Susceptibility of northern corn rootworm (Diabrotica barberi) populations to Cry3Bb1 and Gpp34/Tpp35Ab1 proteins in seedling and diet overlay toxicity assays.

The northern corn rootworm, Diabrotica barberi Smith & Lawrence (Coleoptera: Chrysomelidae) is a major pest of maize in the United States Corn Belt. Recently, resistance to Bacillus thuringiensis (Bt) maize was reported in North Dakota and increased use of Bt maize hybrids could facilitate resistance evolution in other maize-producing states. In this study, susceptibility to Bt proteins was evaluated in wild D. barberi populations from 8 fields collected in 5 different states (Minnesota, Missouri, Nebraska, Iowa, and North Dakota). Field populations were compared to a susceptible D. barberi colony in seedling and diet toxicity assays conducted with 3 concentrations of Cry3Bb1 (0.4, 4.0, and 40.0 µg/cm2) and Gpp34/Tpp35Ab1 (previously called Cry34/35Ab1; 1.4, 14.0, and 140.0 µg/cm2). The 2019 population from Meeker Co., Minnesota (MN-2019), exhibited the lowest mortality to Cry3Bb1 and also had nominally lowest mortality to Gpp34/Tpp35Ab1 at the highest concentrations tested in diet toxicity assays. Percent second instar was also highest for larvae of the Minnesota population surviving Cry3Bb1. In seedling assays, MN and IA-2018 populations exhibited the highest proportion survival and dry weight to both proteins expressed in corn. No significant differences in mortality, percent second instar, and dry weight were observed at the highest concentration for both proteins among the populations collected in in 2020. Most D. barberi populations were still highly susceptible to Cry3Bb1 and Gpp34/Tpp35Ab1 proteins based on diet and seedling assays, but resistance appears to be developing in some D. barberi populations. Now that methods are available, resistance monitoring may also be needed for D. barberi in some regions.

Host search behaviors of specialist and generalist root feeding herbivores (Diabrotica spp.) on host and non-host plants.

Western, northern, Mexican, and southern corn rootworms (WCR, NCR, MCR, and SCR) are serious corn pests. We evaluated host search behavior of these pests on six plant species using a video tracking system. After a 5-min exposure to plant roots, behavioral parameters were automatically recorded and used to quantify the search behavior. The search behavior was not observed for sorghum since no neonates survived after contacting sorghum roots. After exposures to corn roots, all neonates exhibited the localized search behaviors (i.e., shortening total distance traveled, lowering movement speed, increasing turn angle, moving farther from origin) which are used to stay in and search within root systems. When larvae contacted roots of wheat, barley, oats, soybean, or controls, they expanded the search area by extending the travel path, increasing velocity, and reducing turn angles and total distance moved. The intensity of the search expansion is highly associated with the host preferences known for the four rootworm species and subspecies. Neonates of each corn rootworm exhibited distinct search behaviors. In fact, NCR larvae had the highest speed, the greatest travel path, and the lowest turn angle, whereas MCR larvae had the highest turn angle and moved faster than WCR and SCR larvae.

An improved bioassay for the testing of entomopathogenic nematode virulence to the western corn rootworm (Diabrotica virgifera virgifera) (Coleoptera: Chrysomelidae): with focus on neonate insect assessments.

Bioassays involving newly hatched larval insects can be limited by the larvae's feeding state. Assays attempting to monitor mortality effects can be negatively affected by starvation effects on the larvae. Neonate western corn rootworms have significant reductions in viability if not provided food within 24 h post hatch. The recent development of an improved artificial diet for western corn rootworm larvae provides a new bioassay type for evaluating entomopathogenic nematodes that also makes the testing arena easy to observe. Here, we evaluated four species of entomopathogenic nematodes including Heterorhabditis bacteriophora Poinar, Steinernema carpocapsae (Weiser), Steinernema diaprepesi Nguyen & Duncan, and Steinernema rarum (de Doucet) against neonate western corn rootworm, Diabrotica virgifera virgifera LeConte, in 96-well plate diet bioassays. Nematode inoculation levels were 0, 15, 30, 60, and 120 nematodes per larva. Percentage mortality increased for each species as the rate of inoculation increased. Overall, H. bacteriophora and S. carpocapsae caused the greatest amount of larval mortality. The diet-based bioassays were shown to be an effective method for nematode exposure to insect pests. The assays provided adequate moisture to keep nematode from desiccating while also allowing freedom of movement around the arenas. Both rootworm larvae and nematodes were contained within the assay arenas. The addition of nematodes did not cause any significant deterioration of the diet within the three-day period of testing. Overall, the diet bioassays worked well as a measure of entomopathogenic nematode virulence.

Maize Inbred Mp708 is Highly Susceptible to Western Corn Rootworm, Diabrotica virgifera virgifera (Coleoptera: Chrysomelidae), in Field and Greenhouse Assays.

The western corn rootworm (WCR), Diabrotica vifgirera virgifera LeConte, (Coleoptera: Chrysomelidae) causes significant economic damage in corn production each year. Resistance to insecticides and transgenic corn with Bacillus thuringiensis (Bt), Berliner toxins have been reported throughout the United States Corn Belt. Corn breeding programs for natural resistance against WCR larvae could potentially assist in rootworm management. Root damage and root regrowth of eight corn lines were evaluated in field assays at three different locations. Results indicated the inbred 'Mp708' had the greatest root damage and was significantly greater than damage for the susceptible control, B37×H84. In greenhouse assays, we evaluated four of these lines plus a hybrid expressing the mCry3A Bt toxin for damage. The results indicated that Mp708 had significantly higher root damage when compared to 'CRW3(S1)C6' and 'MIR604'. Despite previous work suggesting otherwise, we conclude that Mp708 is highly susceptible to the WCR larvae based on root damage in field and greenhouse plant assays.

Characterization of Thermal and Time Exposure to Improve Artificial Diet for Western Corn Rootworm Larvae.

The western corn rootworm (WCR), Diabrotica virgifera LeConte, is the most serious pest of maize in the United States. In pursuit of developing a diet free of antibiotics for WCR, we characterized effects of thermal exposure (50-141 °C) and length of exposure on quality of WCRMO-2 diet measured by life history parameters of larvae (weight, molting, and survival) reared on WCRMO-2 diet. Our results indicated that temperatures had non-linear effects on performance of WCRMO-2 diet, and no impacts were observed on the length of time exposure. The optimum temperature of diet processing was 60 °C for a duration less than 30 min. A significant decline in development was observed in larvae reared on WCRMO-2 diet pretreated above 75 °C. Exposing WCRMO-2 diet to high temperatures (110-141 °C) even if constrained for brief duration (0.9-2.3 s) caused 2-fold reduction in larval weight and significant delays in larval molting but no difference in survival for 10 days compared with the control diet prepared at 65 °C for 10 min. These findings provide insights into the effects of thermal exposure in insect diet processing.

Development of a nondiapausing strain of northern corn rootworm with rearing techniques for both diapausing and nondiapausing strains.

The northern corn rootworm, Diabrotica barberi Smith & Lawrence, has a univoltine life cycle that typically produces one generation a year. When rearing the northern corn rootworm in the laboratory, in order to break diapause, it is necessary to expose eggs to a five month cold period before raising the temperature. By selective breeding of the small fraction of eggs that hatched without cold within 19-32 days post oviposition, we were able to develop a non-diapausing colony of the northern corn rootworm within five generations of selection. Through selection, the percentages of adult emergence from egg hatch without exposure to cold treatment significantly increased from 0.52% ± 0.07 at generation zero to 29.0% ± 2.47 at generation eight. During this process, we developed an improved method for laboratory rearing of both the newly developed non-diapausing strain as well as the diapausing strain. The development of the non-diapausing colony along with the improvements to the rearing system will allow researchers to produce up to six generations of the northern corn rootworm per year, which would facilitate research and advance our knowledge of this pest at an accelerated rate.

Optimizing Egg Recovery From Wild Northern Corn Rootworm Beetles (Coleoptera: Chrysomelidae).

The northern corn rootworm, Diabrotica barberi Smith & Lawrence (Coleoptera: Chrysomelidae), is one of the most important insect pests in the U.S. Corn Belt. Efforts to obtain eggs from wild northern corn rootworm populations using techniques developed for other rootworm species have been unsuccessful due to lack of oviposition. In 2016, we evaluated four oviposition media in choice tests within each of three female densities in 30.5 × 30.5 × 30.5 cm BugDorm cages. The number of eggs laid per female was significantly affected by female density and the interaction of female density × oviposition media, but oviposition was relatively poor in all oviposition media (1.2 eggs per female when averaging the three female densities and all oviposition media). Single females were also evaluated in nonchoice assays in 6 cm × 6 cm × 8 cm clear plastic boxes and averaged up to 108 eggs per female depending on the oviposition media. In 2017, the cumulative number of eggs laid per female in boxes with one female was not significantly different from the number of eggs laid per female in boxes with 3 females. In 2018, the cumulative number of eggs laid per female was not significantly different between female densities of 1, 3, 5, or 10 females per box. Total egg production per box therefore increased as female density increased. More than 27,000 wild northern corn rootworm eggs were collected from just 190 females when collected relatively early in the field season. We now have an efficient and robust system for obtaining eggs from wild northern corn rootworm females.

Effects of Cold Storage on Nondiapausing Eggs of the Western Corn Rootworm (Coleoptera: Chrysomelidae).

Western corn rootworm, Diabrotica virgifera virgifera LeConte, became much easier to research with the development of a nondiapausing rootworm strain. In the event that the eggs cannot be used immediately researchers have been known to delay egg hatch by storing the eggs at low temperatures. It is not well known how this technique could affect egg hatch or larval development, which could alter the results of an experiment. To test for this nondiapausing eggs of the western corn rootworm were stored at low temperatures to test for potential negative effects on hatch and larval development. Eggs were stored in either soil or agar and placed in refrigerators set to 4 or 8.5°C. Nondiapausing eggs were exposed to the cold for 1, 2, or 4 wk and then placed in a chamber set to 25°C. Eggs were then tested for average hatch percentage in Petri dishes and average larval recovery from containers with seedling corn. Results showed a significant reduction in percent hatch for eggs stored at 4°C for 4 wk. Larval recovery was significantly reduced in eggs stored for 4 wk at both 4 and 8.5°C. Within the treatments tested, egg storage for less than 4 wk in soil at 8.5°C provided the best hatch and larval recovery. Researchers wishing to store eggs may use these results to improve their rearing or testing of western corn rootworm.

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.

Western Corn Rootworm (Coleoptera: Chrysomelidae) Larval Movement in eCry3.1Ab+mCry3A Seed Blend Scenarios.

Corn fields planted with plant-incorporated Bacillus thuringiensis (Bt) proteins must have a portion of the field planted with non-Bt, isoline, plants that serve as a refuge for susceptible insects. In the Corn Belt, refuge seeds are now blended in the bag with Bt seeds for corn hybrids containing two or more toxins targeted toward the western corn rootworm, Diabrotica virgifera virgifera LeConte. Syngenta's corn hybrid, Agrisure Duracade, containing the eCry3.1Ab (event 5307) and mCry3a (event MIR604) rootworm-targeted toxins were registered as a seed blend in 2014. Western corn rootworm larval movement between the refuge plants and the Duracade plants was assessed to determine western corn rootworm survival and amount of root damage on these plants when planted in all possible seed blend scenarios. In this study, western corn rootworm larvae moved between isoline and Bt plants and adult survival was greater on Bt plants if movement from a neighboring infested isoline plant had occurred. However, root damage to these Bt plants did not reach economic levels. The low numbers of western corn rootworm larvae that did move from an infested Bt plant to an isoline plant could potentially select for resistance if they survived to adulthood.

Evaluation of Potential Fitness Costs Associated With eCry3.1Ab Resistance in Diabrotica virgifera virgifera (Coleoptera: Chrysomelidae).

Both an eCry3.1Ab-selected and paired control western corn rootworm, Diabrotica virgifera virgifera LeConte, colony were tested for adult longevity, egg oviposition, egg viability, and larval development in order to evaluate the potential fitness costs associated with eCry3.1Ab resistance. Adult longevity experiments were conducted by pairing virgin males and females together in plastic boxes supplied with food, water, and ovipositional medium and observed for survival time. Eggs were also collected from the ovipositional medium once a week to determine average egg oviposition and egg viability. Larval development time experiments were conducted by infesting seedling assays with 25 neonate larvae and recording larval recovery after several days. Adult longevity, average egg oviposition, and larval development time results indicated a lack of fitness costs associated with eCry3.1Ab resistance in the western corn rootworm. Results of egg viability indicated a fitness advantage for the eCry3.1Ab-selected colony with a significantly higher egg hatch than the control.

Tolerance of eCry3.1Ab in Reciprocal Cross Offspring of eCry3.1Ab-Selected and Control Colonies of Diabrotica virgifera virgifera (Coleoptera: Chrysomelidae).

Two new insect colonies were created by separating virgin western corn rootworm, Diabrotica virgifera virgifera LeConte, males and females from both a selected laboratory colony that was being reared on eCry3.1Ab-expressing corn (Zea mays L.) and a control colony reared on its near-isoline corn. Females from the selected colony were paired with males of the control colony and vice versa to create both a selected female by control male colony (Sel♀) and control female by selected male colony (Con♀). Both colonies along with their parental colonies (eCry3.1Ab-selected and control) were evaluated on eCry3.1Ab-expressing corn and its near-isoline in seedling assays. Larvae from each colony were also used in diet toxicity experiments in order to determine the LC50 and EC50 values for the eCry3.1Ab toxin for each. Statistical analysis of seedling assay experiments did not indicate any significant colony×corn interaction but did show a significant main effect of corn type for both larval recovery and larval head capsule widths. Results from the diet toxicity assays showed the control colony to have a significantly lower LC50 value than the selected and cross colonies and a significantly lower EC50 than the selected and Con♀ colonies. Calculations of dominance values (h) of eCry3.1Ab resistance traits from seedling assays indicated that the two reciprocal cross colonies have a dominance value (h) of ∼1, suggesting dominance of the eCry3.1Ab resistance trait.

Frequency and distribution of extended diapause in Nebraska populations of Diabrotica barberi (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.