Cucurbitacins and predation of the spotted cucumber beetle, Diabrotica undecimpunctata howardi

Two hypotheses about the ralationship of diabroticina beetles and plants in the family Cucurbitaceae are tested: (1) evolution of sensory receptors for cucurbitacins by diabroticina beetles was in part due to the predator protection offered by ingestion of these compounds, and (2) commercial varieties of cucurbitacin-producing cucumber offer Diabrotica undecimpunctata howardi Barber chemical protection from some potential sources of natural controlSpotted cucumber beetles fed either Marketmore 70 cucumber which contains cucurbitacin-C or Marketmore 72 which totally lacks cucurbitacin were presented to four species of vertebrate predators that commonly occur in the summer and/or winter habitats of D. u. howardi: Bufo americanus, B. fowleri, Peromyscus maniculatus, Colinus virginianus. None of the four species were significantly deterred from preying on beetles that had eaten Marketmore 70 cucumber. These results do not support either of the two hypotheses. The limitations of these negative results as evidence for refutation of the first hypothesis are discussed.

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Résumé Deux hypothèses concernant les relations entre les Diabroticina et les Cucurbitaceae ont été examinées: (1) l'évolution chez les Diabroticina des récepteurs pour les Cucurbitacines est due en partie à la protection contre les prédateurs apportée par la consommation de ces produits (2) les variétés modernes de concombres produisant de la cucurbitacine fournissent à D. undecimpunctata howardi Barber une protection chimique contre quelques sources potentielles de lutte biologique.Des D. undecimpunctata howardi, ayant consommé soit des concombres Marketmore 70 contenant de la cucurbitacine C, soit des concombres Marketmore 72 qui ont totalement perdu leur cucurbitacine, ont été présentés à quatre espèces de vertébrés prédateurs, qui s'observent fréquemment dans les habitats d'été ou d'hiver de D. undecimpunctata howardi soient Bufo americanus, B. fowleri, Peromyscus maniculatus, Colinus virginianus. Aucune de ces quatre espèces n'est significativement dissuadée d'attaquer les coléoptères qui on consommé les concombres Marketmore 70. Ces résultats n'étayent aucune des deux hypothèses. La discussion porte sur les limites de ces résultats négatifs comme preuve de la réfutation de l'hypothèse 1.

     

The early embryology of Diabrotica undecimpunctata howardi (coleoptera: Chrysomelidae)

Early embryogenesis is described for the southern corn rootworm, Diabrotica undecimpunctata Howardi Barber, at 24 ± 1°C. During the first four hours following oviposition, the maturation divisions and syngamy are completed. Morphological changes in the second polar body accompany syngamy. Cleavage divisions and energid migration occur during the fourth to the tenth hour. The vitellophags, which appear during cleavage divisions, are distinguished from the blastema-bound nuclei by having smaller, more densely staining nuclei. After completion of a uniform blastoderm (11-14 hour), cell division ceases until the completion of the germ band and the formation of the embryonic membranes (22 hour). This species has a pattern of amnion formation that is different from most Coleoptera but is shared with a few other chrysomelids, some Isoptera, and some Odonata.     

Effects of a potato cysteine proteinase inhibitor on midgut proteolytic enzyme activity and growth of the southern corn rootworm, Diabrotica undecimpunctata howardi (Coleoptera: Chrysomelidae)

The major proteinase activity in extracts of larval midguts from the southern corn rootworm (SCR), Diabrotica undecimpunctata howardi, was identified as a cysteine proteinase that prefers substrates containing an arginine residue in the P1 position. Gelatin-zymogram analysis of the midgut proteinases indicated that the artificial diet-fed SCR, corn root-fed SCR, and root-fed western corn rootworms (Diabrotica virgifera virgifera) possess a single major proteinase with an apparent molecular mass of 25kDa and several minor proteinases. Similar proteinase activity pH profiles were exhibited by root-fed and diet-fed rootworms with the optimal activity being slightly acidic. Rootworm larvae reared on corn roots exhibited significantly less caseinolytic activity than those reared on the artificial diet. Midgut proteolytic activity from SCR was most sensitive to inhibition by inhibitors of cysteine proteinases. Furthermore, rootworm proteinase activity was particularly sensitive to inhibition by a commercial protein preparation from potato tubers (PIN-II). One of the proteins, potato cysteine proteinase inhibitor-10', PCPI-10', obtained from PIN-II by ion-exchange chromatography, was the major source of inhibitory activity against rootworm proteinase activity. PCPI-10' and E-64 were of comparable potency as inhibitors of southern corn rootworm proteinase activity (IC(50) =31 and 35nM, respectively) and substantially more effective than chicken egg white cystatin (IC(50) =121nM). Incorporation of PCPI-10' into the diet of SCR larvae in feeding trials resulted in a significant increase in mortality and growth inhibition. We suggest that expression of inhibitors such as PCPI-10' by transgenic corn plants in the field is a potentially attractive method of host plant resistance to these Diabrotica species.     

Structure and formation of the peritrophic membrane in the larva of the southern corn rootworm, Diabrotica undecimpunctata

The peritrophic membrane (PM) in larvae of the southern corn rootworm Diabrotica undecimpunctata (Coleoptera:Chrysomelidae) forms along the full length of the midgut epithelium, defining D. undecimpunctata as a Type I insect with respect to PM formation. PM formation occurs in three phases: organization of a continuous lamella of matrix from material secreted into the interstices between the microvilli, maturation and apical movement of the lamella along the microvilli, and shedding of the lamella from the tips of the microvilli into the midgut lumen. Subsequent cycles of synthesis and shedding give rise to multiple, concentric lamellae which surround the food in the gut lumen. PM lamellae are 0.2 mum in profile width and consist of a core of bundles of 5 nm-diameter microfibers encased in a finely-granular homogeneous material. The microfiber bundles are arranged in an orthogonal grid-like array with dimensions consistent with formation around the microvilli. The homogeneous material separates from the PM lamellae to enclose food particles suggesting it may contain digestive enzymes. The PM, microvilli and intracellular vesicles in the midgut epithelium stain intensely with wheat germ agglutinin reflecting the presence and sites of secretion and synthesis of chitin.     

Gregarina coronata N. Sp. (Apicomplexa: Eugregarinida) Described from Adults of the Southern Corn Rootworm, Diabrotica undecimpunctata howardi (Coleoptera: Chrysomelidae)

ABSTRACT. Gregarina coronata n. sp. (Apicomplexa: Eugregarinida) is described from the adults of the Southern Corn Root Worm, Diabrotica undecimpunctata howardi (Coleoptera: Chrysomelidae). Measurements given are means, in micrometers, taken from mature gamonts in association. Primite: protomerite hemi-ellipsoidal with basal tumidus, length 47.6, width 44.0, with cytoplasmic granule, apical crown apparent; deutomerite elongate ellipsoidal, length 227.9, width 81.3; epimerite absorbed into anterior in gamont, globular in trophozoite. Satellite: protomerite hemi-ellipsoidal, truncated at association interface to appear trapezoidal, length 39.2, width 49.6, with cytoplasmic granule; deutomerite elongate ellipsoidal, length 240.6, width 80.2; epimerite absorbed into anterior in gamont. Association caudofrontal and often precocious, occurring during growth of trophozoites. Gametocysts spherical, 115.3 in diameter, 132.9 with hyaline coat; producing multiple oocyst chains under moist storage in 24–36 h. Oocysts very uniform in shape and size, dorsad: doliform, length 6.4, equatorial width 3.4, polar width 2.9; pleuron: dorso-ventrally flattened, corpus concave with bicondylic termina; corpus height 0.98, width 4.44; terminus height 1.96, width 0.98.     

Predicting the temperature-dependent natural population expansion of the western corn rootworm, Diabrotica virgifera

The western corn rootworm, Diabrotica virgifera virgifera Leconte (Coleoptera: Chrysomelidae), was accidentally introduced near Belgrade in Serbia just before 1992 and from there its expansion into Europe started. We have estimated its mean rate of expansion from 1992 to 2000 to be approximately 33 km year^?1, using data from the annual surveys of the European and Mediterranean Plant Protection Organisation. We investigated whether or not D. virgifera can establish itself at certain places in Europe, taking its temperature‐dependent development into account. We also estimated the time it will take D. virgifera to reach the Netherlands, considering only its dispersal by flight. All life stage transitions of D. virgifera were simulated with the program INSIM to assess whether or not it could establish itself in particular places. In the simulations, we used the available laboratory data on its life history characteristics, as well as a time series (2–6 years) of daily minimum and maximum temperatures from weather stations across Europe. The temperature‐dependent net reproduction resulting from the simulations showed that D. virgifera populations cannot establish themselves at latitudes above 55°N. The overall expected velocity of D. virgifera's range expansion was computed with van den Bosch et al.'s formula [ van den Bosch F, Hengeveld R & Metz JAJ (1992) Analysing the velocity of animal range expansion. Journal of Biogeography 19: 135–150], where the expansion velocity was based on dispersal characteristics and demographic parameters. We predicted that D. virgifera will not reach the Netherlands by flight from the border of its 2000 range before 2018 using this overall expected velocity. The explanation of this late arrival is that the velocity of population expansion decreases in all directions from the centre of its 2000 range due to unfavourable temperatures. Our analysis is an improvement on former analyses in that it uses temperature‐dependent life history characteristics. We stress, however, that the lack of knowledge on dispersal behaviour and on the values of life history characteristics in field situations might severely limit the applicability of the predicted velocity.     

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.     

Independent Action between DvSnf7 RNA and Cry3Bb1 Protein in Southern Corn Rootworm,Diabrotica undecimpunctata howardi and Colorado Potato Beetle,Leptinotarsa decemlineata

In recent years, corn rootworm (CRW)-resistant maize events producing two or more CRW-active Bt proteins have been commercialized to enhance efficacy against the target pest(s) by providing multiple modes of action (MoA). The maize hybrid MON 87411 has been developed that produces the CRW-active Cry3Bb1 Bt protein (hereafter Cry3Bb1) and expresses a RNAi-mediated MoA that also targets CRW. As part of an environmental risk assessment for MON 87411, the potential for an interaction between the CRW-active DvSnf7 RNA (hereafter DvSnf7) and Cry3Bb1 was assessed in 12-day diet incorporation bioassays with the southern corn rootworm (SCR, Diabrotica undecimpunctata howardi). The potential for an interaction between DvSnf7 and Cry3Bb1 was evaluated with two established experimental approaches. The first approach evaluated each substance alone and in combination over three different response levels. For all three response levels, observed responses were shown to be additive and not significantly different from predicted responses under the assumption of independent action. The second approach evaluated the potential for a fixed sub-lethal concentration of Cry3Bb1 to decrease the median lethal concentration (LC₅₀) of DvSnf7 and vice-versa. With this approach, the LC50 value of DvSnf7 was not altered by a sub-lethal concentration of Cry3Bb1 and vice-versa. In addition, the potential for an interaction between the Cry3Bb1 and DvSnf7 was tested with Colorado potato beetle (CPB, Leptinotarsa decemlineata), which is sensitive to Cry3Bb1 but not DvSnf7. CPB assays also demonstrated that DvSnf7 does not alter the activity of Cry3Bb1. The results from this study provide multiple lines of evidence that DvSnf7 and Cry3Bb1 produced in MON 87411 have independent action.     

Partial characterization of digestive tract proteinases from western corn rootworm larvae,Diabrotica virgifera

We have partially characterized the proteolytic activity within luminal contents of the digestive tracts of larvae of the western corn rootworm, Diabrotic virgifera LeConte. At least 15 proteinases were detected based on chromatographic behavior on ion exchange high-performance liquid chromatography and their mobility on sodium dodecyl sulfate gels containing gelatin. Inhibitors of proteolytic activity indicated that these enzymes are primarily sulfhydryl proteinases. Native polycrylamide gel electrophoresis revealed that a single proteinaceous inhibitor, egg white cystatin, was capable of abolishing a substantial part of the proteolytic acitivity. Our data suggest, accordingly, that gene transfer experiments utilizing the cystatin gene may generate lines of maize which have increased resistance to western corn rootworm larvae. © 1992 Wiley-Liss, Inc.     

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.     

Binary toxins from Bacillus thuringiensis active against the western corn rootworm, Diabrotica virgifera virgifera LeConte

The western corn rootworm, Diabrotica virgifera virgifera LeConte, is a significant pest of corn in the United States. The development of transgenic corn hybrids resistant to rootworm feeding damage depends on the identification of genes encoding insecticidal proteins toxic to rootworm larvae. In this study, a bioassay screen was used to identify several isolates of the bacterium Bacillus thuringiensis active against rootworm. These bacterial isolates each produce distinct crystal proteins with approximate molecular masses of 13 to 15 kDa and 44 kDa. Insect bioassays demonstrated that both protein classes are required for insecticidal activity against this rootworm species. The genes encoding these proteins are organized in apparent operons and are associated with other genes encoding crystal proteins of unknown function. The antirootworm proteins produced by B. thuringiensis strains EG5899 and EG9444 closely resemble previously described crystal proteins of the Cry34A and Cry35A classes. The antirootworm proteins produced by strain EG4851, designated Cry34Ba1 and Cry35Ba1, represent a new binary toxin. Genes encoding these proteins could become an important component of a sustainable resistance management strategy against this insect pest.     

Selection of entomopathogenic fungi for the control of the western corn rootworm Diabrotica virgifera virgifera

Abstract:  The western corn rootworm Diabrotica virgifera virgifera Le Conte (Col., Chrysomelidae), a serious pest of maize, has been recently introduced into Europe. Several approaches for its control are presently under investigation including microbial agents. During a field survey in Hungary in 2005, naturally occurring entomopathogenic fungi were found to attack this pest. These novel isolates together with standard isolates were tested for virulence against D. v. virgifera larvae and adults. Twenty strains of Metarhizium anisopliae , Beauveria bassiana and Beauveria brongniartii were used in bioassays in the laboratory. Larvae and adults were dipped into a spore suspension with a concentration of 1 × 10⁷ conidia (con.)/ml. They were kept for 14 days at 22°C (±2°C) and 70% relative humidity. The number of infected larvae and adults were counted and infection rates were calculated. Adults were significantly more susceptible to entomopathogenic fungi than larvae. The most virulent isolate infected about 47% of larvae ( M. anisopliae Ma2277), whereas the infection rate in adults was up to 97% ( M. anisopliae Ma2275). Isolates of M. anisopliae caused significantly higher mortalities than isolates of B. brongniartii and B. bassiana . Most of the adult beetles were killed within 12 days. Isolates from D. v. virgifera were more virulent than those from other hosts.     

Spatio-temporal dynamics of corn rootworm, Diabrotica spp., adults and their spatial association with environment

Multiyear and multilocation studies were conducted to investigate the within‐ and the between‐year spatial dynamics of corn rootworms Diabrotica spp. (Coleoptera: Chrysomelidae), adults and their spatial associations with environmental factors in cornfields. Grid‐based spatial sampling was conducted by trapping adults emerging from the soil (i.e., ‘emergence‐trap counts’) and by counting the number of adults in the ear zone of corn plant (i.e., ‘ear‐zone count’). Spatial analysis with distance indices (SADIE) was used to determine spatial distribution patterns and to investigate spatial associations. Ear‐zone counts and emergence‐trap counts were spatially dynamic within a year and more frequently aggregated in the middle of growing seasons and random early and late in the season. However, during the peak population periods, spatial distribution of ear‐zone counts and emergence‐trap counts were significantly consistent between years, indicating predictability of future spatial distributions. Spatial distribution of emergence‐trap counts and ear‐zone counts were positively associated with soil moisture and presence of corn plants with silks, respectively. This study demonstrated that within‐year spatial distribution patterns were dynamic and that there was between‐year spatial consistency of corn rootworm adult distributions. Such information can be used to improve preventative management of corn rootworms.     

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).     

Antixenosis in maize reduces feeding by western corn rootworm larvae (Coleoptera: Chrysomelidae)

SUM2162 is the first known example of a naturally occurring maize, Zea mays L., genotype with antixenosis (nonpreference) resistance to western corn rootworm, Diabrotica virgifera virgifera LeConte (Coleoptera: Chrysomelidae), larval feeding. Behavioral responses of neonate western corn rootworm larvae were evaluated in laboratory bioassays with seven maize genotypes selected for native resistance to rootworm feeding damage. Two susceptible maize genotypes and one transgenic (Bacillus thuringiensis) maize genotype were included as controls. In soil bioassays with cut roots, no larvae entered the roots of the resistant variety SUM2162, but at least 75% of the larvae entered the roots of every other maize type. Larvae made significantly fewer feeding holes in the roots of SUM2162 than in all the other maize genotypes, except the isoline control. In feeding bioassays, larval feeding varied significantly among maize genotypes, but there was no significant difference between the resistant varieties and the susceptible controls. There were no significant differences among any of the genotypes in host recognition (search) behavior of larvae after exposure to the roots. Little variation in feeding stimulant blends was observed among maize genotypes, indicating minimal contribution to the observed antixenosis.