Microbial technologies are increasingly adopted to improve sustainable agriculture amid escalating economic, regulatory, and ecological pressures, yet few, if any, are supported by mechanistic understanding and are translated into products with demonstrated real-world performance and adoption. Pink-pigmented facultative methylotrophs (PPFMs) are known to promote plant growth and enhance pathogen resistance, but their impact on insect defense remains largely unexplored. Here, we report on a PPFM species, Methylorubrum extorquens strain NLS0042, developed as TS201, a U.S. EPA-registered bioinsecticide that mitigates maize pest damage and improves yield. Across 7 years (2016 to 2022) of field trials at 22 Midwest U.S. locations, TS201 increased maize yield by 220 kg/ha under moderate to high pressure of corn rootworms, the most destructive insect pest complex of maize in North America. Additionally, TS201 improved root architecture and reduced plant lodging. Large-scale on-farm evaluations across 81 sites in eight U.S. states (2023 to 2024) validated its agronomic benefit under diverse production conditions. Transcriptomic and volatile metabolite analyses revealed that TS201 induces anthranilate biosynthesis and accumulation of methyl anthranilate, a volatile compound that repels western corn rootworm larvae. Insect choice assays demonstrated significant larval avoidance of TS201-treated roots and confirmed the repellent effect of methyl anthranilate on corn rootworm larvae. These findings demonstrate that TS201 primes maize defense through multiple modes of action, offering a novel and effective microbial strategy to enhance crop resilience and support sustainable pest management.Copyright (c) 2026 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Mexican corn rootworm, Diabrotica virgifera zeae (Krysan & Smith), and western corn rootworm, D. v. virgifera LeConte, are both subspecies of D. virgifera. Knowledge of Mexican corn rootworm is extremely limited and biological and management assumptions for it, including resistance management programs, are based on western corn rootworm. To better understand the degree of similarity to western corn rootworm and potential management options, Mexican corn rootworm from a Texas field population were exposed to different transgenic corn hybrids, reared on an artificial diet designed for western corn rootworm, and tested for their response to varying doses of Gpp34Ab1/Tpp35Ab1. The results of the field test revealed corn hybrids that contained a single Cry protein were less protected than hybrids expressing two or more proteins with the most protection provided by a hybrid expressing two Cry toxins and a double-stranded RNA (dvSnf7). Mexican corn rootworm larvae grew normally on the artificial diet, though they ultimately weighed less when compared to western corn rootworm larvae. Lastly, Mexican corn rootworm larvae from this population were significantly less susceptible to Gpp34Ab1/Tpp35Ab1 than a susceptible western corn rootworm colony, but the insects responded similarly in terms of weight inhibition. Together, the field and diet toxicity data suggest that the Texas field population of Mexican corn rootworm was tolerant to Gpp34Ab1/Tpp35Ab1 and likely the other Cry proteins designed to control corn rootworms. Currently, fields with similar issues to this situation should incorporate a pyramided corn hybrid expressing dsRNA and Bt proteins.

Pink-pigmented facultative methylotrophs (PPFMs) are known to promote plant growth and enhance pathogen resistance, but their role in insect defense remains largely unexplored. Here, we report that TS201, a U.S. EPA-registered bioinsecticide composed of Methylorubrum extorquens , enhances maize defense against corn rootworms (CRW) and improves yield through plant-mediated mechanisms. Over seven years of small-plot trials at 22 U.S. Midwest locations, TS201 increased yield by 220 kg/ha under moderate to high CRW pressure. Additional trials demonstrated rooting and lodging benefits. Large-scale trials across 81 sites in eight U.S. states (2023–2024) validated its agronomic benefit. Transcriptomic and metabolomic analyses revealed activation of anthranilate production and increased accumulation of methyl anthranilate, a volatile CRW-repellent compound. Insect assays indicated significant larval avoidance of TS201-treated roots. These findings reveal a novel mode of action for PPFM-induced plant defense and demonstrate TS201 as a strategic microbial tool for integration into broad-spectrum integrated pest management programs. ### Competing Interest Statement Anne Phillips, Allison Jack, and Natalie Breakfield are employees of NewLeaf Symbiotics, St. Louis, MO, USA. All other authors declare no competing interests. Funding for this project was provided by NewLeaf Symbiotics.

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.

The western corn rootworm (WCR), Diabrotica virgifera virgifera LeConte, has evolved resistance to nearly every management tactic utilized in the field. This study investigated the resistance mechanisms in a WCR strain resistant to the Bacillus thuringiensis (Bt) protein eCry3.1Ab using dsRNA to knockdown WCR midgut genes previously documented to be associated with the resistance. ATP-binding cassette transporter (ABCC4), aminopeptidase-N, cadherin, and cathepsin-B were previously found to be differentially expressed in eCry3.1Ab-resistant WCR larvae when compared to susceptible larvae after feeding on maize expressing eCry3.1Ab and its near-isoline. Here we compared the susceptibility of resistant and susceptible WCR larvae to eCry3.1Ab protein in presence or absence of dsRNA targeting the above genes using 10-day diet overlay toxicity assays. Combining ABCC4 dsRNA with eCry3.1Ab protein increased susceptibility to Bt protein in WCR-resistant larvae, but the other three genes had no such effect. Among 65 ABC transport genes identified, several were expressed differently in resistant or susceptible WCR larvae, fed on eCry3.1Ab-expressing maize versus its isoline, that may be involved in Bt resistance. Our findings provide strong evidence that ABCC4 is indirectly involved in WCR resistance to eCry3.1Ab protein by enhancing the effects of Bt-induced toxicity.

We identified a single diet formulation that can be used for three Diabrotica species including southern (SCR), western (WCR), and northern corn rootworm (NCR) by evaluating the performance of these pests on specialized diets (F9800B diet for SCR, WCRMO-2 diet for WCR, and NCRMO-1 diet for NCR) and a larval diet (F9772 diet) widely used for lepidopteran species. After 10 days of rearing, the WCRMO-2 diet yielded better or equal larval growth and development of all three rootworm species compared to other diets. For SCR larvae, the WCRMO-2 diet outperformed other diets. Larval fresh weight, percent molt to 2nd instar, and percent molt to 3rd instar on the WCRMO-2 diet were 12-fold, 2.7-fold, and 14-fold increases, respectively compared to that of the F9800B diet. Significantly more SCR larvae survived on the WCRMO-2 diet (98.9%) than on the F9800B diet (90.6%). The WCRMO-2 diet supported WCR and NCR larvae equal to the NCRMO-1 diet and better than other diets. The F9772 diet was the worst diet of all examined species. The availability of a universal diet (the WCRMO-2 diet) for the three Diabrotica species would facilitate research programs to monitor resistance development and develop new control tactics targeting these important pests.

Sustainable agricultural management practices aimed at improving soil health can alter the soil microbiome, which can influence plant health and defenses against insects. The western corn rootworm (WCR), Diabrotica virgifera virgifera LeConte, is a major belowground pest of corn. Pest management relies heavily on the planting of transgenic crops expressing Bacillus thuringiensis ( Bt ) toxins. In this study, we ask how corn-WCR interactions via the soil microbiome are affected by a conservation management system (extended corn-soybean-wheat rotation with cover crops under no-till) compared with a conventional management system (corn-soybean rotation under mulch tillage and no cover crops) when combined with transgenic Bt corn. To do this, we applied soil microbes from the conservation and conventional management systems to two corn lines, one producing Bt and one non-Bt. We then reared Bt-resistant and Bt-susceptible WCR on inoculated seedlings to examine plant and insect changes in fitness. We found that Bt was effective against susceptible larvae in both soil treatments. Bt-resistant larvae were ~ 20% smaller when reared in the presence of soil microbes from the conservation management system. Thus, control of Bt-resistant WCR may be improved in a conservation system without sacrificing Bt effectiveness in susceptible insects. Comparing the microbial communities using 16S rRNA sequencing revealed that management practices influenced the microbiomes associated with the soil and the plant rhizosphere, but not WCR. Our findings suggest value for growers in utilizing conservation management practices, such as no-till and cover crops, in agricultural systems through bottom-up changes to plant–insect interactions via the soil microbiome.

Artificial diets are widely used to produce insects for research and education programs. Completed diets, in which the diets are fully made from individual ingredients and ready to use, often have high water activity, making them vulnerable to degradation. Proper storage is critical to maintaining diet quality, yet the storage conditions are not well investigated. In this study, we characterized the effects of storage conditions (temperatures and storage duration) on the quality of a diet capable of rearing both specialist and generalist insect species. The completed diet, produced by both private industry and a USDA-Agricultural Research Service laboratory, was exposed to varying temperatures during a 24-hour transit over 1600 km. After transit, it was stored at 4°C for a total storage period of 28 days. In a separate experiment, the completed diet was stored immediately after diet production at five fixed temperatures (-20, 4, 22, 25, and 33°C) for up to 28 days. For both experiments, at 5 intervals after storage (1, 7, 14, 21, and 28 days), diet quality was accessed by life history parameters (survival, molting, and weight) of western corn rootworm (Diabrotica virgifera virgifera LeConte) larvae, the most serious maize pest in the United States. Our results showed that exposure to varying temperatures between -2°C and 27°C for 24 hours had no significant impact on diet quality. However, extended storage (beyond 24 hours) at any of the fixed temperatures negatively affected diet quality. Insects reared on diets stored for over 24 hours at fixed temperatures ranging from -20°C to 33°C had significant declines in performance. Among the tested temperatures, -20°C and 4°C were found to be the most effective for preserving diet quality. At these low temperatures, there were no significant changes in insect weight and survival for diets stored within 21 and 28 days, respectively, though molting was significantly reduced within 7 days of storage. These findings provide the base of information on the storage conditions for completed diets, supporting the production of healthy insects.

Insect resistance to toxins derived from Bacillus thuringiensis (Bt) is a major issue in agriculture. Resistance to Bt has been linked to the loss of toxin binding sites within the insect, changes within the gut microbiota, and midgut tissue regeneration. Histopathological documentation of intoxication and resistance to Bt is lacking for rootworms in the genus Diabrotica (Coleoptera: Chrysomelidae), a major target of Bt corn. Here, we document the morphological response of both Bt-resistant and Bt-susceptible larval western corn rootworm, Diabrotica virgifera virgifera LeConte, to intoxication with eCry3.1Ab. Gut lumen structural differences are subtle between the two colonies when feeding on non-Bt corn. However, upon ingestion of Bt-corn roots, susceptible larvae develop symptoms indicative of gut disruption by Bt, whereas resistant larvae incur milder effects. Mild disruption of the peritrophic matrix and gut lumen is accompanied by stem cell proliferation that may lead to midgut tissue regeneration. These results help contextualize the multifaceted nature of Bt-resistance in western corn rootworm for the first time from a histopathological perspective.

Western corn rootworm, Diabrotica virgifera virgifera (LeConte) (Coleoptera: Chrysomelidae), is the most serious economic pest of maize, Zea mays (L.) (Poales: Poaceae), in the U.S. Corn Belt and also threatens production in Europe. Traditional management options have repeatedly failed over time as western corn rootworm rapidly develops resistance to insecticides, transgenic maize and even crop rotation. Traits that improve host plant resistance and tolerance are highly sought after by plant breeders for crop protection and pest management. However, maize resistance to western corn rootworm appears to be highly complex and despite over 75 yr of breeding efforts, there are no naturally resistant hybrids available commercially. Using phenotypic data from field and greenhouse experiments on a highly diverse collection of 282 inbred lines, we screened and genetically mapped western corn rootworm-related traits to identify genetic loci which may be useful for future breeding or genetic engineering efforts. Our results confirmed that western corn rootworm resistance is complex with relatively low heritability due in part to strong genotype by environment impacts and the inherent difficulties of phenotyping below ground root traits. The results of the Genome Wide Associated Study identified 29 loci that are potentially associated with resistance to western corn rootworm. Of these loci, 16 overlap with those found in previous transcription or mapping studies indicating a higher likelihood they are truly involved in maize western corn rootworm resistance. Taken together with previous studies, these results indicate that breeding for natural western corn rootworm resistance will likely require the stacking of multiple small effect loci.

Abstract 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.

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.

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.

The western corn rootworm (WCR) Diabrotica virgifera virgifera (Coleoptera: Chrysomelidae) remains one of the economically most important pests of maize ( Zea mays ) due to its adaptive capabilities to pest management options. This includes the ability to develop resistance to some of the commercial pesticidal proteins originating from different strains of Bacillus thuringiensis . Although urgently needed, the discovery of new, environmentally safe agents with new modes of action is a challenge. In this study we report the discovery of a new family of binary pesticidal proteins isolated from several Chryseobacterium species. These novel binary proteins, referred to as GDI0005A and GDI0006A, produced as recombinant proteins, prevent growth and increase mortality of WCR larvae, as does the bacteria. These effects were found both in susceptible and resistant WCR colonies to Cry3Bb1 and Cry34Ab1/Cry35Ab1 (reassigned Gpp34Ab1/Tpp35Ab1). This suggests GDI0005A and GDI0006A may not share the same binding sites as those commercially deployed proteins and thereby possess a new mode of action. This paves the way towards the development of novel biological or biotechnological management solutions urgently needed against rootworms.

Adenanthera pavonina, an underutilized tropical tree, is being promoted as an alternative food source for meeting the nutritional needs of human and animals. In this study, we have shown that trypsin inhibitors as one of the predominant proteins in the seeds of A. pavonina. DE-52 column chromatography resulted in the identification of four peaks with trypsin inhibitor activity. SDS-PAGE and immunoblot analyses revealed DE-52 peaks A and B were enriched in 17 and 15 kDa proteins and these proteins cross-reacted against soybean trypsin inhibitor antibodies. Simulated gastric fluid digestion revealed that the 15-17 kDa proteins are resistant to pepsin digestion. Roasting the seeds lowered the trypsin inhibitor activity while boiling intact seeds elevated the enzyme activity. However, the trypsin inhibitor activity was completely abolished when the seeds were boiled without their seed coats. Immunohistochemical detection and confocal microscopy demonstrated that trypsin inhibitors were localized in the cell cytosol.

The development of new biopesticides to control the western corn rootworm (WCR), Diabrotica virgifera virgifera LeConte, is urgent due to resistance evolution to various control methods. We tested an air-dried non-live preparation of Chromobacterium species Panama ( Csp_P ), against multiple corn rootworm species, including Bt-resistant and -susceptible WCR strains, northern (NCR, D. barberi Smith & Lawrence), and southern corn rootworm (SCR, D. undecimpunctata howardi Barber), in diet toxicity assays. Our results documented that Csp_P was toxic to all three corn rootworms species based on lethal (LC 50 ), effective (EC 50 ), and molt inhibition concentration (MIC 50 ). In general, toxicity of Csp_P was similar among all WCR strains and ~ 3-fold less toxic to NCR and SCR strains. Effective concentration (EC 50 ) was also similar among WCR and SCR strains, and 5-7-fold higher in NCR strains. Molt inhibition (MIC 50 ) was similar among all corn rootworm strains except NCR diapause strain that was 2.5–6-fold higher when compared to all other strains. There was no apparent cross-resistance between Csp_P and any of the currently available Bt proteins. Our results indicate that Csp_P formulation was effective at killing multiple corn rootworm strains including Bt-resistant WCR and could be developed as a potential new management tool for WCR control.

Microbial communities associated with animals vary based on both intrinsic and extrinsic factors. Of many possible determinants affecting microbiome composition, host phylogeny, host diet, and local environment are the most important. How these factors interact across spatial scales is not well understood. Here, we seek to identify the main influences on microbiome composition in a specialist insect, the western corn rootworm (WCR; Diabrotica virgifera virgifera), by analyzing the bacterial communities of adults collected from their obligate host plant, corn (Zea mays), across several geographic locations and comparing the patterns in communities to its congeneric species, the northern corn rootworm (NCR; Diabrotica barberi). We found that bacterial communities of WCR and NCR shared a portion of their bacterial communities even when collected from disparate locations. However, within each species, the location of collection significantly influenced the composition of their microbiome. Correlations of geographic distance between sites with WCR bacterial community composition revealed different patterns at different spatial scales. Community similarity decreased with increased geographic distance at smaller spatial scales (~25 km between the nearest sites). At broad spatial scales (>200 km), community composition was not correlated with distances between sites, but instead reflected the historical invasion path of WCR across the United States. These results suggest bacterial communities are structured directly by dispersal dynamics at small, regional spatial scales, while landscape-level genetic or environmental differences may drive community composition across broad spatial scales in this specialist insect.

We studied thyroid hormone (TH) conjugation in fasted trout by incubating isolated hepatocytes with either [125I]T4or [125I]T3, and by analyzing bile from trout injected with either [125I]T4or [125I]T3. Glucuronide conjugates were identified by hydrolysis with β-glucuronidase and sulfate conjugates by acid solvolysis with ethyl acetate/trifluoroacetic acid (1%). We used Sephadex LH-20 chromatography to concentrate the conjugate fractions from hepatocyte incubates prior to HPLC analysis. Glucuronide conjugates of T4and T3were producedin vitroand glucuronides of T4, T3, and 3,3′-T2were foundin vivo.Sulfation of T4occurredin vitroandin vivo.T3sulfation was not establishedin vitro,but sulfate conjugates of T3and T2were found in bile. Significant proportions of unconjugated T4and T3also occurred in the bile. We conclude that (i) as in other vertebrates, iodothyronines undergo hepatic glucuronidation and are excreted as such in the bile, (ii) T4and T3undergo sulfation, and in contrast to mammals, are excreted in significant amounts in the bile, (iii) 3,3′-T2, a prominent deiodination product of T3, is excreted as both glucuronide and sulfate conjugates, and (iv) the isolated hepatocyte system is appropriate for studying aspects of TH metabolism in trout.

The western corn rootworm (WCR), Diabrotica virgifera virgifera LeConte, is the most serious pest of maize ( Zea mays L.) in the U.S. Corn Belt and parts of Europe. Transgenic maize hybrids expressing at least one of the four currently available insecticidal toxins from Bacillus thuringiensis (Bt) Berliner, currently the most widely adopted control method in continuous maize, have faltered due to the emergence of resistance. The resistance mechanisms of WCR to Bt toxins are not fully understood. We identified metabolic profiles of susceptible and resistant WCR larvae fed on maize hybrids expressing each of three available Cry3 proteins (eCry3Ab1, mCry3A, and Cry3Bb1) targeting corn rootworms and a control non-Bt maize via an untargeted metabolomics approach. Over 580 unique metabolites found in WCR larvae were classified into different pathways (amino acids, carbohydrates, cofactors and vitamins, energy, lipid, nucleotide, peptide, and xenobiotics). By exploring shifts in WCR larval metabolome exclusively by Bt toxins, several candidate metabolites and metabolic pathways were identified in susceptible and resistant larvae that may be involved in defense against or recovery from Bt ingestion by these larvae. These findings would provide mechanistic insights into altered metabolic pathways associated with the resistance mechanisms of WCR to Bt toxins.

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.