Purification and kinetic analysis of acetylcholinesterase from western corn rootworm,Diabrotica virgifera virgifera (Coleoptera: Chrysomelidae)

Acetylcholinesterase (AchE, EC 3.1.1.7) was purified from western corn rootworm (WCR, Diabrotica virgifera virgifera) beetles by affinity chromatography. The purification factor reached over 20,000-fold with a specific activity of 169.5 μmol/min/mg and a yield of 23%. The V_max values for hydrolyzing acetylthiocholine (ATC), acetyl-(β-methyl)thiocholine (AβMTC), propionylthiocholine (PTC), and S-butyrylthiocholine (BTC) were 184.8, 140.5, 150.2, and 18.8 μmol/min/mg, respectively, and K_m values were 19.7, 18.5, 14.1, and 11.0 μM, respectively. The first three substrates showed significant inhibition to the AchE at higher concentrations, whereas BTC showed inhibition at the concentrations of 0.25–2 mM but activation at >4 mM. AchE activity was almost completely inhibited by 1 μM eserine and BW284C15, respectively, but only 12% of AchE activity were inhibited by ethopropazine at the same concentration. These results suggested that the purified AchE from WCR was a typical insect AchE. Insecticides or their oxidative metabolites, chlorpyrifos-methyl oxon, carbofuran, carbaryl, malaoxon, and paraoxon, used in in vitro kinetic study exhibited high inhibition to AchE purified from WCR. However, chlorpyrifos-methyl oxon and carbofuran showed at least 36- and 4-fold, respectively, higher inhibitory potency than the remaining insecticides examined. Results from our in vitro inhibition of AchE agreed quite well with the previously published in vivo bioassay data. Arch. Insect Biochem. Physiol. 39:118–125, 1998. © 1998 Wiley-Liss, Inc.     

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.     

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.     

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.     

Impact of cysteine proteinase inhibition in midgut fluid and oral secretion on fecundity and pollen consumption of western corn rootworm (Diabrotica virgifera virgifera)

Cysteine proteinases predominate in the midgut fluid (MF) and oral secretion (OS) of adult western corn rootworm (WCR) based on their mild acidic pH optima (pH 6.0), enhanced activities after treatment with thiol reducing agents, and inhibition by selective cysteine proteinase inhibitors (PIs). Four cysteine PIs including E-64, calpeptin, calpain inhibitor II, and leupeptin (also a serine PI) strongly inhibited azocaseinolytic activity in a dose-dependent manner in both the MF and OS. The most significant effect on adult female WCR of cysteine PI consumption with corn pollen was the reduction in fecundity, but female survival was not apparently affected. Mean fresh weights for all PI-fed females were also lower than control groups. All PI-fed groups [E-64, calpain inhibitor I (Cal I) and leupeptin] had a significantly lower daily egg production than respective corn pollen-fed controls. E-64 was more potent than leupeptin and Cal I on inhibiting fecundity, which correlates with their relative anti-proteinase potency in vitro. E-64, Cal I, and leupeptin at 1.5-2 nmol/beetle/day reduced fecundity down to 25-45% of control values. Reduced egg production by PI-fed beetles results from a combination of the direct inhibition of protein digestion and a post-ingestive negative feedback mechanism, which reduces food intake. The supplement of ten essential amino acids into the E-64-treated pollen enhanced up to 3.7-fold the number of eggs laid compared to the E-64-fed group without these amino acids, suggesting that egg production is dependent on the supply of essential amino acids from corn pollen proteolysis.     

Polymorphic microsatellite loci from the western corn rootworm (Insecta: Coleoptera: Chrysomelidae) and cross‐amplification with other Diabrotica spp

Corn rootworms (Diabrotica spp.) make up the major insect pest complex of corn in the US and Europe, and there is a need for molecular markers for genetics studies. We used an enrichment strategy to develop microsatellite markers from the western corn rootworm (Diabrotica virgifera virgifera). Of 54 loci isolated, 25 were polymorphic, and of these, 17 were surveyed for variability in 59 wild individuals. In addition, the potential for cross‐amplification of these microsatellites was surveyed for Mexican, northern, and southern corn rootworms. Nine microsatellite loci showed Mendelian inheritance and are likely to be useful in population genetics studies.     

Predator responses to novel haemolymph defences of western corn rootworm (Diabrotica virgifera) larvae

Many herbivorous arthropods use defensive chemistry to discourage predators from attacking. This chemistry relies on the ability of predators to rapidly learn to recognize and avoid offensive stimuli. Western corn rootworm (WCR), Diabrotica virgifera virgifera LeConte (Coleoptera: Chrysomelidae), employs multifaceted chemical defences in its haemolymph, which may contribute significantly to its success as a major economic pest. Here, we test the hypothesis that agrobiont predators can rapidly learn to recognize and avoid WCR larvae, and will thereby reduce their contribution to WCR suppression. In controlled feeding assays, the effectiveness of WCR haemolymph defences varied across three predator taxa (crickets, centipedes, and ants). Centipedes (Chilopoda: Lithobiidae) were minimally affected by WCR defences, but crickets [Gryllus pennsylvanicus Burmeister (Orthoptera: Gryllidae)] spent less time feeding on WCR than on an undefended control prey, house fly maggots. However, we uncovered no evidence indicating that experienced crickets rapidly learn to avoid WCR larvae, indicating that haemolymph defences offer few, if any, survival benefits for WCR. Colonies of ants [Lasius neoniger Emery (Hymenoptera: Formicidae)] switched from low worker participation in initial attacks on WCR to higher worker participation in subsequent attacks, indicating an attempt to overcome, rather than avoid, WCR haemolymph defences. These results suggest that a diverse assemblage of natural enemies will show a diverse array of behavioural responses to toxic pest prey, and highlight the importance of behavioural diversity in driving the function of natural enemy assemblages.     

Tracking predation of subterranean pests: digestion of corn rootworm DNA by a generalist mite

qPCR is a useful tool for understanding predator–prey relationships. We investigated rootworm DNA digestion by male and female predatory mites. Males and females initially consumed comparable amounts of DNA, which was digested at similar rates. Field-collected mites need to be preserved quickly for best qPCR results.     

Performance of a model for egg hatching of the western corn rootworm,Diabrotica virgifera virgifera LeConte,using measured and modelled soil temperatures as input

A model for egg hatching of the western corn rootworm,Diabrotica virgifera virgifera LeConte, was tested at several locations in Ontario, Canada, during the 1989 and 1990 seasons. The model required soil temperatures as input and was tested using measured and modelled data. Modelled soil temperatures at 5 and 10 cm depths were obtained from empirically and physically based models. The physically based model provided better estimates of soil temperatues, but both models slightly underestimated the temperatures. Predicted egg hatching, using measured and modelled soil temperature at 5 and 10 cm depths for all locations, compared reasonably well with the observations of egg hatching. When using modelled soil temperatures, the egg developmental model performed better using soil temperatures from the physically based model. However, both soil temperature models provided sufficiently accurate temperature values for use in the egg developmental model. Unlike the empirically based model, the physically based model was not site-specific and its application to larger areas appeared feasible.     

Development and economic evaluation of spatial sampling plans for corn rootworm Diabrotica spp. (Col., Chrysomelidae) adults

Abstract  To develop spatial sampling plans for corn rootworm ( Diabrotica spp.) adults, their spatial distributions were characterized and economics of sampling plans were evaluated by comparing sampling costs between spatial and conventional (non-spatial) sampling plans. Semivariogram modelling and spatial by with distance indices showed that corn rootworm adults were significantly (P < 0.05) aggregated at peak population densities and any two samples were spatially correlated within approximately 45 m, with 39–90% of the variability explained by spatial dependence. Sampling costs for spatial sampling plans linearly increased as the sampling distance decreased and exponentially increased as the field size increased. Although sampling costs for non-spatial sampling plans were generally lower, spatial sampling plans could be more economical when the mean insect density became lower and the field size became smaller. This study demonstrated that spatial sampling plans could be optimized to minimize the sampling costs and maximize the spatial resolution.     

Evolutionary directional asymmetry and shape variation in Diabrotica virgifera virgifera (Coleoptera: Chrysomelidae): an example using hind wings

The western corn rootworm Diabrotica virgifera virgifera LeConte is a pest of maize in the USA and Europe and especially a problem in particular regions of Croatia. In the present study, patterns of variation in hind wing shape were examined. The first objective was to examine the influence of soil type on 10 populations of D. v. virgifera sampled from three regions in Croatia that differed according to edaphic factors and climate. The second objective was to investigate the potential evolutionary presence of directional asymmetry on hind wings. Geometric morphometrics was used to examine these objectives by quantifying the morphological variation within and among individuals and populations. Overall, D. v. virgifera hind wing shape changed according to major soil type classifications in Croatia. The three hind wing morphotypes found varied because of basal radial vein differences, related to landmarks 1, 3, 7, and 14. The findings of the present study show that hind wing shape in D. v. virgifera can be used to differentiate populations based on edaphic factors and may have application as a monitoring tool in the integrated management of D. v. virgifera. In an evolutionary context, the presence of directional asymmetry in the hind wings of D. v. virgifera adds to the ever growing data on the evolution of insect wings. © 2013 The Linnean Society of London, Biological Journal of the Linnean Society, 2014, 111 , 110–118.     

Polymorphic microsatellite loci from northern and Mexican corn rootworms (Insecta: Coleoptera: Chrysomelidae) and cross-amplification with other Diabrotica spp

The northern corn rootworm (Diabrotica barberi) and Mexican corn rootworm (Diabrotica virgifera zeae) are significant agricultural pests. For the northern corn rootworm, and to a lesser extent, the Mexican corn rootworm, high resolution molecular markers are needed. Here we present 14 polymorphic microsatellite loci isolated from libraries constructed using pooled northern and Mexican corn rootworm genomic DNA. Polymorphism in other Diabrotica, including the banded cucumber beetle, southern corn rootworm and western corn rootworm, is described.     

Screening of entomopathogenic nematodes for virulence against the invasive western corn rootworm, Diabrotica virgifera virgifera (Coleoptera: Chrysomelidae) in Europe

Entomopathogenic nematode species available in Europe were screened for their efficacy against both the root-feeding larvae and silk-feeding adults of the western corn rootworm, Diabrotica virgifera virgifera LeConte. Laboratory screening tests were aimed at the selection of candidate biological control agents for the management of this invasive alien pest in Europe. Steinernema glaseri, S. arenarium, S. abassi, S. bicornutum, S. feltiae, S. kraussei, S. carpocapsae and Heterorhabditis bacteriophora were studied to determine their virulence against third instar larvae and adults of D. v. virgifera in small-volume arenas (using nematode concentrations of 0.5, 0.8, 7.9 and 15.9 infective juveniles cm-2). All nematode species were able to invade and propagate in D. v. virgifera larvae, but adults were rarely infected. At concentrations of 7.9 and 15.9 cm-2, S. glaseri, S. arenarium, S. abassi and H. bacteriophora caused the highest larval mortality of up to 77%. Steinernema bicornutum, S. abassi, S. carpocapsae and H. bacteriophora appeared to have a high propagation level, producing 5970+/-779, 5595+/-811, 5341+/-1177 and 4039+/-1025 infective juveniles per larva, respectively. Steinernema glaseri, S. arenarium, S. feltiae, S. kraussei and H. bacteriophora were further screened at a concentration of 16.7 nematodes cm-2 against third instar larvae in medium-volume arenas (sand-filled trays with maize plants). Heterorhabditis bacteriophora, S. arenarium and S. feltiae caused the highest larval mortality with 77+/-16.6%, 67+/-3.5%, and 57+/-17.1%, respectively. In a next step, criteria for rating the entomopathogenic nematode species were applied based on results obtained for virulence and propagation, and for current production costs and availability in Europe. These criteria were then rated to determine the potential of the nematodes for further field testing. Results showed the highest potential in H. bacteriophora, followed by S. arenarium and S. feltiae, for further testing as candidate biological control agents.     

Toxicity and neurophysiological effects of fipronil and fipronil sulfone on the western corn rootworm (Coleoptera: Chrysomelidae)

Fipronil is a member of the relatively new phenylpyrazole insecticide class that is active at the neuro‐inhibitory gamma amino butyric acid (GABA)‐gated chloride channel/ionopore complex. The toxicity and neurophysiological effects of fipronil and its oxidative sulfone metabolite [5‐amino‐1‐(2,6‐dichloro‐4‐trifluoromethylphenyl)‐3‐cyano‐4‐trifluoromethylsulfonylpyrazole] were investigated by using an insecticide‐susceptible western corn rootworm population. In topical bioassays using adult rootworms, fipronil was toxic at very low doses (LD₅₀ = 0.07; LD₉₀ = 0.33 ng/mg). At the LD₉₀, pre‐treatment with the cytochrome P450 monooxygenase inhibitor piperonyl butoxide led to mild antagonism of fipronil toxicity (LD₉₀ = 0.42 ng/mg), while the sulfone analog had greater toxicity (LD₉₀ = 0.22 ng/mg). In neurophysiological studies of spontaneous electrical activity, adult and larval rootworms were equally affected by fipronil and the sulfone analog at 10 μM (in the presence of 5 mM GABA) in comparison to GABA‐treated baselines. Using larval rootworms, insensitivity of the GABA receptor to binding by picrotoxinin or dieldrin (10 μM ) was not apparent in the presence of 5 mM GABA. Further neurophysiological investigation using a range of concentrations (0.625–20.0 μM) on larval rootworms indicated concentration‐dependent effects on bursting activity for both fipronil and the sulfone analog; however, subtle differences were observed between these two compounds. Results indicate that both fipronil and its oxidative sulfone metabolite have similar toxicological and neurological effects on rootworms. Arch. Insect Biochem. Physiol. 40:150–156, 1999. © 1999 Wiley‐Liss, Inc.     

Characterization of western corn rootworm (Coleoptera: Chrysomelidae) population dynamics in relation to landscape attributes

Abstract 1 The western corn rootworm (WCR), Diabrotica virgifera virgifera Leconte (Coleoptera: Chrysomelidae), creates economic and environmental concerns in the Corn Belt region of the U.S.A. To supplement the population control tactics of the Areawide Pest Management Program in Brookings, South Dakota, Geographical Information Systems (GIS) were used to examine the spatial relationships from 1997 to 2001 between WCR population dynamics, habitat structure, soil texture and elevation. 2 Using the inverse distance weighted interpolation technique, WCR population density maps were created from georeferenced emergence and post‐emergence traps placed in maize fields. For each year, these maps were overlaid with vegetation, soil and elevation maps to search for quantitative relationships. 3 Through visual interpretation and correlation analysis, shifts in landscape structure, such as size, number and arrangement of patches, were shown to associate with WCR population abundance and distribution in varying degrees. Contingency analysis showed that WCR population abundance is associated with soil texture and elevation. 4 An understanding of the interactions between WCR population dynamics and landscape variables provides information to pest managers, and this can be used to identify patterns in the landscape that promote high insect population density patches to improve pest management strategies.     

Toxic and behavioural effects of free fatty acids on western corn rootworm (Coleoptera: Chrysomelidae) larvae

Feeding behaviour, feeding intensity and staying behaviour of neonate western corn rootworm (Diabrotica virgifera virgifera LeConte) larvae were evaluated in response to synthetic feeding stimulant blends. All of the treatments contained a 3‐sugar blend (glucose : fructose : sucrose, 30 : 4 : 4 mg/ml) and one of twelve free fatty acids. Each free fatty acid was tested in this blend at three different concentrations. The addition of the 12 : 0, 16 : 0, 16 : 1, 18 : 0, 18 : 1, 18 : 2 and 18 : 3 free fatty acids to the sugar blend significantly (P < 0.05) increased the percentage of larvae feeding, but did not increase food consumption per larva. Most of the free fatty acids elicited staying behaviour. At the lowest dose (0.1 mg/ml), all of the free fatty acids except the 18 : 0 and the 20 : 0 elicited staying by significantly more larvae than the sugar blend, and at the highest dose (1.0 mg/ml), eight free fatty acids (8 : 0, 10 : 0, 12 : 0, 14 : 0, 16 : 1, 18 : 1, 18 : 2 and 18 : 3) caused more larvae to stay compared to the sugar blend. Larvae were visibly impaired after exposure to some of the free fatty acids. At the highest dose, the 8 : 0, 10 : 0, 12 : 0, 14 : 0, 16 : 1, 18 : 1 and 18 : 2 free fatty acids were toxic to the larvae. At least 60% of larvae were impaired after exposure to the 12 : 0, 16 : 1 and 18 : 2 free fatty acids and the 8 : 0 and 10 : 0 free fatty acids caused 100% impairment or death. Synthetic blends were compared with liquid from crushed maize roots and with a methanol extract of maize roots. Feeding intensity and staying behaviour on the root liquid and the root extract were significantly greater than on any of the synthetic blends, suggesting the presence of additional compounds in maize roots that serve as feeding cues for western corn rootworm larvae.