupae, in female pupae and in adults, suggesting multiple post-embryonic roles. We found that silencing HSP90 expression by RNA interference was lethal within 10 days at all developmental stages. Titration experiments revealed that larvae were more susceptible than pupae or beetles. Interestingly, HSP90 silencing in final instar larvae resulted in abnormal pupal phenotypes lacking compound eyes and exhibiting prepupal features, suggesting developmental arrest at the prepupal stage. Our results suggest that HSP90 functions can be expanded beyond the known ones in insect embryogenesis to include roles in post-embryonic development such as the regulation of compound eye development.

MMPs regulate both development and immunity in the tribolium model insect.

BACKGROUND: Matrix metalloproteinases (MMPs) are evolutionarily conserved and multifunctional effector molecules in development and homeostasis. In spite of previous, intensive investigation in vitro and in cell culture, their pleiotrophic functions in vivo are still not well understood. METHODOLOGY/PRINCIPAL FINDINGS: We show that the genetically amenable beetle Tribolium castaneum represents a feasible model organism to explore MMP functions in vivo. We silenced expression of three insect-type Tribolium MMP paralogs and their physiological inhibitors, TIMP and RECK, by dsRNA-mediated genetic interference (RNAi). Knock-down of MMP-1 arrested development during pupal morphogenesis giving phenotypes with altered antennae, compound eyes, wings, legs, and head. Parental RNAi-mediated knock-down of MMP-1 or MMP-2 resulted in larvae with non-lethal tracheal defects and with abnormal intestines, respectively, implicating additional roles of MMPs during beetle embryogenesis. This is different to findings from the fruit fly Drosophila melanogaster, in which MMPs have a negligible role in embryogenesis. Confirming pleiotrophic roles of MMPs our results also revealed that MMPs are required for proper insect innate immunity because systemic knock-down of Tribolium MMP-1 resulted in significantly higher susceptibility to the entomopathogenic fungus Beauveria bassiana. Moreover, mRNA levels of MMP-1, TIMP, and RECK, and also MMP enzymatic activity were significantly elevated in immune-competent hemocytes upon stimulation. To confirm collagenolytic activity of Tribolium MMP-1 we produced and purified recombinant enzyme and determined a similar collagen IV degrading activity as observed for the most related human MMP, MMP-19. CONCLUSIONS/SIGNIFICANCE: This is the first study, to our knowledge, investigating the in vivo role of virtually all insect MMP paralogs along with their inhibitors TIMP and RECK in both insect development and immunity. Our results from the Tribolium model insect indicate that MMPs regulate tracheal and gut development during beetle embryogenesis, pupal morphogenesis, and innate immune defense reactions thereby revealing the evolutionarily conserved roles of MMPs.

Beetle immunity: Identification of immune-inducible genes from the model insect Tribolium castaneum.

The red flour beetle, Tribolium castaneum, is an established genetically tractable model insect for evolutionary and developmental studies. Therefore, it may also represent a valuable model for comparative analysis of insect immunity. Here, we used the suppression subtractive hybridization method to identify Tribolium genes that are transcriptionally induced in response to injection of crude lipopolysaccharide (LPS). Determined genes encode proteins that share sequence similarities with counterparts from other insects known to mediate sensing of infection (e.g. Toll and PGRP) or to represent potential antimicrobial effectors (e.g. ferritin, c-type lysozyme, serine proteinase inhibitors, and defensins). Especially significant is the identification of thaumatin-like peptides, representing ancient antifungal peptides originally reported from plants, that are absent from the genomes of many other insects such as Drosophila, Anopheles, and Apis. We produced recombinant thaumatin-1 in bacteria and we found that it represents an antimicrobial peptide against filamentous fungi in Tribolium. Additionally, septic injury induces expression of genes involved in stress adaptation (e.g. heat-shock proteins) or insecticide resistance (e.g. cytochrome P450s) in Tribolium, suggesting that there may be crosstalk between the immune and stress responses.