Bruce E. Hibbard

Direct and indirect plant defenses are not suppressed by endosymbionts of a specialist root herbivore.

Insect endosymbionts influence many important metabolic and developmental processes of their host. It has been speculated that they may also help to manipulate and suppress plant defenses to the benefit of herbivores. Recently, endosymbionts of the root herbivore Diabrotica virgifera virgifera have been reported to suppress the induction of defensive transcripts in maize roots, which may explain the finding of another study that once attacked plants become more susceptible to subsequent D. v. virgifera attack. To test this hypothesis, we cured D. v. virgifera from its major endosymbiont Wolbachia and tested whether endosymbiont-free individuals elicit different defense responses in maize roots. The presence of Wolbachia did not alter the induction of defense marker genes and resistance in a susceptible maize hybrid and a resistant line. Furthermore, attacked maize plants emitted the same amount of (E)-β-caryophyllene, a volatile signal that serves as foraging cue for both entomopathogenic nematodes and D. v. virgifera. Finally, the effectiveness of the entomopathogenic nematode Heterorhabditis bacteriophora to infest D. v. virgifera was not changed by curing the larvae from their endosymbionts. These results show that the defense mechanisms of maize are not affected by Wolbachia. Consequently, D. v. virgifera does not seem to derive any plant-defense mediated benefits from its major endosymbiont.

Evaluation of Conventional Resistance to European Corn Borer (Lepidoptera: Crambidae) and Western Corn Rootworm (Coleoptera: Chrysomelidae) in Experimental Maize Lines Developed from a Backcross Breeding Program

Abstract Plant resistance is a promising control method for the two mostdamaging insect pests of maize, Zea mays L.: the Europeancorn borer, Ostrinia nubilalis (Hübner), and thewestern corn rootworm, Diabrotica virgifera virgiferaLeConte. Fifteen experimental lines of maize, derived from a backcrossbreeding program designed to introgress resistance to European cornborer from Peruvian maize into two U.S. Corn Belt adapted inbred lines,were evaluated for resistance to European corn borer and western cornrootworm. The experimental lines were in the second generation ofbackcrossing. All experimental lines were resistant to leaf bladefeeding by European corn borer. These lines had low levels of2,4-dihydroxy-7-methoxy-1,4-benzoxazin-3-one, a chemical commonlyassociated with leaf blade feeding resistance, indicating that this wasnot the mechanism of resistance to leaf blade feeding in these lines.Eleven experimental lines were resistant to leaf sheath and collarfeeding by European corn borer. Useful sources of European corn borerovipositional nonpreference and root feeding resistance to western cornrootworm were not identified. Some of the lines evaluated in this studymay provide useful sources of resistance to both leaf blade and leafsheath and collar feeding by European corn borer.

A new artificial diet for western corn rootworm larvae is compatible with and detects resistance to all current Bt toxins

Insect resistance to transgenic crops is a growing concern for farmers, regulatory agencies, the seed industry, and researchers. Since 2009, instances of field-evolved Bt resistance or cross resistance have been documented for each of the four Bt proteins available for western corn rootworm (WCR), a major insect pest. To characterize resistance, WCR populations causing unexpected damage to Bt maize are evaluated in plant and/or diet toxicity assays. Currently, it is not possible to make direct comparisons of data from different Bt proteins due to differing proprietary artificial diets. Our group has developed a new, publicly available diet (WCRMO-1) with improved nutrition for WCR larvae. For the current manuscript, we tested the compatibility of all Bt proteins currently marketed for WCR on the WCRMO-1 diet and specific proprietary diets corresponding to each toxin using a susceptible colony of WCR. We also tested WCR colonies selected for resistance to each protein to assess the ability of the diet toxicity assay to detect Bt resistance. The WCRMO-1 diet is compatible with each of the proteins and can differentiate resistant colonies from susceptible colonies for each protein. Our diet allows researchers to monitor resistance without the confounding nutritional differences present between diets.

Diet improvement for western corn rootworm (Coleoptera: Chrysomelidae) larvae

The western corn rootworm (WCR), Diabrotica virgifera virgifera LeConte, is the most serious insect pest of corn (Zea mays L.) in the United States and parts of Europe, and arguably one of the world’s most expensive pests to control. Several diet formulations are currently used by industry and public researchers to evaluate WCR larvae in diet-toxicity bioassays. However, a publicly available diet that produces normative insects that are physiologically similar to WCR larvae reared on corn roots will accelerate development of management technologies. We report a new diet formulation that supports improved weight gain, larval development and survival compared with the only public diet for WCR that is currently available in the refereed literature. The formulation was created by using response surface methods combined with n-dimensional mixture designs to identify and improve the formulation of key ingredients. Weight gain increased two-fold, and survival and molting rates increased from 93% and 90%, respectively when reared on the public diet, to approximately 99% for both survival and molting at 11 days when reared on our new formulation. This new formulation provides a standardized growth medium for WCR larvae that will facilitate comparison of research results from various working groups and compliance with regulatory requirements.

Development and Characterization of MIR604 Resistance in a Western Corn Rootworm Population (Coleoptera: Chrysomelidae)

Abstract mCry3A is one of only four proteins licensed for commercial use in Diabrotica control. Utilizing a colony of western corn rootworm, Diabrotica virgifera virgifera LeConte, selected for resistance to mCry3A, we evaluated how mCry3A resistance was inherited and whether fitness costs were associated with mCry3A resistance. Reciprocal crosses between a selected colony and a control colony were performed; resulting progeny along with parent colonies were evaluated in dose toxicity assays, greenhouse assays, and seedling assays. Dose toxicity assay results were inconclusive, as the highest dose of protein tested did not produce sufficient mortality for accurate LC50 calculation. In whole-plant greenhouse assays on mCry3A-expressing corn, larval relative survival of the selected female × control male reciprocal cross was similar to that of the selected colony, while that of the control female × selected male was intermediate between the mCry3-selected colony and the control colony. However, when adult relative survival in whole-plant greenhouse assays was examined, no significant difference between the reciprocal crosses and the two parent colonies was detected. Heritability calculations based on both larval (0.66) and adult (1.03) survival data indicate that resistance to mCry3A is not inherited in a recessive manner. The selected colony was removed from selection pressure and evaluated after three or eight generations of removal. At three generations of removal from selection, a slight decrease in larval relative survival was detected compared with the selected colony. At eight generations of removal from selection, larval relative survival was comparable with that of the selected colony.

Survey of bacteria associated with western corn rootworm life stages reveals no difference between insects reared in different soils

Western corn rootworm (Diabrotica virgifera virgifera LeConte) is a serious pest of maize (Zea mays L.) in North America and parts of Europe. With most of its life cycle spent in the soil feeding on maize root tissues, this insect is likely to encounter and interact with a wide range of soil and rhizosphere microbes. Our knowledge of the role of microbes in pest management and plant health remains incomplete. An important component of an effective pest management strategy is to know which microorganisms are present that could play a role in life history or management. For this study, insects were reared in soils from different locations. Insects were sampled at each life stage to determine the possible core bacteriome. Additionally, soil was sampled at each life stage and resulting bacteria were identified to determine the contribution of soil to the rootworm bacteriome, if any. We analyzed the V4 hypervariable region of bacterial 16S rRNA genes with Illumina MiSeq to survey the different species of bacteria associated with the insects and the soils. The bacterial community associated with insects was significantly different from that in the soil. Some differences appear to exist between insects from non-diapausing and diapausing colonies while no significant differences in community composition existed between the insects reared on different soils. Despite differences in the bacteria present in immature stages and in male and female adults, there is a possible core bacteriome of approximately 16 operational taxonomic units (i.e., present across all life stages). This research may give insights into how resistance to Bt develops, improved nutrition in artificial rearing systems, and new management strategies.

Author Correction: A new artificial diet for western corn rootworm larvae is compatible with and detects resistance to all current Bt toxins

A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has not been fixed in the paper.

Comparison of Six Artificial Diets for Western Corn Rootworm Bioassays and Rearing

The western corn rootworm, Diabrotica virgifera virgifera LeConte (Coleoptera: Chrysomelidae), is considered the most important maize (Zea mays L.) pest in the U.S. Corn Belt. Bioassays testing susceptibility to Bacillus thuringiensis Berliner (Bt) and other toxins of corn rootworm larvae often rely on artificial diet formulations. Successful bioassays on artificial diet for corn rootworm have sometimes been challenging because of microbial contamination. Toward the long-term goal of developing a universal artificial diet for western corn rootworm larvae, we compared larval survival, dry weight, and percentage of molt in 10-d bioassays from six current diets of which we were aware. In addition, as part of longer term rearing efforts, we recorded molting over an extended period of development (60 d). Six different artificial diets, including four proprietary industry diets (A, B, C, and D), the first published artificial diet for western corn rootworm (Pleau), and a new diet (WCRMO-1) were evaluated. Western corn rootworm larval survival was above 90% and contamination was 0% on all diets for 10 d. Diet D resulted in the greatest dry weight and percentage molting when compared with the other diets. Although fourth-instar western corn rootworm larvae have not been documented previously (only three instars have been previously documented), as many as 10% of the larvae from Diet B molted into a fourth instar prior to pupating. Overall, significant differences were found among artificial diets currently used to screen western corn rootworm. In order for data from differing toxins to be compared, a single, reliable and high-quality western corn rootworm artificial diet should eventually be chosen by industry, academia, and the public as a standard for bioassays.

Comparative Assessment of Four Steinernematidae and Three Heterorhabditidae Species for Infectivity of Larval Diabrotica Virgifera Virgifera

Larval Diabrotica virgifera virgifera LeConte (Coleoptera: Chrysomelidae) were exposed to seven different entomopathogenic nematode species to test their potential infectivity in a laboratory setting. Known D. virgifera-infecting nematode species Heterorhabditis bacteriophora Poinar, Heterorhabditis megidis Poinar, Jackson & Klein, Steinernema feltiae Filipjev, and Steinernema carpocapsae Weiser were tested in a concerted experiment alongside Steinernema diaprepesi Nguyen & Duncan, Steinernema riobrave Cabanillas, Poinar & Raulston, and a Missouri wild-type H. bacteriophora which have not been previously tested on D. virgifera. The species S. rarum Doucet was tested separately for D. virgifera infectivity. Third-instar D. virgifera were exposed to either 60 or 120 nematodes per larva for 6 d. Following exposure, mortality was recorded and larvae were examined to determine the presence of active nematode infections. Results indicated a significantly higher proportion of larvae with active infections from the Heterorhabditidae species and S. diaprepesi than the other Steinernematidae species for both exposure rates; mortality data indicated a similar trend. Steinernema rarum showed almost no infectivity in laboratory experiments.