Host resistance to Bacillus thuringiensis is linked to altered bacterial community within a specialist insect herbivore.

Evolution of resistance to transgenic crops producing toxins from Bacillus thuringiensis (Bt) threatens the sustainability of the technology. Examination of resistance mechanisms has largely focused on characterization of mutations in proteins serving as Bt toxin binding sites. However, insect microbial communities have the potential to provide host resistance to pesticides in a myriad of ways. Previous findings suggest the killing mechanism of Bt relies on enteric bacteria becoming pathogenic in the disrupted gut environment of the insect following Bt intoxication. Thus, here we hypothesized that resistance to Bt would alter the microbiome composition of the insect. Previous studies have manipulated the microbiome of susceptible insects and monitored their response to Bt. In our study, we characterized the associated bacterial communities of Bt-resistant and -susceptible western corn rootworms, a widespread pest of maize in the United States. We found resistant insects harbor a bacterial community that is less rich and distinct from susceptible insects. After feeding on Bt-expressing maize, susceptible insects exhibited dysbiosis of the associated bacterial community, whereas the community within resistant insects remained relatively unchanged. These results suggest resistance to Bt produces alterations in the microbiome of the western corn rootworm that may contribute to resistance. We further demonstrated that by itself, feeding on Bt toxin-expressing seedlings caused a shift in the microbiota. This work provides a broader picture of the effect stressors have on microbiome composition, and the potential heritable changes induced as a result of intense selection.

Expression Profiles of Digestive Genes in the Gut and Salivary Glands of Tarnished Plant Bug (Hemiptera: Miridae).

Host plant preference of agricultural pests may shift throughout the growing season, allowing the pests to persist on wild hosts when crops are not available. Lygus Hahn (Hemiptera: Miridae) bugs are severe pests of cotton during flowering and fruiting stages, but can persist on alternative crops, or on weed species. Diversity of digestive enzymes produced by salivary glands and gut tissues play a pivotal role in an organism's ability to utilize various food sources. Polyphagous insects produce an array of enzymes that can process carbohydrates, lipids, and proteins. In this study, the digestive enzyme repertoire of the tarnished plant bug, Lygus lineolaris (Palisot de Beauvois), was identified by high-throughput sequencing followed by cDNA cloning and sequencing. This study identified 87 digestive genes, including 30 polygalacturonases (PG), one ß-galactosidase, three α-glucosidases, six ß-glucosidases, 28 trypsin-like proteases, three serine proteases, one apyrase-like protease, one cysteine protease, 12 lipases, and two transcripts with low similarity to a xylanase A-like genes. RNA-Seq expression profiles of these digestive genes in adult tarnished plant bugs revealed that 57 and 12 genes were differentially expressed in the salivary gland and gut (≥5-fold, P ≤ 0.01), respectively. All polygalacturonase genes, most proteases, and two xylanase-like genes were differentially expressed in salivary glands, while most of the carbohydrate and lipid processing enzymes were differentially expressed in the gut. Seven of the proteases (KF208689, KF208697, KF208698, KF208699, KF208700, KF208701, and KF208702) were not detected in either the gut or salivary glands.

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.

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.

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.

eCry3.1Ab-resistant Western Corn Rootworm Larval Midgut Epithelia Respond Minimally to Bt Intoxication.

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.

Toxicometabolomic profiling of resistant and susceptible western corn rootworm larvae feeding on Bt maize seedlings.

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.

Chromobacterium Csp_P biopesticide is toxic to larvae of three Diabrotica species including strains resistant to Bacillus thuringiensis.

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 (LC50), effective (EC50), and molt inhibition concentration (MIC50). In general, toxicity of Csp_P was similar among all WCR strains and ~ 3-fold less toxic to NCR and SCR strains. Effective concentration (EC50) was also similar among WCR and SCR strains, and 5-7-fold higher in NCR strains. Molt inhibition (MIC50) 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.

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 [...].

Restoration of susceptibility following removal of selection for Cry34/35Ab1 resistance documents fitness costs in resistant population of western corn rootworm, Diabrotica virgifera virgifera.

BACKGROUND: Management of the corn pest, western corn rootworm (WCR), Diabrotica virgifera virgifera (LeConte) (Coleoptera: Chrysomelidae), relies heavily on the planting of transgenic corn expressing toxins produced by the bacterium Bacillus thuringiensis (Bt). This has resulted in the evolution of resistance to all of the four commercially available Bt toxins targeting coleopteran insects. In this study, we evaluated the susceptibility of a Cry34/35Ab1-resistant WCR colony in seedling and diet toxicity assays after removal from selection for six and nine generations. In addition, female fecundity, egg fertility, adult lifespan, larval development, and adult emergence were evaluated in two Cry34/35Ab1-resistant and two susceptible WCR colonies to assess fitness costs. RESULTS: Susceptibility to Cry34/35Ab1 was restored in a colony removed from selection after six and nine generations based on diet toxicity assays and comparisons of relative survival, head capsule width, and dry weight in plant assays. Thus, pronounced fitness costs associated with resistance to Cry34/35Ab1 were documented by susceptibility being restored within six generations. In separate studies evaluating specific fitness costs, larval fitness when reared on isoline corn did not differ between resistant and susceptible colonies. However, beetles from susceptible colonies lived longer than resistant beetles which resulted in females from susceptible colonies producing significantly more eggs than resistant colonies, with no differences in egg fertility. CONCLUSIONS: The presence of a fitness cost that may contribute to the restoration of susceptibility to Bt has not been documented in other Cry3-resistant WCR populations and could have significant impact on the deployment of resistance management practices. Published 2021. This article is a U.S. Government work and is in the public domain in the USA.

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.

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.

Analysis of ESTs generated from immune-stimulated hemocytes of larval Heliothis virescens.

Heliothis virescens immunome components responding to baculoviral and bacterial infection were identified from expressed sequence tags (ESTs) generated from an immune-stimulated larval hemocyte cDNA library. A total of 5548 ESTs were generated comprising 448 contigs and 1114 singletons, totaling 1606 putative transcripts 1101 of which had BLAST scores, including many known orthologs from other insect species. Orthologs of known or putative immune function were identified among them melanization pathway components, proteases, antibacterial proteins, lectins, bacteria-binding proteins, ferritins, scavenger receptors, cell surface receptors, signaling pathway components, and stress response enzymes. Additionally, many enzymes of central metabolism, cytoskeletal, mitochondrial, and ribosomal components, as well as transcriptional and translational regulators were identified. The effect of bacterial and baculoviral infection upon transcript levels of three identified immunome targets from among the ESTs was quantitated using real time PCR. Scolexin-B, C-type lectin and growth-blocking peptide binding protein transcripts were significantly elevated by bacterial infection. Per os infection with the baculovirus Helicoverpa zea single nucleopolyhedrovirus however did not significantly alter transcript levels of these three genes. The ESTs reported here are the first large scale report of the H. virescens immunome responding to entomopathogens, and represent a first step to a more complete transcriptome for this pest moth.

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.

Differential gene expression in response to eCry3.1Ab ingestion in an unselected and eCry3.1Ab-selected western corn rootworm (Diabrotica virgifera virgifera LeConte) population.

Diabrotica virgifera virgifera LeConte, the western corn rootworm (WCR) is one of the most destructive pests in the U.S. Corn Belt. Transgenic maize lines expressing various Cry toxins from Bacillus thuringiensis have been adopted as a management strategy. However, resistance to many Bt toxins has occurred. To investigate the mechanisms of Bt resistance we carried out RNA-seq using Illumina sequencing technology on resistant, eCry3.1Ab-selected and susceptible, unselected, whole WCR neonates which fed on seedling maize with and without eCry3.1Ab for 12 and 24 hours. In a parallel experiment RNA-seq experiments were conducted when only the midgut of neonate WCR was evaluated from the same treatments. After de novo transcriptome assembly we identified differentially expressed genes (DEGs). Results from the assemblies and annotation indicate that WCR neonates from the eCry3.1Ab-selected resistant colony expressed a small number of up and down-regulated genes following Bt intoxication. In contrast, unselected susceptible WCR neonates expressed a large number of up and down-regulated transcripts in response to intoxication. Annotation and pathway analysis of DEGs between susceptible and resistant whole WCR and their midgut tissue revealed genes associated with cell membrane, immune response, detoxification, and potential Bt receptors which are likely related to eCry3.1Ab resistance. This research provides a framework to study the toxicology of Bt toxins and mechanism of resistance in WCR, an economically important coleopteran pest species.

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.

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.

Cloning and characterization of the secreted hemocytic prophenoloxidases of Heliothis virescens.

The plasma enzyme phenoloxidase plays an important role in host resistance against viral, bacterial, fungal, filarial, and parasitoid challenge. Two Heliothis virescens prophenoloxidase transcripts, HvPPO-1 and HvPPO-2, were assembled from ESTs derived from a hemocyte cDNA library. The 2,363-bp HvPPO-1 contig encoded a 696-amino acid protein. The 3,255-bp HvPPO-2 contig encoded a 684-amino acid protein. Hemocyte and fat body transcript levels of HvPPO-1 were slightly elevated by bacterial infection in 5th instar larvae; however, HvPPO-2 expression was not significantly elevated above controls by bacterial infection. Per os infection of 4th instar larvae with the baculovirus Helicoverpa zea SNPV (HzSNPV) had a mild but significant suppressive effect upon fat body and hemocytic HvPPO-1 expression when compared to expression in same-aged controls. HvPPO-2 expression levels in fat bodies and hemocytes from 4th instar larvae was not significantly altered by HzSNPV infection. HzSNPV infection of 5th instar larvae caused no significant alteration of HvPPO-1 or of HvPPO-2 expression in either fat bodies or hemocytes. Thus, even though prophenoloxidase subunits are constitutively expressed at high levels in larval H. virescens hemocytes and fat bodies, the subunit HvPPO-1 is differentially regulated by bacterial and baculoviral infection.