Frequency and Distribution of Extended Diapause in Nebraska Populations of \u3ci\u3eDiabrotica barberi\u3c/i\u3e (Coleoptera: Chrysomelidae)

The frequency of extended diapause in populations of the northern corn rootworm, Diabrotica barberi Smith & Lawrence (Coleoptera: Chrysomelidae), was measured in eastern Nebraska. Adult collections were made during late summer in 2008 and 2009 from eight sites each year (seven sites were consistent over years). Eggs were obtained from 12 to 20 females per site and were held on moist soil under appropriate temperature profiles to facilitate egg survival, diapause development, and diapause termination. Percentage egg hatch was recorded after the first and second year for the 2008 and 2009 collections. Additional extended diapause expression was estimated for the 2008 and 2009 collections by counting remaining live eggs after the second-year egg hatch was completed. These data also were used to estimate each site’s maximum egg viability. Results collectively indicate that the extended diapause trait was present in all eastern Nebraska populations; however, significant variation in the frequency of extended diapause was observed within and among D. barberi populations (first-year mean percentage egg hatch range: 2008 collections: 4.5–38.6%; 2009 collections: 10.7–42.5%). Geographically, the highest incidence of extended diapause was found along a north-south transect in eastern counties of Nebraska that parallels the Missouri River. In general, the frequency of extended diapause was lower in sites found west of the north-south transect. This study contributes to the knowledge base needed to develop appropriate D. barberi management strategies in Nebraska. These data can be used to pinpoint areas of Nebraska that may have the greatest risk of economic injury in first-year corn (Zea mays L.) when D. barberi densities are high

Reduced susceptibility of western corn rootworm (\u3ci\u3eDiabrotica virgifera virgifera\u3c/i\u3e LeConte) populations to Cry34/35Ab1‑expressing maize in northeast Nebraska

The western corn rootworm (WCR; Diabrotica virgifera virgifera LeConte) is a significant pest of maize (Zea mays L.) across the United States Corn Belt. Transgenic maize hybrids expressing insecticidal proteins derived from Bacillus thuringiensis (Bt) have been used to manage WCR since 2003. Widespread resistance to Cry3Bb1 (and associated cross-resistance to mCry3A and eCry3.1Ab) has placed increased selection pressure on Cry34/35Ab1 in single-protein and pyramided transgenic maize hybrids. Data on the susceptibility of Nebraska WCR populations to Cry34/35Ab1 has not been published since 2015 and plant-based bioassays conducted in 2017–2018 confirmed resistance to Cry3Bb1 + Cry34/35Ab1 maize, suggesting resistance to Cry34/35Ab1 has evolved in the Nebraska landscape. Therefore, plant-based bioassays were conducted on F1 progeny of WCR populations collected from northeast Nebraska in 2018 and 2019. Larval survival and development were used to classify resistance to Cry34/35Ab1 in each WCR population. Bioassays confirmed incomplete resistance to Cry34/35Ab1 maize in 21 of 30 WCR populations; 9 of 30 WCR populations remained susceptible to Cry34/35Ab1. Collectively, results indicate that northeast Nebraska WCR populations were in the initial stages of resistance evolution to Cry34/35Ab1 during 2018–2019. Appropriate resistance management strategies are needed to mitigate resistance and preserve efficacy of rootworm-active products containing Cry34/35Ab1

Frequency and Distribution of Extended Diapause in Nebraska Populations of \u3ci\u3eDiabrotica barberi\u3c/i\u3e (Coleoptera: Chrysomelidae)

The frequency of extended diapause in populations of the northern corn rootworm, Diabrotica barberi Smith & Lawrence (Coleoptera: Chrysomelidae), was measured in eastern Nebraska. Adult collections were made during late summer in 2008 and 2009 from eight sites each year (seven sites were consistent over years). Eggs were obtained from 12 to 20 females per site and were held on moist soil under appropriate temperature profiles to facilitate egg survival, diapause development, and diapause termination. Percentage egg hatch was recorded after the first and second year for the 2008 and 2009 collections. Additional extended diapause expression was estimated for the 2008 and 2009 collections by counting remaining live eggs after the second-year egg hatch was completed. These data also were used to estimate each site’s maximum egg viability. Results collectively indicate that the extended diapause trait was present in all eastern Nebraska populations; however, significant variation in the frequency of extended diapause was observed within and among D. barberi populations (first-year mean percentage egg hatch range: 2008 collections: 4.5–38.6%; 2009 collections: 10.7–42.5%). Geographically, the highest incidence of extended diapause was found along a north-south transect in eastern counties of Nebraska that parallels the Missouri River. In general, the frequency of extended diapause was lower in sites found west of the north-south transect. This study contributes to the knowledge base needed to develop appropriate D. barberi management strategies in Nebraska. These data can be used to pinpoint areas of Nebraska that may have the greatest risk of economic injury in first-year corn (Zea mays L.) when D. barberi densities are high

Southern Corn Rootworm (Coleoptera: Chrysomelidae) Adult Emergence and Population Growth Assessment After Selection With Vacuolar ATPase-A double-stranded RNA Over Multiple Generations

The southern corn rootworm, Diabrotica undecimpunctata howardi Barber (Coleoptera: Chrysomelidae), was exposed over multiple generations to vacuolar (v)ATPase-A double-stranded (ds)RNA, first as adults and later, as neonate larvae. During adult selection, high mortality and lower fecundity were observed in the RNAi-selected cages after beetles were exposed to sublethal dsRNA concentrations that varied between LC40 and LC75. During larval selection, a delay in adult emergence and effects on population growth parameters were observed after neonates were exposed to sublethal dsRNA concentrations that varied between LC50 and LC70. Some of the parameters measured for adult emergence such as time to reach maximum linear adult emergence, time elapsed before attaining linear emergence, termination point of the linear emergence, and total days of linear emergence increase, were significantly different between RNAi-selected and control colonies for at least one generation. Significant differences were also observed in population growth parameters such as growth rate, net reproductive rate, doubling time, and generation time. After seven generations of selection, there was no indication that resistance evolved. The sublethal effects caused by exposures of southern corn rootworm to dsRNAs can affect important life history traits and fitness especially through delays in adult emergence and reduction in population growth. Although changes in susceptibility did not occur, the observation of sublethal effects suggests important responses to potential selection pressure. Assuming resistance involves a recessive trait, random mating between susceptible and resistant individuals is an important factor that allows sustainable use of transgenic plants, and delays in adult emergence observed in our studies could potentially compromise this assumption

Effect of Starch-Based Corn Rootworm (Coleoptera: Chrysomelidae) Baits on Selected Nontarget Insect Species: Influence of Semiochemical Composition

Various starch-encapsulated semiochemical-insecticide formulations, developed for potential use in adult corn rootworm (Diabrotica spp.) management programs, were evaluated in the laboratory and field for effectiveness on corn rootworm beetles: a carabid, Harpalus pennsylvanicus DeGeer; and a coccinellid, Coleomegilla maculata lengi Timberlake. Carbaryl was formulated in pregelatinized starch matrices along with Diabroticaspecific semiochemicals. The specific combination of feeding-gustatory stimulants encapsulated within or coating the outside of starch granules significantly influenced effectiveness. Allstarch formulations containing feeding-gustatory stimulants effectively killed Diabrotica virgifera virgifera LeConte adults in laboratory and field bioassays. However, H. pennsylvanicus and C. m. lengi mortality was greatly reduced when presented with starch granules coated with buffalo gourd (Cucurbita foetidissima H.B.K.) root powder (contains cucurbitacin E, I, and E-glycoside) or purified cucurbitacin I. Cucurbitacin I and component(s) of buffalo gourd root powder appear to be C. m. lengi and H. pennsylvanicus antifeedants. In the field, significantly more C. m. lengi and D. v. virgifera were collected at traps baited with pollen-coated than root powder-coated starch granules. When granules were broadcast over plants, mortality of C. m. lengi was greater in plots receiving pollen-coated than root powder-coated granules whereas the opposite was observed for corn rootworm beetles. Data suggest that to optimize the effectiveness of starch baits against D. v. virgifera and to minimize adverse effects on C. m. lengi and H. pennsylvanicus, granules coated with cucurbitacin rather than with starch or pollen should be used in corn rootworm management programs

Molecular phylogeny of Diabrotica beetles (Coleoptera: Chrysomelidae) inferred from analysis of combined mitochondrial and nuclear DNA sequences

The phylogenetic relationships of thirteen Diabrotica (representing virgifera and fucata species groups) and two outgroup Acalymma beetle species (Coleoptera: Chrysomelidae) were inferred from the phylogenetic analysis of a combined data set of 1323 bp of mitochondrial DNA (mtDNA) cytochrome oxidase subunit 1 (COI) and the entire second internal transcribed spacer region (ITS-2) of nuclear ribosomal DNA of 362 characters. Species investigated were D. adelpha, D. balteata, D. barberi, D. cristata, D. lemniscata, D. longicornis, D. porracea, D. speciosa, D. undecimpunctata howardi, D. u. undecimpunctata, D. virgifera virgifera, D. v. zeae, D. viridula, and outgroup A. blandulum and A. vittatum. Maximum parsimony (MP), minimum evolution (ME), and maximum likelihood (ML) analyses of combined COI and ITS-2 sequences clearly place species into their traditional morphological species groups with MP and ME analyses resulting in identical topologies. Results generally confer with a prior work based on allozyme data, but within the virgifera species group, D. barberi and D. longicornis strongly resolve as sister taxa as well as monophyletic with the neotropical species, D. viridula, D. cristata and D. lemniscata also resolve as sister taxa. Both relationships are not in congruence with the prior allozyme-based hypothesis. Within the fucata species group, D. speciosa and D. balteata resolve as sister taxa. Results also strongly supported the D. virgifera and D. undecimpunctata subspecies complexes. Our proposed phylogeny provides some insight into current hypotheses regarding distribution status and evolution of various life history traits for Diabrotica

Western corn rootworm pyrethroid resistance confirmed by aerial application simulations of commercial insecticides

The western corn rootworm (Diabrotica virgifera virgifera LeConte) (WCR) is a major insect pest of corn (Zea mays L.) in the United States (US) and is highly adaptable to multiple management tactics. A low level of WCR field-evolved resistance to pyrethroid insecticides has been confirmed in the US western Corn Belt by laboratory dose-response bioassays. Further investigation has identified detoxification enzymes as a potential part of the WCR resistance mechanism, which could affect the performance of insecticides that are structurally related to pyrethroids, such as organophosphates. Thus, the responses of pyrethroid-resistant and -susceptible WCR populations to the commonly used pyrethroid bifenthrin and organophosphate dimethoate were compared in active ingredient bioassays. Results revealed a relatively low level of WCR resistance to both active ingredients. Therefore, a simulated aerial application bioassay technique was developed to evaluate how the estimated resistance levels would affect performance of registered rates of formulated products. The simulated aerial application technique confirmed pyrethroid resistance to formulated rates of bifenthrin whereas formulated dimethoate provided optimal control. Results suggest that the relationship between levels of resistance observed in dose-response bioassays and actual efficacy of formulated product needs to be further explored to understand the practical implications of resistance

Fipronil metabolism, oxidative sulfone formation and toxicity among organophosphate- and carbamate-resistant and susceptible western corn rootworm populations

Fipronil toxicity and metabolism were studied in two insecticide-resistant, and one susceptible western corn rootworm (Diabrotica virgifera virgifera, LeConte) populations. Toxicity was evaluated by exposure to surface residues and by topical application. Surface residue bioassays indicated no differences in fipronil susceptibility among the three populations. Topical bioassays were used to study the relative toxicity of fipronil, fipronil. the mono-oxygenase inhibitor piperonyl butoxide, and fipronil\u27s oxidative sulfone metabolite in two populations (one resistant with elevated mono-oxygenase activity). Fipronil and fipronil-sulfone exhibited similar toxicity and application of piperonyl butoxide prior to fipronil resulted in marginal effects on toxicity. Metabolism of [14C]fipronil was evaluated in vivo and in vitro in the three rootworm populations. In vivo studies indicated the dominant pathway in all populations to be formation of the oxidative sulfone metabolite. Much lower quantities of polar metabolites were also identified. In vitro studies were performed using sub-cellular protein fractions (microsomal and cytosolic), and glutathione-agarose purified glutathione-Stransferase. Oxidative sulfone formation occurred almost exclusively in in vitro microsomal reactions and was increased in the resistant populations. Highly polar metabolites were formed exclusively in in vitro cytosolic reactions. In vitro reactions performed with purified, cytosolic glutathione-Stransferase (MW=27kDa) did not result in sulfone formation, although three additional polar metabolites not initially detectable in crude cytosolic reactions were detected. Metabolism results indicate both cytochromes P450 and glutathione-S-transferases are important to fipronil metabolism in the western corn rootworm and that toxic sulfone formation by P450 does not affect net toxicity