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.     

Novel Bacillus thuringiensis Binary Insecticidal Crystal Proteins Active on Western Corn Rootworm, Diabrotica virgifera virgifera LeConte

A new family of insecticidal crystal proteins was discovered by screening sporulated Bacillus thuringiensis cultures for oral activity against western corn rootworm (WCR) larvae. B. thuringiensis isolates PS80JJ1, PS149B1, and PS167H2 have WCR insecticidal activity attributable to parasporal inclusion bodies containing proteins with molecular masses of ca. 14 and 44 kDa. The genes encoding these polypeptides reside in apparent operons, and the 14-kDa protein open reading frame (ORF) precedes the 44-kDa protein ORF. Mutagenesis of either gene in the apparent operons dramatically reduced insecticidal activity of the corresponding recombinant B. thuringiensis strain. Bioassays performed with separately expressed, biochemically purified 14- and 44-kDa polypeptides also demonstrated that both proteins are required for WCR mortality. Sequence comparisons with other known B. thuringiensis insecticidal proteins failed to reveal homology with previously described Cry, Cyt, or Vip proteins. However, there is evidence that the 44-kDa polypeptide and the 41.9- and 51.4-kDa binary dipteran insecticidal proteins from Bacillus sphaericus are evolutionarily related. The 14- and 44-kDa polypeptides from isolates PS80JJ1, PS149B1, and PS167H2 have been designated Cry34Aa1, Cry34Ab1, and Cry34Ac1, respectively, and the 44-kDa polypeptides from these isolates have been designated Cry35Aa1, Cry35Ab1, and Cry35Ac1, respectively.     

Secondary Contact and Admixture between Independently Invading Populations of the Western Corn Rootworm,Diabrotica virgifera virgifera in Europe

The western corn rootworm, Diabrotica virgifera virgifera (Coleoptera: Chrysomelidae), is one of the most destructive pests of corn in North America and is currently invading Europe. The two major invasive outbreaks of rootworm in Europe have occurred, in North-West Italy and in Central and South-Eastern Europe. These two outbreaks originated from independent introductions from North America. Secondary contact probably occurred in North Italy between these two outbreaks, in 2008. We used 13 microsatellite markers to conduct a population genetics study, to demonstrate that this geographic contact resulted in a zone of admixture in the Italian region of Veneto. We show that i) genetic variation is greater in the contact zone than in the parental outbreaks; ii) several signs of admixture were detected in some Venetian samples, in a Bayesian analysis of the population structure and in an approximate Bayesian computation analysis of historical scenarios and, finally, iii) allelic frequency clines were observed at microsatellite loci. The contact between the invasive outbreaks in North-West Italy and Central and South-Eastern Europe resulted in a zone of admixture, with particular characteristics. The evolutionary implications of the existence of a zone of admixture in Northern Italy and their possible impact on the invasion success of the western corn rootworm are discussed.     

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.     

Western corn rootworm (Diabrotica virgifera virgifera LeConte) population dynamics

1 The western corn rootworm Diabrotica virgifera virgifera LeConte is a major insect pest of field maize, Zea mays L. Larvae can cause substantial injury by feeding on maize roots. Larval feeding may destroy individual roots or root nodes, and reduce plant growth, stability, and yield. Costs associated with managing corn rootworms in continuous maize are annually one of the largest expenditures for insect management in the United States Corn Belt.
2 Even though D. virgifera virgifera has been studied intensively for over 50 years, there is renewed interest in the biology, ecology, and genetics of this species because of its ability to rapidly adapt to management tactics, and its aggressive invasive nature.
3 This article provides a comprehensive review of D. virgifera virgifera population dynamics, specifically: diapause, larval and adult development, seasonality, spatial and temporal dynamics at local and landscape scales, invasiveness in North America and Europe, and non-trophic interactions with other arthropods.
4 Gaps in current knowledge are identified and discussed especially within the context of challenges that scientists in North America and Europe are currently facing regarding pest dynamics and the need to develop appropriate management strategies for each geographic area.     

Validation of Reference Housekeeping Genes for Gene Expression Studies in Western Corn Rootworm (Diabrotica virgifera virgifera)

Quantitative Real-time PCR (qRT-PCR) is a powerful technique to investigate comparative gene expression. In general, normalization of results using a highly stable housekeeping gene (HKG) as an internal control is recommended and necessary. However, there are several reports suggesting that regulation of some HKGs is affected by different conditions. The western corn rootworm (WCR), Diabrotica virgifera virgifera LeConte (Coleoptera: Chrysomelidae), is a serious pest of corn in the United States and Europe. The expression profile of target genes related to insecticide exposure, resistance, and RNA interference has become an important experimental technique for study of western corn rootworms; however, lack of information on reliable HKGs under different conditions makes the interpretation of qRT-PCR results difficult. In this study, four distinct algorithms (Genorm, NormFinder, BestKeeper and delta-CT) and five candidate HKGs to genes of reference (β-actin; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; β-tubulin; RPS9, ribosomal protein S9; EF1a, elongation factor-1α) were evaluated to determine the most reliable HKG under different experimental conditions including exposure to dsRNA and Bt toxins and among different tissues and developmental stages. Although all the HKGs tested exhibited relatively stable expression among the different treatments, some differences were noted. Among the five candidate reference genes evaluated, β-actin exhibited highly stable expression among different life stages. RPS9 exhibited the most similar pattern of expression among dsRNA treatments, and both experiments indicated that EF1a was the second most stable gene. EF1a was also the most stable for Bt exposure and among different tissues. These results will enable researchers to use more accurate and reliable normalization of qRT-PCR data in WCR experiments.     

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.     

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.     

Western corn rootworm (Diabrotica virgifera virgifera LeConte) (Coleoptera: Chrysomelidae) oviposition can be released by decapitation

Nervous system control of western corn rootworm (Diabrotica virgifera virgifera LeConte, WCR) (Coleoptera: Chrysomelidae) oviposition was studied using decapitation. Gravid females (n = 364) were decapitated with scissors and floated in water-filled Petri dishes. Oviposition by individuals was observed at 30 min intervals for 4 hr after decapitation; cumulative oviposition was tallied at 48 hr post-treatment. Overall, 82.7% of females laid eggs within 48 hr. Oviposition commenced quickly; 78.4% of females laid eggs during the observation period, 81.8% of these began egg laying within 30 min of decapitation. Egg-laying females deposited a total of 66.8 ± 2.1 eggs (mean ± SEM); this was 85.1% of the total mature egg load. Dissection revealed that 31.9% of n = 301 laying females and 14.3% of 63 non-laying females had a rupture of the common oviduct, manifest as an egg-filled hernia containing a mean of 8.17 ± 1.3 eggs (range: 1–83). Among n = 237 females that laid eggs during observations, females with hernias laid significantly fewer eggs (35.8 ± 4.2) than intact females (48.0 ± 2.7) during the 4-hr interval. There was no difference in the mean proportion of hatch for eggs collected from the same n = 10 females before (0.88 ± 0.03) or after decapitation (0.84 ± 0.04). Rapid oviposition following decapitation suggests that WCR egg laying is under constant descending neural inhibition; the motor programme controlling egg laying must reside posterior to the head. Decapitation can be used to quickly collect mature, fertilized WCR eggs.     

Behaviour and ecology of the western corn rootworm (Diabrotica virgifera virgifera LeConte)

1 The western corn rootworm (WCR) is a historic pest with a legacy of resistance and behavioural plasticity. Its behaviour and nutritional ecology are important to rootworm management. The success of the most effective and environmentally benign rootworm management method, annual crop rotation, was based on an understanding of rootworm behaviour and host–plant relationships. Enthusiastic adoption of crop rotation, provided excellent rootworm management, but also selected for behavioural resistance to this cultural control.
2 Though well-studied, significant gaps in WCR biology remain. Understanding the topics reviewed here (mating behaviour, nutritional ecology, larval and adult movement, oviposition, alternate host use, and chemical ecology) is a starting point for adapting integrated pest management and insect resistance management (IRM) to an expanding WCR threat. A presentation of significant questions and areas in need of further study follow each topic.
3 The expansion of WCR populations into Europe exposes this pest to new environmental and regulatory conditions that may influence its behaviour and ecology. Reviewing the state of current knowledge provides a starting point of reference for researchers and pest management decision-makers in North America and Europe.
4 The trend toward increasing adoption of transgenic maize will place an increasing premium on understanding WCR behaviour. IRM plans designed to promote sustainable deployment of transgenic hybrids are grounded on assumptions about WCR movement, mating and ovipositional behaviour. Preserving the utility of new and old management options will continue to depend on a thorough understanding of WCR biology, even as the ecological circumstances and geography of WCR problems become more complex.     

Field-Cage Studies of Beauveria bassiana (Hyphomycetes: Moniliaceae) for the Suppression of Adult Western Corn Rootworm, Diabrotica virgifera virgifera (Coleoptera: Chrysomelidae)

The efficacy of the entomopathogenic fungus Beauveria bassiana (Balsamo) Vuillemin, was tested as a control agent for adult western corn rootworm, Diabrotica virgifera virgifera LeConte, in walk-in field cages. Suspensions of B. bassiana conidia were applied to corn plants in cages into which laboratory-reared beetles had been released. Beetles were collected at 3 and 5 days post-application and evaluated in the laboratory for mortality. Mortality was 10, 29 and 50%, at rate equivalents of 7 X 10 12 , 2 X 10 13 (two applications), and 5 X 10 13 conidia/ ha, respectively. There was no significant difference in mortality of beetles collected at 3 days compared with 5 days post-application. Mortality due to B. bassiana was 24% when beetles were released into field cages 24 h post-application (5 X 10 13 conidia/ha) compared with 50% when beetles were present during the application. Beetle mortality declined significantly with increasing time from application in feeding assays carried out with leaf samples removed from plants at 0, 12, 24 and 72 h post-application. Mortality of beetles collected from treated plants within cages and maintained in the laboratory was found to overestimate the population decline by 10% when compared with beetle estimates from treated plants within field cages.     

Morphological integration and modularity in Diabrotica virgifera virgifera LeConte (Coleoptera: Chrysomelidae) hind wings

The morphological integration of the hind wings of the western corn rootworm Diabrotica virgifera virgifera LeConte was investigated to get a better insight of the undergone by this invasive species. Geometric morphometric methods were used to test two modularity hypotheses associated with the wing development and function (hypothesis H1: anterior/posterior or H2: distal/proximal wing parts). Both hypotheses were rejected and the results showed the integrated behavior of the hind wings of D. v. virgifera. The hypothesized modules do not represent separate units of variation, so in a similar fashion as exhibited by the model species Drosophila melanogaster, the hind wings of D. v. virgifera act as a single functional unit. The moderate covariation strength found between anterior and posterior and distal and proximal parts of the hind wing of D. v. virgifera confirms its integrated behavior. We conclude that the wing shape shows internal integration, which could enable flexibility and thus enhance flight maneuverability. This study contributes to the understanding of morphological integration and modularity on a non-model organism. Additionally, these findings lay the groundwork for future flight performance and biogeographical studies on how wing shape and size vary across the endemic and expanded/invaded range in the USA and Europe infested with D. v. virgifera.     

Greenhouse-Selected Resistance to Cry3Bb1-Producing Corn in Three Western Corn Rootworm Populations

Transgenic corn producing the Bacillus thuringiensis (Bt) toxin Cry3Bb1 has been useful for controlling western corn rootworm, Diabrotica virgifera virgifera LeConte, one of the most economically important crop pests in the United States. However, rapid evolution of resistance by this beetle to Bt corn producing Cry3Bb1 has been reported previously from the laboratory, greenhouse, and field. Here we selected in the greenhouse for resistance to Cry3Bb1 corn in three colonies of WCR derived from Kansas, Minnesota, and Wisconsin, respectively. Three generations of rearing on Cry3Bb1 corn significantly increased larval survival on Cry3Bb1 corn, resulting in similar survival in the greenhouse for selected colonies on Cry3Bb1 corn and isoline corn that does not produce Bt toxin. After four to seven generations of rearing on Cry3Bb1 corn, survival in the field on Cry3Bb1 corn relative to isoline corn more than doubled for selected colonies (72%) compared with control colonies (33%). For both selected and control colonies, survival in the field was significantly lower on Cry3Bb1 corn than on isoline corn. On isoline corn, most fitness components were similar for selected colonies and control colonies. However, fecundity was significantly lower for selected colonies than control colonies, indicating a fitness cost associated with resistance. The rapid evolution of resistance by western corn rootworm to Bt corn reported here and previously underlines the importance of effective resistance management for this pest.     

A review of resistance breeding options targeting western corn rootworm (Diabrotica virgifera virgifera LeConte)

1 This review presents the latest research regarding maize resistance breeding against western corn rootworm (WCR) in the U.S.A. and Europe.
2 Investigations in Europe on the development of maize cultivars possessing resistant mechanisms against WCR are just beginning. In 2003, the European Commission implemented measures aimed at slowing down the spread of the WCR in Europe. Nevertheless, this pest has already been found in 20 countries of the European region. To establish a sustainable production system, the evaluation of native (nontransgenic) resistance in maize cultivars is essential.
3 This review emphasizes the future challenges involved in the research of native resistance breeding in maize against the insect.     

Quick scouting of eggs of western corn rootworm (Diabrotica virgifera virgifera LeConte, 1868) from soil

Main method to control the American Corn Rootworm is crop rotation (Camprag et. al., 1994) but we don't know how to determine the possible number of larvae under fall so we cannot use autumn cereals to change the row of cultivated plants. The pest spends almost 10 months in soil in egg and larval state (Chiang, 1973). There are two methods for scouting Diabrotica eggs and larval instars from soil over the winter. One of the two most important methods is holding soil samples on fixed temperature (Fromm et al., 1999). This method takes more than one and a half month but its result is highly reliable. The conventional egg-washing technique takes fewer days to count the number of Diabrotica eggs in soil but it has lower effectiveness than the other one because the eggs in a sample cannot be counted correctly. Our results show that the effectiveness of egg washing with high concentrated salty water (NaCl) is high and the method is quick enough to help planning the crop rotation even under the autumn period (Takács et al., 2004).