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.

Differential Proteomics Analysis Unraveled Mechanisms of Arma chinensis Responding to Improved Artificial Diet.

The development of artificial diets could considerably simplify and reduce the cost of mass rearing of natural enemies compared to conventional rearing methods. However, improvement of artificial diets can be tedious, convoluted and often uncertain. For accelerating diet development, a better method that can offer informative feedback to target deficiencies in diet improvement is required. Our previous research demonstrated several biological characteristics were diminished in the insect predator, Arma chinensis Fallou, fed on an artificial diet formulated with the aid of transcriptomic methods compared to the Chinese oak silk moth pupae. The present study reports differential proteomic analysis by iTRAQ-PRM, which unravels the molecular mechanism of A. chinensis responding to improvements in the artificial diet. Our study provides multivariate proteomic data and provides comprehensive sequence information in studying A. chinensis. Further, the physiological roles of the differentially expressed proteins and pathways enable us to explain several biological differences between natural prey-fed and improved diet-fed A. chinensis, and subsequent proposed reformulation optimizations to artificial diets.

Recent Advances in Insect Rearing Methodology to Promote Scientific Research and Mass Production.

The benefits obtained from our ability to produce insects have encompassed a wide array of applications, from the early stages of examining different species, to the present day of mass production for multiple purposes [...].

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.

Assessing the Single and Combined Toxicity of the Bioinsecticide Spear and Cry3Bb1 Protein Against Susceptible and Resistant Western Corn Rootworm Larvae (Coleoptera: Chrysomelidae).

The western corn rootworm (WCR), Diabrotica virgifera virgifera LeConte (Coleoptera: Chrysomelidae), poses a serious threat to maize (Zea mays L.) growers in the U.S. Corn Belt. Transgenic corn expressing Bacillus thuringiensis (Bt) Berliner is the major management tactic along with crop rotation. Bt crops targeting WCR populations have been widely planted throughout the Corn Belt. Rootworms have developed resistance to nearly all management strategies including Bt corn. Therefore, there is a need for new products that are not cross-resistant with the current Bt proteins. In this study, we evaluated the susceptibility of WCR strains resistant and susceptible to Cry3Bb1 to the biological insecticide Spear-T (GS-omega/kappa-Hexatoxin-Hv1a) alone and combined with Cry3Bb1 protein. The activity of Hv1a alone was similar between Cry3Bb1-resistant and susceptible strains (LC50s = 0.95 mg/cm2 and 1.50 mg/cm2, respectively), suggesting that there is no cross-resistance with Cry3Bb1 protein. Effective concentration (EC50), molt inhibition concentration (MIC50), and inhibition concentration (IC50) values of Hv1a alone were also similar between both strains, based on non-overlapping confidence intervals. Increased mortality (64%) was observed on resistant larvae exposed to Hv1a (0.6 mg/cm2) + Cry3Bb1 protein (170.8 µg/cm2) compared to 0% mortality when exposed to Cry3Bb1 alone and 34% mortality to Hv1a alone (0.3 mg/cm2). The time of larval death was not significantly different between Hv1a alone (3.79 mg/cm2) and Hv1a (0.6 mg/cm2) + Cry3Bb1 (170.8 µg/cm2). New control strategies that are not cross-resistant with current insecticides and Bt proteins are needed to better manage the WCR, and Hv1a together with Cry3Bb1 may fit this role.

Effect of Prey Species and Prey Densities on the Performance of Adult Coenosia attenuata.

Mass production of Coenosia attenuata Stein at low cost is very important for their use as a biological control agent. The present study reports the performance of C. attenuata adults when reared on Drosophila melanogaster Meigen or Bradysia impatiens (Johannsem). Different densities (6, 9, 15, 24 and 36 adults per predator) of D. melanogaster or (6, 12, 24, 36 and 48 adults per predator) of B. impatiens were used at 26 ± 1 °C, 14:10 (L:D) and 70 ± 5% RH. The results concluded that C. attenuata adults had higher fecundity, longer longevity and less wing damage when reared on B. impatiens adults compared to D. melanogaster adults. Additionally, C. attenuata adults demonstrated greater difficulty catching and carrying heavier D. melanogaster adults than lighter B. impatiens adults. In this case, 12 to 24 adults of B. impatiens daily per predator were considered optimal prey density in the mass rearing of adult C. attenuata.

Baseline Susceptibility of a Laboratory Strain of Northern Corn Rootworm, Diabrotica barberi (Coleoptera: Chrysomelidae) to Bacillus thuringiensis Traits in Seedling, Single Plant, and Diet-Toxicity Assays.

The northern corn rootworm (NCR), Diabrotica barberi Smith & Lawrence, is an economic pest of maize in the U.S. Corn Belt. The objective of this study was to determine the baseline susceptibility of a laboratory NCR strain to Bt proteins eCry3.1Ab, mCry3A, Cry3Bb1, and Cry34/35Ab1 using seedling, single plant, and diet-toxicity assays. Plant assays were performed in greenhouse using corn hybrids expressing one of the Bt proteins and each respective near-isoline. Diet-toxicity assays, consisting of Bt proteins overlaid onto artificial diet were also conducted. In both plant assays, significantly more larvae survived Cry34/35Ab1-expressing corn compared with all other Bt-expressing corn, and larvae that survived eCry3.1Ab-expressing corn had significantly smaller head capsule widths compared with larvae that survived Cry34/35Ab1-expressing corn. In seedling assays, larvae surviving eCry3.1Ab-expressing corn also had significantly smaller head capsule widths compared with larvae that survived mCry3A-expressing corn. Additionally, larvae that survived mCry3A-expressing corn weighed significantly more than larvae surviving eCry3.1Ab- and Cry34/35Ab1-expressing corn. In single plant assays, no significant differences in larval dry weight was observed between any of the Bt-expressing corn. In diet assays, LC50s ranged from 0.14 (eCry3.1Ab) to 10.6 µg/cm2 (Cry34/35Ab1), EC50s ranged from 0.12 (Cry34/35Ab1) to 1.57 µg/cm2 (mCry3A), IC50s ranged from 0.08 (eCry3.1Ab) to 2.41 µg/cm2 (Cry34/35Ab1), and MIC50s ranged from 2.52 (eCry3.1Ab) to 14.2 µg/cm2 (mCry3A). These results establish the toxicity of four Bt proteins to a laboratory diapausing NCR strain established prior to the introduction of Bt traits and are important for monitoring resistance evolution in NCR field populations.

Author Correction: Multidimensional approach to formulating a specialized diet for northern corn rootworm larvae.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Development of an improved and accessible diet for western corn rootworm larvae using response surface modeling.

The western corn rootworm (WCR), Diabrotica virgifera virgifera LeConte, is an important pest of maize (Zea mays L.). Published WCR diets contain corn root powder, which is not available for purchase, thereby limiting the practical use of diets containing this ingredient. We applied response surface modeling combined with mixture designs to formulate a WCR diet that does not require corn root powder. We developed the new formulation by systematically exploring eight protein ingredients from animal, plant, and yeast sources based on simultaneous evaluation of three life history parameters (weight, molting, and survival). This formulation (WCRMO-2) without corn root powder supported approximately 97% of larval survival and successful molting. Larval weight gain after 10 days of feeding on WCRMO-2 was 4-fold greater than that of larvae feeding on the current best published WCR diet. Additionally, there was no significant difference in these larval performance traits when larvae were reared on WCRMO-2 and the best proprietary WCR diet. A commercial version of WCRMO-2 was tested and found to perform comparably for these traits. These improvements met our goal of a diet comprised of available ingredients that supports performance of WCR larvae equal to or better than publicly available formulations and proprietary formulations.

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 woefully incomplete, yet that knowledge could play an important role in effective pest management strategies. For this study, insects were reared on maize in soils from different locations. Insects from two different laboratory colonies (a diapausing and a non-diapausing colony) 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 possible 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 provide insights into Bt resistance development, improved nutrition in artificial rearing systems, and new management strategies.

Characterization of Corn Root Factors to Improve Artificial Diet for Western Corn Rootworm (Coleoptera: Chrysomelidae) Larvae.

The western corn rootworm, Diabrotica virgifera virgifera LeConte (Coleoptera: Chrysomelidae), is an important economic pest of maize (Zea mays L.) in North America and Europe. Previous efforts to formulate an artificial diet for western corn rootworm larvae highlighted an important role of corn root powder, which had a significant positive impact on several larval developmental traits. Unfortunately, this ingredient is not available for purchase. Toward the goal of developing an artificial diet for western corn rootworm larvae with all ingredients readily accessible, we conducted research to isolate essential growth factors for larval development from corn root powder to improve the performance of diet without corn root powder. For all experiments, multiple life history parameters (survival, weight, and molting) were recorded from 15-d diet bioassays. Corn roots may contain factors that assist in larval growth, but some of these factors were not fully extracted by methanol and remained in the extracted root. Methanolic extracts significantly increased molting to second instar, but did not significantly increase survival, dry weight, or molting to third instar, suggesting the primary corn root substituents affecting these factors cannot be extracted or other extraction methods may be required to extract the essential factors from corn roots. We showed that whole corn root powder was best when used in combination with all the other nutrient sources in the published western corn rootworm formulation. Corn root powder made from proprietary seed and Viking seed has similar value.

Multidimensional approach to formulating a specialized diet for northern corn rootworm larvae.

The northern corn rootworm (NCR), Diabrotica barberi Smith & Lawrence, is a major pest of maize (Zea mays L.). This pest has developed resistance to insecticides and adapted to crop rotation and may already be in the early stages of adaptation to toxins produced by Bacillus thuringiensis (Bt). Toxicity bioassays using artificial diet have proven to be valuable for monitoring resistance in many species, but no artificial diet has been developed specifically for NCR larvae. Toward this end, we first evaluated known Diabrotica diets to identify a starting media. We then developed a specialized diet for NCR using an iterative approach. Screening designs including 8 diet components were performed to identify the principal nutritional components contributing to multiple developmental parameters (survival, weight, and molting). We then applied mixture designs coupled with response surface modeling to optimize a blend of those components. Finally, we validated an improved NCR diet formulation that supports approximately 97% survival and molting, and a 150% increase in larval weight after 10 days of feeding compared with the best previously published artificial diet. This formulation appears suitable for use in diet bioassays as a tool for evaluating the resistance of NCR populations to insecticides.

Comparative Susceptibility of Western Corn Rootworm (Coleoptera: Chrysomelidae) Neonates to Selected Insecticides and Bt Proteins in the Presence and Absence of Feeding Stimulants.

The susceptibility of western corn rootworm, Diabrotica virgifera virgifera LeConte, larvae to nine insecticides from five different classes and to Bt proteins eCry3.1Ab and mCry3A in the presence or absence of feeding stimulants, was estimated in filter paper and diet toxicity assays, respectively. The use of a synthetic feeding stimulant blend of the sugars glucose, sucrose, and fructose plus linoleic acid at a ratio of 30:4:4:0.3 mg/ml of distilled water was evaluated to determine whether they increase the efficacy of insecticides and Bt proteins. The efficacy of thiamethoxam diluted in solutions with feeding stimulants was significantly increased when compared to thiamethoxam dilutions in water (>60-fold). Differences in the efficacy of the other insecticide classes when diluted in feeding stimulant solutions were no greater than fivefold when compared to the insecticides diluted in water. The presence of corn root juice as a natural feeding stimulant diminished toxicity of the insecticides, except for thiamethoxam, even though larval fresh weight was higher when fed on root juice compared to feeding stimulant or water. The use of feeding stimulants in diet toxicity assays did not enhance efficacy of eCry3.1Ab nor mCry3A proteins. Feeding stimulants can be recommended in combination with thiamethoxam to increase larval mortality. These results are discussed in terms of applicability of feeding stimulants to improve susceptibility of western corn rootworm larvae to pesticides in general.

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.

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.

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.

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.

Nutritional Value of Pupae Versus Larvae of Tenebrio molitor (Coleoptera: Tenebrionidae) as Food for Rearing Podisus maculiventris (Heteroptera: Pentatomidae).

Life-table analysis yielded demographic parameter values that indicate that Tenebrio molitor (L.) pupae are potentially more suitable factitious prey to mass-produce the predator Podisus maculiventris (Say) and are more suitable prey than the larvae. P. maculiventris developed faster (23.2 vs. 25.5 d), weighed more (females 80.9 vs. 66.6 mg and males 64.7 vs. 53.7 mg), and had a higher survival rate (0.88 vs. 0.7), fecundity, and reproductive output (87.1 vs. 22.8 eggs/female) when reared on pupae compared with larvae of T. molitor. The total protein content and soluble protein content were significantly higher in pupae (60.2 and 23%, respectively) than larvae (53.1 and 14.4%, respectively). Lipid content was significantly lower in pupae (32.1%) than larvae (35.9%), and larvae had more polyunsaturated fatty acids (83.6 vs. 56.6 mg/g) and less oleic (0.1 mg/g) and steric (6.1 mg/g) acids than pupae (37.3 and 12.3 mg/g, respectively). The total sugar content was not significantly different between pupae and larvae. However, larvae had significantly more fructose than pupae, but pupae had more galactose, glucosamine, glucose, mannose, and trehalose than larvae. Differences in nutritional composition and its impact on predator demographic parameters are potential factors that make the pupal stage a better food source.

Biotransfer, bioaccumulation and effects of herbivore dietary Co, Cu, Ni, and Zn on growth and development of the insect predator Podisus maculiventris (Say).

Increased metal availability in the environment can be detrimental for the growth and development of all organisms in a food web. In part, this toxicity is due to biotransfer or bioaccumulation of metals between trophic levels. We evaluated the survival, growth, and development of a generalist Hemipteran predator (Podisus maculiventris) when fed herbivorous prey (Spodoptera exigua) reared on artificial diet amended with Cu, Zn, Ni, and Co. Predator nymphs were fed S. exigua larvae raised on diet amended with sublethal (Minimum Sublethal Concentration or MSC) or lethal (Minimum Lethal Concentration or MLC) concentrations of each metal, as well as control diet. We determined if metals were biotransferred or bioaccumulated from the diet to herbivore and predator, as well as if predator growth or survival was affected by herbivore diet. Podisus maculiventris fed herbivores raised on MLC levels of both Cu and Zn took significantly longer to mature to adults, whereas their overall survival was not affected by prey diet metal concentration for any metal. Adult weights were significantly reduced for predators raised on herbivores reared on diets amended with the MLC of Cu and Zn. Copper and Zn were bioaccumulated from diet to herbivore and from herbivore to predator, whereas Ni was biotransferred (although concentrations decreased as trophic level increased). The pattern for Co was more complex, with biotransfer the main outcome. Our results show that availability of metals in a food web can affect growth and development of a hemipteran predator, and that metals are transferred between trophic levels, with metal-specific biotransfer and bioaccumulation outcomes.