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.

Extended Sentinel Monitoring of Helicoverpa zea Resistance to Cry and Vip3Aa Toxins in Bt Sweet Corn: Assessing Changes in Phenotypic and Allele Frequencies of Resistance.

Transgenic corn and cotton that produce Cry and Vip3Aa toxins derived from Bacillus thuringiensis (Bt) are widely planted in the United States to control lepidopteran pests. The sustainability of these Bt crops is threatened because the corn earworm/bollworm, Helicoverpa zea (Boddie), is evolving a resistance to these toxins. Using Bt sweet corn as a sentinel plant to monitor the evolution of resistance, collaborators established 146 trials in twenty-five states and five Canadian provinces during 2020-2022. The study evaluated overall changes in the phenotypic frequency of resistance (the ratio of larval densities in Bt ears relative to densities in non-Bt ears) in H. zea populations and the range of resistance allele frequencies for Cry1Ab and Vip3Aa. The results revealed a widespread resistance to Cry1Ab, Cry2Ab2, and Cry1A.105 Cry toxins, with higher numbers of larvae surviving in Bt ears than in non-Bt ears at many trial locations. Depending on assumptions about the inheritance of resistance, allele frequencies for Cry1Ab ranged from 0.465 (dominant resistance) to 0.995 (recessive resistance). Although Vip3Aa provided high control efficacy against H. zea, the results show a notable increase in ear damage and a number of surviving older larvae, particularly at southern locations. Assuming recessive resistance, the estimated resistance allele frequencies for Vip3Aa ranged from 0.115 in the Gulf states to 0.032 at more northern locations. These findings indicate that better resistance management practices are urgently needed to sustain efficacy the of corn and cotton that produce Vip3Aa.

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.

Influence of Holding Conditions and Storage Duration of Halyomorpha halys (Hemiptera: Pentatomidae) Eggs on Adventive and Quarantine Populations of Trissolcus japonicus (Hymenoptera: Scelionidae) Behavior and Parasitism Success.

Halyomorpha halys (Stål) is an invasive pest in the United States and other countries. In its native range, H. halys eggs are parasitized by a co-evolved parasitoid, Trissolcus japonicus (Ashmead). In the United States, T. japonicus, a classical biological control candidate, is being redistributed in many states where adventive populations exist. To establish if H. halys egg holding conditions affect T. japonicus foraging behavior or successful parasitism, naïve, female parasitoids from an adventive population were allowed to forage in laboratory bioassay arenas with either fresh or frozen (-20 or -80°C) egg masses, the latter held for five durations ranging from 1 h to 112 d. Parasitoid movements were recorded for 1 h. Thereafter, parasitoids were transferred with the same egg mass for 23 h. Additionally, female parasitoids from a quarantine colony were exposed to: 1) pairs of fresh egg masses and egg masses frozen at -40°C (>24 h) or 2) a single fresh egg mass or egg mass frozen at -40°C (<1 h). All exposed egg masses were held to assess progeny emergence. In the foraging bioassay, holding temperature and storage duration appeared to influence host-finding and host quality. Egg masses held at -80°C and fresh egg masses resulted in significantly greater levels of parasitism and progeny emergence compared with eggs held at -20°C. No differences were recorded between egg masses held at -40°C for ≤1 h and fresh egg masses. These results will help refine methods for preparation of egg masses for sentinel monitoring and parasitoid mass rearing protocols.

Integrating Trissolcus japonicus (Ashmead, 1904) (Hymenoptera: Scelionidae) into Management Programs for Halyomorpha halys (Stål, 1855) (Hemiptera: Pentatomidae) in Apple Orchards: Impact of Insecticide Applications and Spray Patterns.

Halyomorpha halys (Stål, 1855) (Hemiptera: Pentatomidae) is an invasive species in the United States, where it has caused significant damage to specialty crops, including apples. While integrated pest management techniques have been developed for H. halys in apple, including spray application techniques, it is unknown how these techniques affect foraging, adventive Trissolcus japonicus (Ashmead, 1904) (Hymenoptera: Scelionidae), and its offspring. In this study, egg masses (unparasitized and 2 and 7 day parasitized pre-treatment) were placed in apple orchards in treated and untreated locations that received full block insecticide applications or reduced application techniques, including border row or alternate row middle applications. Bifenthrin, thiamethoxam + λ-cyhalothrin, clothianidin, and methomyl were evaluated. Egg masses were retrieved 24 h after spray applications. For 2 and 7 day parasitized pre-treatment, adult T. japonicus emergence was recorded from each egg mass. For unparasitized egg masses, T. japonicus females were given 24 h to forage and oviposit on post-treatment egg masses with female survivorship, and adult emergence from egg masses was recorded. Female survivorship was significantly lower on post-treatment egg masses retrieved from areas receiving bifenthrin applications. Emergence from post-treatment egg masses was affected by thiamethoxam + λ-cyhalothrin, bifenthrin, and methomyl in some treated areas, whereas less impact was observed on 2 and 7 day pre-treatment parasitized egg masses in general. These data provide further insights into H. halys management and the potential impact of T. japonicus in sprayed orchard agroecosystems.

Development of Behaviorally Based Monitoring and Biosurveillance Tools for the Invasive Spotted Lanternfly (Hemiptera: Fulgoridae).

The spotted lanternfly, Lycorma delicatula White, is an invasive planthopper (Hemiptera: Fulgoridae) that was first detected in the United States in Berks County, PA, in 2014, and has since spread in the mid-Atlantic region. This phloem-feeding pest has a broad host range, including economically important crops such as grape where their feeding causes dieback of infested plants. Monitoring the presence and abundance of L. delicatula is of utmost importance to develop pest management approaches. Current monitoring practices include sticky bands deployed on tree trunks, sometimes paired with commercially available methyl salicylate lures. A drawback associated with sticky bands is the high numbers of nontarget captures. Here, we developed traps for L. delicatula based on a circle trap originally designed for weevils. These traps are comprised of a screen funnel that wraps around the trunk of a tree and guides individuals walking up the trunk into a collection device. In 2018 and 2019, we compared circle trap designs with sticky bands in Pennsylvania and Virginia. In both years, circle trap designs yielded captures that were equivalent to or exceeded captures of L. delicatula on sticky bands. Nontarget captures were significantly lower for circle traps compared with sticky bands. Presence of a methyl salicylate lure in association with traps deployed on host trees or vertical tree-mimicking posts did not increase L. delicatula captures compared with unbaited traps. Circle traps, modified using vinyl screen and a larger collection device, present an alternative to the current approach with reduced nontarget capture for monitoring L. delicatula.

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.

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.

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.

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.