Effects of age, sex, and dietary history on response to cucurbitacin in Acalymma vittatum

The chrysomelid Acalymma vittatum is stenophagous, subsisting almost entirely on plants in the Cucurbitaceae, which generally contain cucurbitacins. Cucurbitacins are extremely bitter tetracyclic triterpenoids that are toxic to most organisms. As do other diabroticite beetles, A. vittatum sequester cucurbitacins, which have been shown to act as phagostimulants and arrestants. Our results reveal, however, that for A. vittatum the response to cucurbitacin diminishes with continued sequestration. Colony-reared A. vittatum were fed only roots (as larvae) and foliage of either `Marketmore 76' (which contains a normal amount of cucurbitacin, `bitter') or `Marketmore 80' (a near isogenic line that contains no cucurbitacin, `non-bitter') cucumber. Over 1200 individual beetles from the day of adult emergence to 15 days following emergence were placed in choice and no-choice arenas containing potted cotyledons of the two cucumber varieties for 24 h. In choice tests, overall preference for the bitter cucumber cultivar was maintained, but degree of preference changed with age and became significantly less for beetles reared on bitter diets. Furthermore, in no-choice tests, age, sex, dietary history, and interactions among these variables all significantly affected the feeding response to cucurbitacin. For A. vittatum reared without cucurbitacin, total consumption of the bitter cultivar increased over time. For beetles reared with cucurbitacin, total foliage consumption of the bitter cultivar declined, within nine days, to equal that of the non-bitter cultivar. Feral A. vittatum, unexpectedly, consumed more of the non-bitter than the bitter cultivar in no-choice tests. Ecological and applied implications of this variation in response to cucurbitacin are discussed.     

Effects of host plant and cucurbitacin on growth of larval Diabrotica undecimpunctata howardi

The effects of host plant and dietary cucurbitacin on the growth of larval southern corn rootworm (SCR), Diabrotica undecimpunctata howardi Barber (Chrysomelidae: Luperini), were investigated. SCR were reared on four hosts: corn, Zea mays; peanuts, Arachis hypogaea; and two squash varieties, Cucurbita pepo cv. Ambassador (containing cucurbitacin D (0.08 mg g^–1 fr.wt.) = bitter), and C. pepo cv. Early Yellow Crookneck (lacking cucurbitacin = non-bitter). Larval growth was significantly greater on corn and peanuts than on either squash variety. After four weeks, adults had emerged from corn and peanut plants, while squash-reared larvae had not yet entered the pupal stage. There was no difference in larval growth on the two varieties of squash. Primary metabolite measurements showed no nutritional differences between the two squash varieties. Artificial diet experiments were used to test the effect of three concentrations of cucurbitacin D (0.0, 0.1, and 0.6 mg g^–1 diet) on growth of larval SCR. Larvae reared on diet containing 0.6 mg g^–1 cucurbitacin weighed significantly less than larvae reared on diet containing 0.1 mg g^–1 or no cucurbitacin after 10 d. No significant difference in growth was measured between the 0.1 mg g^–1 diet and the 0.0 mg g^–1 diet. Results are discussed relative to theories about the relationship between diabroticites and cucurbitacins.     

Toxicity of Chromobacterium subtsugae to southern green stink bug (Heteroptera: Pentatomidae) and corn rootworm (Coleoptera: Chrysomelidae)

Diabrotica spp. (Coleoptera: Chrysomelidae) beetles and southern green stink bugs, Nezara viridula (L.) (Heteroptera: Pentatomidae), are pests on corn, Zea mays L., and soybean, Glycine max (L.) Merr., as well as on cucurbits. Control of these insects has depended on chemicals. An alternative to chemical control is the use of biologicals. Use of bacteria, fungi, viruses, pheromones, and metabolites to control these insects can potentially improve resistance management and reduce pesticide use. Other than Bacillus thuringiensis Berliner, few bacteria have been discovered that are lethal to either of these pests. Chromobacterium subtsugae Martin et al., a newly described bacterium that is known to be toxic to Colorado potato beetle, Leptinotarsa decemlineata (Say), larvae, was found to be toxic to both diabroticite adult beetles and southern green stink bug adults. In laboratory assays, toxins produced by these bacteria kill 80-100% of the adults of two species of diabroticite beetles, Diabrotica undecimpunctata howardi Barber and Diabrotica virgifera virgifera LeConte, and 100% of southern green stink bug adults within 6 d. For green stink bug, live bacteria were not needed for toxicity.     

Effect of a phloxine B-cucurbitacin bait on diabroticite beetles (Coleoptera: Chrysomelidae)

Cucurbitacin E glycoside, extracted from a bitter mutant of Hawkesbury watermelon [Citrulls lanatus (Thunb.) Matsum. & Nakai (Syn. Citrullus vulgaris Schrad)] is the active ingredient of a feeding stimulant for the corn rootworm complex. It is the primary component of a water-soluble bait that can be combined with toxins for adult diabroticite control. Studies were conducted using phloxine B (D&C Red 28), a xanthene dye, as the toxin. This dye was efficacious against Diabrotica undecimpunctata howardi Barber, spotted cucumber beetle, and Acalymma vittatum (F.), striped cucumber beetle, in cucumber plots and could be recovered from cucumber leaves for 8 d after treatment. The average amount of dye recovered per dead spotted cucumber beetle at 8 d after treatment was 0.173 microg. Concentrated and sugar-free fermented forms of the watermelon extract were developed and compared with the fresh juice in field applications on cucumber plants. There was no significant difference in mortality of beetles from phloxine B-bait prepared with fresh, fermented, or concentrated extract, although in laboratory studies, fermented juice had higher feeding stimulant activity.     

Evaluation of cucurbitacin-based gustatory stimulant to facilitate cucumber beetle (Coleoptera: Chrysomelidae) management with foliar insecticides in melons

The bitter plant-derived compounds cucurbitacins are known to stimulate feeding of adult cucumber beetles (Coleoptera: Chrysomelidae). A cucurbitacin-based gustatory stimulant applied as a flowable bait combined with either spinosad or carbaryl was compared with foliar sprays of spinosad and carbaryl for controlling two cucumber beetle species (Diabrotica undecimpunctata undecimpunctata Mannerheim and Acalymma trivittatum Mannerheim) in honeydew melons (Cucumis melo L.). Field studies were conducted on the University of California-Davis plant pathology farm in 2008 and 2009. Beetle densities after applications and fruit damage from beetle feeding were compared among treatments. In addition, beetle survival was compared within field cages placed over the treated foliage infested with beetles. Using all three measures of efficacy, we determined that the addition of cucurbitacin bait had no effect on the level of cucumber beetle control with carbaryl in either 2008 or 2009. In both years, spinosad did not significantly reduce cucumber beetle densities in either field cages or field plots and did not reduce fruit damage relative to the untreated control. The addition of the bait to spinosad did not improve its efficacy. A laboratory bioassay of the spinosad formulation used in the field showed it had significant lethal effects on adults of both cucumber beetle species. Results indicated that the bait formulation used did not improve cucumber beetle control but may benefit from the addition of floral attractants or using a different type of cucurbitacin.     

Effect of Bacillus thuringiensis cry3Bb1 protein on the feeding behavior and longevity of adult western corn rootworms (Coleoptera: Chrysomelidae)

The first transgenic corn hybrids expressing the Bacillus thuringiensis (Bt) Cry3Bb1 protein to control corn rootworm (Diabrotica spp.) larvae were registered for commercial use in 2003. This study was conducted to investigate the effect of Cry3Bb1 protein in combination with a cucurbitacin bait on adult feeding and longevity of both organophosphate-resistant and -susceptible western corn rootworms, Diabrotica virgifera virgifera LeConte (Coleoptera: Chrysomelidae). In choice and no-choice tests, possible repellency to the Bt protein was quantified by comparing beetle consumption of cellulose disks treated with three concentrations of Bt in combination with a feeding stimulant (Invite EC) to disks treated with stimulant alone. A lethal-time assay also was conducted to examine survival of beetles exposed to Bt protein in their diet. Results from these assays indicate that adult rootworms are not significantly deterred by the presence of Cry3Bb1 on the treated discs and that ingestion of toxin does not adversely affect adult longevity.     

Insect feeding on cucumber mediated by rhizobacteria-induced plant resistance

Select strains of plant growth-promoting rhizobacteria (PGPR) were evaluated in greenhouse experiments with cucumber for induction of resistance against cucumber beetle (Diabrotica undecimpunctata howardi Barber) feeding and the beetle-transmitted cucurbit wilt disease. When beetles were given a choice between PGPR-treated and nontreated cucumber, their feeding on stems and cotyledons and the severity of wilt symptoms were significantly lower on PGPR-treated plants. HPLC analysis demonstrated that cotyledons from PGPR-treated plants contained significantly lower concentrations of the cucumber beetle feeding stimulant cucurbitacin than nontreated plants. These results suggest that a mechanism for PGPR-induced resistance against cucumber beetle feeding may involve a change in the metabolic pathway for cucurbitacin synthesis.     

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.     

Adult activity and oviposition of corn rootworms,Diabrotica spp. (Coleoptera: Chrysomelidae), in Miscanthus,corn and switchgrass

The ability of the biomass crop Miscanthus (Miscanthus × giganteus Greef and Deuter ex Hodkinson and Renvoize) to support larval development for both United States and European populations of the western corn rootworm, Diabrotica virgifera virgifera LeConte, suggests an avenue for interactions with corn (Zea mays L.). To provide context to survival of D. v. virgifera on Miscanthus, adult activity and oviposition of Diabrotica spp. were monitored in central Illinois in 2010–2011 in Miscanthus, corn and switchgrass (Panicum virgatum L.). For D. v. virgifera, vial traps within corn plots captured 3–10 times as many adults as in Miscanthus or switchgrass, while soil samples showed females laid approximately 10 times as many eggs in corn as in the perennial grasses. Adult southern corn rootworms, Diabrotica undecimpunctata howardi Barber, were the most abundant species in 2010 and clearly preferred switchgrass as an adult habitat, with vial traps in switchgrass capturing 5–10 times as many D. u. howardi as those in corn or Miscanthus. Based on the small production areas for Miscanthus and switchgrass (and low use of both by D. v. virgifera), it seems likely that there are no current impacts of these perennial grasses on pest status of Diabrotica spp. in corn or other crops. However, adaptations by Diabrotica spp. to pest management practices suggest they could be a source for interactions between biomass and food or feed crops. Early‐season soil samples did not recover eggs of D. u. howardi, but their use of switchgrass as an adult habitat suggests additional research in areas where switchgrass may be grown near peanuts, alfalfa or other hosts may be needed. Also, investigation of other candidate bioenergy crops known to support Diabrotica spp. larval development is needed to better understand the possible effects of a changing agricultural landscape on corn rootworms.     

Control of Larval Northern Corn Rootworm. (Diabrotica barberi) with Two Steinernematid Nematode Species

The entomogenous nematodes Steinerema feltiae and S. bibionis did not penetrate the roots of corn, Zea mays, to infect larval northern corn rootworm (NCR), Diabrotica barberi, feeding within. Laboratory bioassays against first instar NCR indicated that S. feltiae, Mexican strain (LD₅₀ = 49 nematodes/insect) is more virulent than S. bibionis (LD₅₀ = 100). Numbers of NCR larvae in a grain corn crop were reduced by both nematode species applied at corn seeding time at the rate of 10,000 infective-stage juveniles per linear meter of corn row. The chemical insecticide fonofos provided significantly better control than either nematode species.     

Foraging resources of adult Mexican corn rootworm (Coleoptera: Chrysomelidae) in Bell County, Texas

Pollen analyses were used to determine pollen foraging resources of adult Mexican corn rootworms, Diabrotica virgifera zeae Krysan & Smith, captured near Temple, Bell County, TX, in 1996 and 1997. In 1996, adult Mexican corn rootworms were captured in a corn, Zea mays L., field. In 1997, nine locations outside of cornfields were added. Overall, 92% of the beetles (n = 1,323) contained pollen. More than 142,000 pollen grains were counted, representing 45 families, 63 genera, and 27 species. Overall, in 1996, noncorn grass pollen (70%) occurred in the greatest percentage of total pollen followed by corn (17%), then nongrass pollen (13%). In 1997, noncorn grass pollen (76%) had the greatest percentage, then nongrass (18%), and finally corn pollen (6%). Corn pollen was found in 34% of the beetles in 1996 and 26% in 1997. Fifteen Asteraceae taxa were encountered including sunflower, Helianthus annuus L. Thirteen Fabaceae were found including soybean, Glycine max L. Mexican corn rootworm adults foraged mainly on noncorn grass pollen, but also foraged on pollen from a large diversity of plant species, indicating that noncorn pollen may play a role in the food habits of Mexican corn rootworm adults.     

Influence of preceding crop on wireworm (Coleoptera: Elateridae) abundance in the coastal plain of North Carolina

Three studies were conducted to determine the effect of preceding crop on wireworm (Coleoptera: Elateridae) abundance in the coastal plain of North Carolina. In all three studies, samples of wireworm populations were taken from the soil by using oat, Avena sativa L., baits. Treatments were defined by the previous year's crop and were chosen to reflect common crop rotations in the region. Across all three studies, eight wireworm species were recovered from the baits: Conoderus amplicollis (Gyllenhal), Conoderus bellus (Say), Conoderus falli (Lane), Conoderus lividus (Degeer), Conoderus scissus (Schaeffer), Conoderus vespertinus (F.), Glyphonyx bimarginatus (Schaeffer), and Melanotus communis (Gyllenhal). The effect of corn, Zea mays L.; cotton, Gossypium hirsutum L.; fallow; soybean, Clycine max (L.) Merr.; sweet potato, Ipomoea batatas (L.) Lam.; and tobacco (Nicotiana spp.) was evaluated in a small-plot replicated study. M. communis was the most frequently collected species in the small-plot study and was found in significantly higher numbers following soybean and corn. The mean total number of wireworms per bait (all species) was highest following soybean. A second study conducted in late fall and early spring assessed the abundance of overwintering wireworm populations in commercial fields planted to corn, cotton, peanut (Arachis hypogaea L.), soybean, sweet potato, and tobacco in the most recent previous growing season. C. lividus was the most abundant species, and the mean total number of wireworms was highest following corn and soybean. A survey was conducted in commercial sweet potato in late spring and early summer in fields that had been planted to corn, cotton, cucurbit (Cucurbita pepo L.), peanut, soybean, sweet potato, or tobacco in the most recent previous growing season. C. vespertinus was the most abundant species, and the mean total number of wireworms per bait was highest following corn.     

Monitoring cucumber beetles in sweetpotato and cucurbits with kairomone-baited traps

Seven kairomone formulations (Trécé, Inc., Salinas, CA) were evaluated for their effectiveness as attractants for luring three species of cucumber beetles into Pherocon CRW traps (Trécé, Inc.) in cucurbit and sweetpotato fields. The spotted cucumber beetle, Diabrotica undecimpunctata howardi Barber; the banded cucumber beetle, Diabrotica balteata LeConte; and the striped cucumber beetle, Acalymma vittatum (F.), were captured in this study. TRE8276 (TIC mixture: 500 mg of 1,2,4-trimethoxybenzene, 500 mg of indole, and 500 mg of trans-cinnamaldeyde) and TRE8336 (500 mg of 1,2,4-trimethoxybenzene, 500 mg of trans-cinnamaldeyde, 500 mg of 4-methoxyphenethanol) were the most effective lures for spotted and striped cucumber beetles. None of the kairomone lures was very effective for attracting banded cucumber beetles. Three population peaks of spotted cucumber beetles were observed in cucurbit and sweetpotato fields at the U.S. Vegetable Laboratory (Charleston, SC). The efficacy of TRE8276 declined rapidly after 2 wk in the field. An improved design of the Pherocon CRW trap, with a yellow bottom and more-tapered top section, was more effective for capturing cucumber beetles than the original trap design made entirely of clear plastic. Banded cucumber beetles were not captured in sweetpotato fields at inland locations in North Carolina or South Carolina.     

Correspondence between rates of host plant consumption and responses to the Acalymma vittatum male-produced aggregation pheromone

Abstract In field studies, wind-directed traps were used to investigate the resource-based properties of the male-produced aggregation pheromone of Acalymma vittatum (F.) (Coleoptera: Chrysomelidae). Responses to early season overwintered field-collected male A. vittatum without food were compared with those feeding on pollen, and in turn these were compared with those feeding on cucurbit seedlings. These comparisons were modelled after transitions as they would occur at the initiation of A. vittatum host plant colonization.
In another experiment, colony-reared male A. vittatum that had been fed only near isogenic lines of potted cucumber ( Cucumis sativus ) seedlings that either lacked or contained cucurbitacin were placed as bait in the traps. Using the inverse relationship, which is understood for A. vittatum , between previous exposure and the phagostimulant response to cucurbitacin, rates of foliage consumption could be manipulated. The interaction between relative rates of host plant consumption of the males used as lures and responses to the pheromone could thus be tested. The use of A. vittatum reared entirely without dietary cucurbitacin as bait also enabled evaluation of the potential role of cucurbitacin in pheromone biosynthesis. Trap responses were closely correlated with the rates of feeding of the beetles in the trap lures, but cucurbitacin was neither a precursor nor a prerequisite for the pheromone. Implications for early season field colonization and the adaptive significance of this pheromone are discussed.     

Divergence of defensive cucurbitacins in independent Cucurbita pepo domestication events leads to differences in specialist herbivore preference

Crop domestication and improvement often concurrently affect plant resistance to pests and production of secondary metabolites, creating challenges for isolating the ecological implications of selection for specific metabolites. Cucurbitacins are bitter triterpenoids with extreme phenotypic differences between Cucurbitaceae lineages, yet we lack integrated models of herbivore preference, cucurbitacin accumulation, and underlying genetic mechanisms. In Cucurbita pepo, we dissected the effect of cotyledon cucurbitacins on preference of a specialist insect pest (Acalymma vittatum) for multiple tissues, assessed genetic loci underlying cucurbitacin accumulation in diverse germplasm and a biparental F₂ population (from a cross between two independent domesticates), and characterized quantitative associations between gene expression and metabolites during seedling development. Acalymma vittatum affinity for cotyledons is mediated by cucurbitacins, but other traits contribute to whole-plant resistance. Cotyledon cucurbitacin accumulation was associated with population structure, and our genetic mapping identified a single locus, Bi-4, containing genes relevant to transport and regulation – not biosynthesis – that diverged between lineages. These candidate genes were expressed during seedling development, most prominently a putative secondary metabolite transporter. Taken together, these findings support the testable hypothesis that breeding for plant resistance to insects involves targeting genes for regulation and transport of defensive metabolites, in addition to core biosynthesis genes.     

<Emphasis Type="Italic">Trichogramma Brassicae</Emphasis> and SLAM<Superscript>®</Superscript>, an Integrated Approach to Managing European Corn Borer and Corn Rootworms

European corn borer, Ostrinia nubilalis (Hübner) (Lepidoptera: Crambidae); western corn rootworm (WCRW), Diabrotica virgifera virgifera LeConte; and northern corn rootworm (NCRW), Diabrotica barberi (Smith & Lawrence) (Coleoptera: Chrysomelidae) are important pests of corn, Zea mays L., that occur simultaneously in the US Corn Belt. Areawide management strategies for northern and western corn rootworm in Iowa, Kansas, South Dakota, Illinois, and Indiana, and for the Mexican strain in Texas, use SLAM^®, which contains cucurbitacin, a feeding stimulant, and a small amount of the insecticide carbaryl. This management strategy is environmentally sound because it introduces a substantially lesser amount of chemical insecticide into the environment than prophylactic soil insecticide applications for WCRW management. To develop a management program for both corn pests, the compatibility of SLAM^® to control CRW, along with a biological agent to manage European corn borer, was investigated. Laboratory studies were conducted using the egg parasitoid Trichogramma brassicae (Bezdenko) (Hymenoptera: Trichogrammatidae). In experiments in which SLAM^® and T. brassicae were evaluated as components of a management system, SLAM^® did not affect parasitism or parasitoid emergence rates. Trichogramma brassicae displayed vigorous biotic fitness, emergence rates, sex ratios, and fecundity over the duration of the study. Data collected under these laboratory conditions indicated that SLAM^® had no adverse effect on the quality of T. brassicae, indicated by its ability to reproduce in the filial generation.     

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.     

A cucurbitacin glycoside from Picrorhiza kurrooa

A novel cucurbitacin glycoside bitter principle has been isolated from roots extracts of Picrorhiza kurrooa and shown to be 25-acetoxy-2-β-glucosyloxy-3,16,20-trihydroxy-9-methyl-19-norlanosta-5,23-diene-22-one. Two previously described catalpol derivatives were also isolated; neither was as bitter as the cucurbitacin glycoside.     

Prairie grasses as hosts of the northern corn rootworm (Coleoptera: Chrysomelidae)

We evaluated 27 prairie grass species thought to be among those dominant 200 yr ago in the northern midwest as larval hosts of the northern corn rootworm, Diabrotica barberi Smith and Lawrence. Maize (Zea mays L.), spring wheat (Triticum aestivum L.), and grain sorghum (Sorghum bicolor L.) were included as controls for a total of 30 species. Twenty pots of each species were planted in a randomized complete block design. Each pot was infested 5 wk later with 20 neonate northern corn rootworm larvae. Two pots within each species and block were assigned an extraction date of 7 or 14 d after infestation. The remaining two pots from each block were used to monitor adult emergence. The percentage of larvae recovered, change in larval head capsule width, and change in average dry weights varied significantly among the grass species. The highest percentage of larvae was recovered from slender wheatgrass, Elymus trachycaulus (Link), and this was significantly greater than the percentage recovered from all other species including maize for the 14-d sample date. Several additional species were also relatively good hosts, in that the percentage of larvae recovered from these species was not significantly different from maize. The average dry weight of larvae recovered was significantly greater for larvae recovered from maize than for larvae recovered from all other species except slender wheatgrass, when the two samples dates were combined. Overall, adults were produced from only 6 of the 28 species evaluated, and no analysis was performed because of the low numbers. The results of this study are discussed in relation to the potential of alternate hosts of northern corn rootworm to serve as a bridge to survival on transgenic maize.     

Inhibition of seed germination by extracts of bitter Hawkesbury watermelon containing cucurbitacin, a feeding stimulant for corn rootworm (Coleoptera: Chrysomelidae)

Cucurbitacins are feeding stimulants for corn rootworm used in baits to control the adults of this insect pest. Corn rootworm larvae also feed compulsively on cucurbitacins. Cucurbitacins are reported to be gibberellin antagonists that may preclude their use as seed treatments for these soil-dwelling insects. The crude extract of a bitter Hawkesbury watermelon containing cucurbitacin E-glycoside significantly inhibited germination of watermelon, squash, and tomato seeds. Although the germination of corn seed was not significantly inhibited, root elongation was inhibited by crude extracts, but not by high-performance liquid chromatography-purified cucurbitacin E-glycoside. Therefore, the effects of the major components in the bitter watermelon extract (e.g., sugars) on seed germination and root elongation were determined. Pure sugars (glucose and fructose), at concentrations found in watermelon extract, mimicked the inhibition of seed germination and root elongation seen with the crude bitter Hawkesbury watermelon extract. Removal of these sugars may be necessary to use this extract as a bait for corn rootworm larvae as a seed or root treatment.