Development and characterization of the first dsRNA-resistant insect population from western corn rootworm, Diabrotica virgifera virgifera LeConte.

The use of dsRNA to control insect pests via the RNA interference (RNAi) pathway is being explored by researchers globally. However, with every new class of insect control compounds, the evolution of insect resistance needs to be considered, and understanding resistance mechanisms is essential in designing durable technologies and effective resistance management strategies. To gain insight into insect resistance to dsRNA, a field screen with subsequent laboratory selection was used to establish a population of DvSnf7 dsRNA-resistant western corn rootworm, Diabrotica virgifera virgifera, a major maize insect pest. WCR resistant to ingested DvSnf7 dsRNA had impaired luminal uptake and resistance was not DvSnf7 dsRNA-specific, as indicated by cross resistance to all other dsRNAs tested. No resistance to the Bacillus thuringiensis Cry3Bb1 protein was observed. DvSnf7 dsRNA resistance was inherited recessively, located on a single locus, and autosomal. Together these findings will provide insights for dsRNA deployment for insect pest control.

Mechanistic insights into the first Lygus-active ß-pore forming protein.

The cotton pests Lygus hesperus and Lygus lineolaris can be controlled by expressing Cry51Aa2.834_16 in cotton. Insecticidal activity of pore-forming proteins is generally associated with damage to the midgut epithelium due to pores, and their biological specificity results from a set of key determinants including proteolytic activation and receptor binding. We conducted mechanistic studies to gain insight into how the first Lygus-active ß-pore forming protein variant functions. Biophysical characterization revealed that the full-length Cry51Aa2.834_16 was a stable dimer in solution, and when exposed to Lygus saliva or to trypsin, the protein underwent proteolytic cleavage at the C-terminus of each of the subunits, resulting in dissociation of the dimer to two separate monomers. The monomer showed tight binding to a specific protein in Lygus brush border membranes, and also formed a membrane-associated oligomeric complex both in vitro and in vivo. Chemically cross-linking the ß-hairpin to the Cry51Aa2.834_16 body rendered the protein inactive, but still competent to compete for binding sites with the native protein in vivo. Our study suggests that disassociation of the Cry51Aa2.834_16 dimer into monomeric units with unoccupied head-region and sterically unhindered ß-hairpin is required for brush border membrane binding, oligomerization, and the subsequent steps leading to insect mortality.

Environmental RNAi in herbivorous insects.

Environmental RNAi (eRNAi) is a sequence-specific regulation of endogenous gene expression in a receptive organism by exogenous double-stranded RNA (dsRNA). Although demonstrated under artificial dietary conditions and via transgenic plant presentations in several herbivorous insects, the magnitude and consequence of exogenous dsRNA uptake and the role of eRNAi remains unknown under natural insect living conditions. Our analysis of coleopteran insects sensitive to eRNAi fed on wild-type plants revealed uptake of plant endogenous long dsRNAs, but not small RNAs. Subsequently, the dsRNAs were processed into 21 nt siRNAs by insects and accumulated in high quantities in insect cells. No accumulation of host plant-derived siRNAs was observed in lepidopteran larvae that are recalcitrant to eRNAi. Stability of ingested dsRNA in coleopteran larval gut followed by uptake and transport from the gut to distal tissues appeared to be enabling factors for eRNAi. Although a relatively large number of distinct coleopteran insect-processed plant-derived siRNAs had sequence complementarity to insect transcripts, the vast majority of the siRNAs were present in relatively low abundance, and RNA-seq analysis did not detect a significant effect of plant-derived siRNAs on insect transcriptome. In summary, we observed a broad genome-wide uptake of plant endogenous dsRNA and subsequent processing of ingested dsRNA into 21 nt siRNAs in eRNAi-sensitive insects under natural feeding conditions. In addition to dsRNA stability in gut lumen and uptake, dosage of siRNAs targeting a given insect transcript is likely an important factor in order to achieve measurable eRNAi-based regulation in eRNAi-competent insects that lack an apparent silencing amplification mechanism.

Establishing an in vivo assay system to identify components involved in environmental RNA interference in the western corn rootworm.

The discovery of environmental RNA interference (RNAi), in which gene expression is suppressed via feeding with double-stranded RNA (dsRNA) molecules, opened the door to the practical application of RNAi-based techniques in crop pest management. The western corn rootworm (WCR, Diabrotica virgifera virgifera) is one of the most devastating corn pests in North America. Interestingly, WCR displays a robust environmental RNAi response, raising the possibility of applying an RNAi-based pest management strategy to this pest. Understanding the molecular mechanisms involved in the WCR environmental RNAi process will allow for determining the rate limiting steps involved with dsRNA toxicity and potential dsRNA resistance mechanisms in WCR. In this study, we have established a two-step in vivo assay system, which allows us to evaluate the involvement of genes in environmental RNAi in WCR. We show that laccase 2 and ebony, critical cuticle pigmentation/tanning genes, can be used as marker genes in our assay system, with ebony being a more stable marker to monitor RNAi activity. In addition, we optimized the dsRNA dose and length for the assay, and confirmed that this assay system is sensitive to detect well-known RNAi components such as Dicer-2 and Argonaute-2. We also evaluated two WCR sid1- like (sil) genes with this assay system. This system will be useful to quickly survey candidate systemic RNAi genes in WCR, and also will be adaptable for a genome-wide RNAi screening to give us an unbiased view of the environmental/systemic RNAi pathway in WCR.

Ultrastructural changes caused by Snf7 RNAi in larval enterocytes of western corn rootworm (Diabrotica virgifera virgifera Le Conte).

The high sensitivity to oral RNA interference (RNAi) of western corn rootworm (WCR, Diabrotica virgifera virgifera Le Conte) provides a novel tool for pest control. Previous studies have shown that RNAi of DvSnf7, an essential cellular component of endosomal sorting complex required for transport (ESCRT), caused deficiencies in protein de-ubiquitination and autophagy, leading to WCR death. Here we investigated the detailed mechanism leading to larval death by analyzing the ultrastructural changes in midgut enterocytes of WCR treated with double-stranded RNA (ds-DvSnf7). The progressive phases of pathological symptoms caused by DvSnf7-RNAi in enterocytes include: 1) the appearance of irregularly shaped macroautophagic complexes consisting of relatively large lysosomes and multi-lamellar bodies, indicative of failure in autolysosome formation; 2) cell sloughing and loss of apical microvilli, and eventually, 3) massive loss of cellular contents indicating loss of membrane integrity. These data suggest that the critical functions of Snf7 in insect midgut cells demonstrated by the ultrastructural changes in DvSnf7 larval enterocytes underlies the conserved essential function of the ESCRT pathway in autophagy and membrane stability in other organisms.

Characterization of the spectrum of insecticidal activity of a double-stranded RNA with targeted activity against Western Corn Rootworm (Diabrotica virgifera virgifera LeConte).

The sequence specificity of the endogenous RNA interference pathway allows targeted suppression of genes essential for insect survival and enables the development of durable and efficacious insecticidal products having a low likelihood to adversely impact non-target organisms. The spectrum of insecticidal activity of a 240 nucleotide (nt) dsRNA targeting the Snf7 ortholog in Western Corn Rootworm (WCR; Diabrotica virgifera virgifera) was characterized by selecting and testing insects based upon their phylogenetic relatedness to WCR. Insect species, representing 10 families and 4 Orders, were evaluated in subchronic or chronic diet bioassays that measured potential lethal and sublethal effects. When a specific species could not be tested in diet bioassays, the ortholog to the WCR Snf7 gene (DvSnf7) was cloned and corresponding dsRNAs were tested against WCR and Colorado potato beetle (Leptinotarsa decemlineata); model systems known to be sensitive to ingested dsRNA. Bioassay results demonstrate that the spectrum of activity for DvSnf7 is narrow and activity is only evident in a subset of beetles within the Galerucinae subfamily of Chrysomelidae (>90% identity with WCR Snf7 240 nt). This approach allowed for evaluating the relationship between minimum shared nt sequence length and activity. A shared sequence length of ≥ 21 nt was required for efficacy against WCR (containing 221 potential 21-nt matches) and all active orthologs contained at least three 21 nt matches. These results also suggest that WCR resistance to DvSnf7 dsRNA due to single nucleotide polymorphisms in the target sequence of 240 nt is highly unlikely.

Physiological and cellular responses caused by RNAi- mediated suppression of Snf7 orthologue in western corn rootworm (Diabrotica virgifera virgifera) larvae.

Ingestion of double stranded RNA (dsRNA) has been previously demonstrated to be effective in triggering RNA interference (RNAi) in western corn rootworm (WCR, Diabrotica virgifera virgifera LeConte), providing potential novel opportunities for insect pest control. The putative Snf7 homolog of WCR (DvSnf7) has previously been shown to be an effective RNAi target for insect control, as DvSnf7 RNAi leads to lethality of WCR larvae. Snf7 functions as a part of the ESCRT (Endosomal Sorting Complex Required for Transport) pathway which plays a crucial role in cellular housekeeping by internalization, transport, sorting and lysosomal degradation of transmembrane proteins. To understand the effects that lead to death of WCR larvae by DvSnf7 RNAi, we examined some of the distinct cellular processes associated with ESCRT functions such as de-ubiquitination of proteins and autophagy. Our data indicate that ubiquitinated proteins accumulate in DvSnf7 dsRNA-fed larval tissues and that the autophagy process seems to be impaired. These findings suggest that the malfunctioning of these cellular processes in both midgut and fat body tissues triggered by DvSnf7 RNAi were the main effects leading to the death of WCR. This study also illustrates that Snf7 is an essential gene in WCR and its functions are consistent with biological functions described for other eukaryotes.

Characterizing the mechanism of action of double-stranded RNA activity against western corn rootworm (Diabrotica virgifera virgifera LeConte).

RNA interference (RNAi) has previously been shown to be effective in western corn rootworm (WCR, Diabrotica virgifera virgifera LeConte) larvae via oral delivery of synthetic double-stranded RNA (dsRNA) in an artificial diet bioassay, as well as by ingestion of transgenic corn plant tissues engineered to express dsRNA. Although the RNAi machinery components appear to be conserved in Coleopteran insects, the key steps in this process have not been reported for WCR. Here we characterized the sequence of events that result in mortality after ingestion of a dsRNA designed against WCR larvae. We selected the Snf7 ortholog (DvSnf7) as the target mRNA, which encodes an essential protein involved in intracellular trafficking. Our results showed that dsRNAs greater than or equal to approximately 60 base-pairs (bp) are required for biological activity in artificial diet bioassays. Additionally, 240 bp dsRNAs containing a single 21 bp match to the target sequence were also efficacious, whereas 21 bp short interfering (si) RNAs matching the target sequence were not. This result was further investigated in WCR midgut tissues: uptake of 240 bp dsRNA was evident in WCR midgut cells while a 21 bp siRNA was not, supporting the size-activity relationship established in diet bioassays. DvSnf7 suppression was observed in a time-dependent manner with suppression at the mRNA level preceding suppression at the protein level when a 240 bp dsRNA was fed to WCR larvae. DvSnf7 suppression was shown to spread to tissues beyond the midgut within 24 h after dsRNA ingestion. These events (dsRNA uptake, target mRNA and protein suppression, systemic spreading, growth inhibition and eventual mortality) comprise the overall mechanism of action by which DvSnf7 dsRNA affects WCR via oral delivery and provides insights as to how targeted dsRNAs in general are active against insects.

Asymmetrically expressed axin required for anterior development in Tribolium.

Canonical Wnt signaling has been implicated in an AP axis polarizing mechanism in most animals, despite limited evidence from arthropods. In the long-germ insect, Drosophila, Wnt signaling is not required for global AP patterning, but in short-germ insects including Tribolium castaneum, loss of Wnt signaling affects development of segments in the growth zone but not those defined in the blastoderm. To determine the effects of ectopic Wnt signaling, we analyzed the expression and function of axin, which encodes a highly conserved negative regulator of the pathway. We found Tc-axin transcripts maternally localized to the anterior pole in freshly laid eggs. Expression spread toward the posterior pole during the early cleavage stages, becoming ubiquitous by the time the germ rudiment formed. Tc-axin RNAi produced progeny phenotypes that ranged from mildly affected embryos with cuticles displaying a graded loss of anterior structures, to defective embryos that condensed at the posterior pole in the absence of serosa. Altered expression domains of several blastodermal markers indicated anterior expansion of posterior fates. Analysis of other canonical Wnt pathway components and the expansion of Tc-caudal expression, a Wnt target, suggest that the effects of Tc-axin depletion are mediated through this pathway and that Wnt signaling must be inhibited for proper anterior development in Tribolium. These studies provide unique evidence that canonical Wnt signaling must be carefully regulated along the AP axis in an arthropod, and support an ancestral role for Wnt activity in defining AP polarity and patterning in metazoan development.

Parallel duplication and partial subfunctionalization of ß-catenin/armadillo during insect evolution.

ß-Catenin is a multifunctional scaffolding protein with roles in Wnt signaling, cell adhesion, and centrosome separation. Here, we report on independent duplications of the insect ß-Catenin ortholog armadillo (arm) in the red flour beetle Tribolium castaneum and the pea aphid Acyrthosiphon pisum. Detailed sequence analysis shows that in both species, one paralog lost critical residues of the α-Catenin binding domain, which is essential for cell adhesion, and accumulated a dramatically higher number of amino acid substitutions in the central Arm repeat domain. Residues associated with aspects of Wnt signaling, however, are conserved in both paralogs. Consistent with these molecular signatures, the effects of specific and combinatorial knockdown experiments in the Tribolium embryo indicate that the duplication resulted in redundant involvement in Wnt signaling of both ß-Catenin paralogs but differential inheritance of the ancestral cell adhesion and centrosome separation functions. We conclude that the duplicated pea aphid and flour beetle ß-catenin genes experienced partial subfunctionalization, which appears to be evolutionarily favored. Providing first evidence of genetic separability of the cell adhesion and centrosome separation functions, the duplicated Tribolium and Acyrthosiphon arm paralogs offer new inroads for context-specific analyses of ß-Catenin. Our data also revealed the conservation of a C-terminally truncated Arm isoform in both singleton and duplicated homologs, suggesting an as yet unexplored role in Wnt signaling.

Conservation, loss, and redeployment of Wnt ligands in protostomes: implications for understanding the evolution of segment formation.

BACKGROUND: The Wnt genes encode secreted glycoprotein ligands that regulate a wide range of developmental processes, including axis elongation and segmentation. There are thirteen subfamilies of Wnt genes in metazoans and this gene diversity appeared early in animal evolution. The loss of Wnt subfamilies appears to be common in insects, but little is known about the Wnt repertoire in other arthropods, and moreover the expression and function of these genes have only been investigated in a few protostomes outside the relatively Wnt-poor model species Drosophila melanogaster and Caenorhabditis elegans. To investigate the evolution of this important gene family more broadly in protostomes, we surveyed the Wnt gene diversity in the crustacean Daphnia pulex, the chelicerates Ixodes scapularis and Achaearanea tepidariorum, the myriapod Glomeris marginata and the annelid Platynereis dumerilii. We also characterised Wnt gene expression in the latter three species, and further investigated expression of these genes in the beetle Tribolium castaneum. RESULTS: We found that Daphnia and Platynereis both contain twelve Wnt subfamilies demonstrating that the common ancestors of arthropods, ecdysozoans and protostomes possessed all members of all Wnt subfamilies except Wnt3. Furthermore, although there is striking loss of Wnt genes in insects, other arthropods have maintained greater Wnt gene diversity. The expression of many Wnt genes overlap in segmentally reiterated patterns and in the segment addition zone, and while these patterns can be relatively conserved among arthropods and the annelid, there have also been changes in the expression of some Wnt genes in the course of protostome evolution. Nevertheless, our results strongly support the parasegment as the primary segmental unit in arthropods, and suggest further similarities between segmental and parasegmental regulation by Wnt genes in annelids and arthropods respectively. CONCLUSIONS: Despite frequent losses of Wnt gene subfamilies in lineages such as insects, nematodes and leeches, most protostomes have probably maintained much of their ancestral repertoire of twelve Wnt genes. The maintenance of a large set of these ligands could be in part due to their combinatorial activity in various tissues rather than functional redundancy. The activity of such Wnt 'landscapes' as opposed to the function of individual ligands could explain the patterns of conservation and redeployment of these genes in important developmental processes across metazoans. This requires further analysis of the expression and function of these genes in a wider range of taxa.

Loss of Tc-arrow and canonical Wnt signaling alters posterior morphology and pair-rule gene expression in the short-germ insect, Tribolium castaneum.

Wnt signaling has been implicated in posterior patterning in short-germ insects, including the red flour beetle Tribolium castaneum (Bolognesi et al. Curr Biol 18:1624-1629, 2008b; Angelini and Kaufman Dev Biol 283:409-423, 2005; Miyawaki et al. Mech Dev 121:119-130, 2004). Specifically, depletion of Wnt ligands Tc-Wnt1 and Tc-WntD/8 produces Tribolium embryos lacking abdominal segments. Similar phenotypes are produced by depletion of Tc-porcupine (Tc-porc) or Tc-pangolin (Tc-pan), indicating that the signal is transmitted through the canonical Wnt pathway (Bolognesi et al. Curr Biol 18:1624-1629, 2008b). Here we show that RNAi for the receptor Tc-arrow produced similar truncated phenotypes, providing additional evidence supporting canonical signal transduction. Furthermore, since in Tribolium segments are defined sequentially by a pair-rule gene circuit that, when interrupted, produces truncated phenotypes (Choe et al. Proc Natl Acad Sci U S A 103:6560-6564, 2006), we investigated the relationship between loss of Wnt signaling and this pair-rule gene circuit. After depletion of the receptor Tc-arrow, expression of Tc-Wnt1 was noticeably absent from the growth zone, while Tc-WntD/8 was restricted to a single spot of expression in what remained of the posterior growth zone. The primary pair-rule genes Tc-runt (Tc-run) and Tc-even-skipped (Tc-eve) were expressed normally in the anterior segments, but were reduced to a single spot in the remnants of the posterior growth zone. Thus, expression of pair-rule genes and Tc-WntD/8 are similarly affected by depletion of Wnt signal and disruption of the posterior growth zone.

Multiple Wnt genes are required for segmentation in the short-germ embryo of Tribolium castaneum.

wingless (wg)/Wnt family are essential to development in virtually all metazoans. In short-germ insects, including the red flour beetle (Tribolium castaneum), the segment-polarity function of wg is conserved [1]. Wnt signaling is also implicated in posterior patterning and germband elongation [2-4], but despite its expression in the posterior growth zone, Wnt1/wg alone is not responsible for these functions [1-3]. Tribolium contains additional Wnt family genes that are also expressed in the growth zone [5]. After depleting Tc-WntD/8 we found a small percentage of embryos lacking abdominal segments. Additional removal of Tc-Wnt1 significantly enhanced the penetrance of this phenotype. Seeking alternative methods to deplete Wnt signal, we performed RNAi with other components of the Wnt pathway including wntless (wls), porcupine (porc), and pangolin (pan). Tc-wls RNAi caused segmentation defects similar to Tc-Wnt1 RNAi, but not Tc-WntD/8 RNAi, indicating that Tc-WntD/8 function is Tc-wls independent. Depletion of Tc-porc and Tc-pan produced embryos resembling double Tc-Wnt1,Tc-WntD/8 RNAi embryos, suggesting that Tc-porc is essential for the function of both ligands, which signal through the canonical pathway. This is the first evidence of functional redundancy between Wnt ligands in posterior patterning in short-germ insects. This Wnt function appears to be conserved in other arthropods [6] and vertebrates [7-9].

Peritrophic membrane role in enhancing digestive efficiency. Theoretical and experimental models.

The peritrophic membrane (PM) is an anatomical structure surrounding the food bolus in most insects. Rejecting the idea that PM has evolved from coating mucus to play the same protective role as it, novel functions were proposed and experimentally tested. The theoretical principles underlying the digestive enzyme recycling mechanism were described and used to develop an algorithm to calculate enzyme distributions along the midgut and to infer secretory and absorptive sites. The activity of a Spodoptera frugiperda microvillar aminopeptidase decreases by 50% if placed in the presence of midgut contents. S. frugiperda trypsin preparations placed into dialysis bags in stirred and unstirred media have activities of 210 and 160%, respectively, over the activities of samples in a test tube. The ectoperitrophic fluid (EF) present in the midgut caeca of Rhynchosciara americana may be collected. If the enzymes restricted to this fluid are assayed in the presence of PM contents (PMC) their activities decrease by at least 58%. The lack of PM caused by calcofluor feeding impairs growth due to an increase in the metabolic cost associated with the conversion of food into body mass. This probably results from an increase in digestive enzyme excretion and useless homeostatic attempt to reestablish destroyed midgut gradients. The experimental models support the view that PM enhances digestive efficiency by: (a) prevention of non-specific binding of undigested material onto cell surface; (b) prevention of excretion by allowing enzyme recycling powered by an ectoperitrophic counterflux of fluid; (c) removal from inside PM of the oligomeric molecules that may inhibit the enzymes involved in initial digestion; (d) restriction of oligomer hydrolases to ectoperitrophic space (ECS) to avoid probable partial inhibition by non-dispersed undigested food. Finally, PM functions are discussed regarding insects feeding on any diet.

Tribolium Wnts: evidence for a larger repertoire in insects with overlapping expression patterns that suggest multiple redundant functions in embryogenesis.

Wingless (wg)/Wnt family genes encode secreted glycoproteins that function as signalling molecules in the development of vertebrates as well as invertebrates. In a survey of Wnt family genes in the newly sequenced Tribolium genome, we found a total of nine Wnt genes. In addition to wg or Wnt1, Tribolium contains orthologs of the vertebrate Wnt5-7 and Wnt9-11 genes. As in Drosophila, Wnt1, Wnt6 and Wnt10 are clustered in the genome. Comparative genomics indicates that Wnt9 is also a conserved member of this cluster in several insects for which genome sequence is available. One of the Tribolium Wnt genes appears to be a member of the WntA family, members of which have been identified in Anopheles and other invertebrates but not in Drosophila or vertebrates. Careful phylogenetic examination suggests an Apis Wnt gene, previously identified as a Wnt4 homolog, is also a member of the WntA family. The ninth Tribolium Wnt gene is related to the diverged Drosophila WntD gene, both of which phylogenetically group with Wnt8 genes. Some of the Tribolium Wnt genes display multiple overlapping expression patterns, suggesting that they may be functionally redundant in segmentation, brain, appendage and hindgut development. In contrast, the unique expression patterns of Wnt5, Wnt7 and Wnt11 in developing appendages likely indicate novel functions.

Sequences of cDNAs and expression of genes encoding chitin synthase and chitinase in the midgut of Spodoptera frugiperda.

The focus of this study was on the characterization and expression of genes encoding enzymes responsible for the synthesis and degradation of chitin, chitin synthase (SfCHSB) and chitinase (SfCHI), respectively, in the midgut of the fall armyworm, Spodoptera frugiperda. Sequences of cDNAs for SfCHSB and SfCHI were determined by amplification of overlapping PCR fragments and the expression patterns of these two genes were analyzed during insect development by RT-PCR. SfCHSB encodes a protein of 1523 amino acids containing several transmembrane segments, whereas SfCHI encodes a protein of 555 amino acids composed of a catalytic domain, a linker region and a chitin-binding domain. SfCHSB is expressed in the midgut during the feeding stages, whereas SfCHI is expressed during the wandering and pupal stages. Both genes are expressed along the whole midgut. Chitin staining revealed that this polysaccharide is present in the peritrophic membrane (PM) only when SfCHSB is expressed. There is little or no chitin in the midgut when SfCHI is expressed. These results support the hypothesis that SfCHSB is responsible for PM chitin synthesis during the larval feeding stages and SfCHI carries out PM chitin degradation during larval-pupal molting, suggesting mutually exclusive temporal patterns of expression of these genes.