Baseline susceptibility of tobacco budworm (Lepidoptera: Noctuidae) to Cry1F toxin from Bacillus thuringiensis

Transgenic cotton, Gossypium hirsutum L., lines expressing both Cry1F and Cry1Ac insecticidal proteins from Bacillus thuringiensis (Bt) have been commercially available in the United States since 2005. Both Bt proteins are highly effective against tobacco budworm, Heliothis virescens (F.), and other lepidopteran pests of cotton. Although CrylAc has been available in Bt cotton since 1996, the Cry1F component is relatively new. As part of the proactive resistance management program for Cry1F/Cry1Ac cotton, a susceptibility-monitoring program is being implemented. Baseline variation in the susceptibility to Cry1F in field populations of tobacco budworm was measured. There was a three-fold variation in the amount of Cry1F needed to kill 50% of the neonates from 15 different field populations from the southern and central United States. Future variation in susceptibility of tobacco budworm populations to Cry1F or even resistance evolution could be documented based on this baseline data. A candidate diagnostic concentration was determined that may be efficiently used to identify individuals that potentially carry major alleles conferring field-relevant resistance to Cry1F before such alleles spread through field populations.     

Susceptibility of bollworm and tobacco budworm (Lepidoptera: Noctuidae) to Cry2Ab2 insecticidal protein

Susceptibilities of 82 bollworm, Helicoverpa zea (Boddie), and 44 tobacco budworm, Heliothis virescens (F.) (Lepidoptera: Noctuidae), populations to Cry2Ab2 protein were measured in diet incorporated assays at the University of Arkansas from 2002 to 2005. Resulting data were used to calculate overall (pooled data) estimates of species susceptibility for future benchmarks of resistance. Variabilities among populations also were studied by comparing regressions for individual populations and calculating mean susceptibilities for different subgroups of the colonies studied. Individual lethal concentration (LC50) estimates for nine laboratory, seven laboratory-cross, and 28 field populations of H. virescens varied up to 48-fold when adjusted for the response of the most susceptible laboratory colony studied. Mean susceptibilities of all laboratory, laboratory-cross, or field colonies varied only two-fold. When grouped by host plants, populations collected on tobacco, Nicotiana tabacum (L.), seemed to be less susceptible than those collected on other host plants. Individual LC50 values for 82 laboratory, laboratory-cross and field populations of H. zea varied up to 37-fold. Mean LC50 values of all laboratory, laboratory-cross, or field populations varied only three-fold. Susceptibilities of populations from Bollgard cotton were up to four-fold less than those from Bacillus thuringiensis corn, Zea mays L. Field populations collected during late season were generally less susceptible than those collected early in the season. Across the two species, H. zea was less sensitive to Cry2Ab2 than H. virescens. Both species seem to be less sensitive to Cry2Ab2 than to CrylAc.     

Examination of the F2 screen for rare resistance alleles to Bacillus thuringiensis toxins in the diamondback moth (Lepidoptera: Plutellidae)

A synthetic laboratory population of the diamondback moth, Plutella xylostella (L.), was used to test the F2 screen developed for detecting the frequency of rare resistance alleles to Cry1Ac and Cry1C toxins of Bacillus thuringiensis (Bt). Of the 120 single-pair matings set up, 106 produced enough F2 families for screening of Cry1Ac or Cry1C resistance alleles using both transgenic broccoli and an artificial diet overlay assay with a diagnostic dose. When using Bt broccoli plants as the F2 screen method, only one F2 family was detected for Cry1Ac resistance and no family was detected for Cry1C resistance. Six families were detected for either Cry1Ac or Cry1C resistance using the diet assay. The survivors in the diagnostic diet assay were crossed with the resistant individuals to confirm their resistance genotypes. Four F2 families were confirmed to contain one copy of an allele resistant to Cry1Ac in the original single-pairs and four other F2 families contained an allele resistant to Cry1C. Our results suggest that using transgenic plants expressing a high level of a Bt toxin in an F2 screen may underestimate the frequency of resistance alleles with high false negatives, or fail to detect true resistance alleles. The diagnostic diet assay was a better F2 screen method to detect alleles, especially for the Cry1Ac resistance with monogenic inheritance in the diamondback moth. The estimated probabilities of false positives and false negatives were 33 and 1%, respectively, for detecting Cry1Ac resistance at the allele frequency of 0.012 using the diagnostic diet assay. Careful validation of the screening method for each insect-crop system is necessary before the F2 screen can be used to detect rare Bt resistance alleles in field populations.     

Regulation of sex pheromone biosynthesis in the oriental tobacco budworm,Helicoverpa assulta (Lepidoptera: Noctuidae)

The five components, Z9-16:Ald, 16:Ald, Z11-16:Ald, Z9-16:Ac and Z11-16:Ac, of the sex pheromone in Helicoverpa assulta were mostly detected during the scotophase, with their titer peaking at the 4th hour during the scotophase under a 15L/9D regime. They were not detected during the photophase, but were produced during the photophase when decapitated females were injected with extracts of virgin female (FHE), male heads (MHE), homogenates of the brain-suboesophageal ganglion complex (Br-SOG), or synthetic Hez-PBAN. Production of Z9-16:Ald increased during the first 45min after FHE injection and then declined to a very low level after 2h during the photophase. Synthetic Hez-PBAN stimulated the sex pheromone glands for at least 2h and the effect was more or less proportional to the concentration of the peptide. From the present results, we suggest the following: PBAN is released continuously into the haemolymph to stimulate pheromone biosynthesis at least during the first half of the scotophase, PBAN is synthesized and accumulated independent of photoperiod or sex, and the release starts just prior (about 1h) to the beginning of the scotophase.     

Selective feeding of tobacco budworm and bollworm (Lepidoptera: Noctuidae) on meridic diet with different concentrations of Bacillus thuringiensis proteins

Laboratory experiments were conducted to evaluate the behavior of bollworm, Helicoverpa zea (Boddie), and tobacco budworm, Heliothis virescens (F.), larvae on meridic diet with different concentrations of the Cry1Ac and Cry2Ab proteins from Bacillus thuringiensis subsp. kurstaki Berliner. The proteins used in these experiments are the ones in commercially available Bollgard and Bollgard II cotton. Both bollworms and tobacco budworms selectively fed on nontreated diet compared with diet treated with Cry1Ac. In addition, bollworms exhibited a concentration response with Cry1Ac. In general, bollworms selected diet with low concentrations of Cry1Ac compared with diet with higher concentrations of Cry1Ac. For Cry2Ab, the avoidance was not as prominent as that observed for Cry1Ac. Based on results from no-choice assays, the Cry1Ac and Cry2Ab concentrations used in choice assays represented a wide range of biological activity on both species. The lower concentrations provided low levels of mortality, whereas the higher concentrations provided high levels of mortality. Also, the developmental times of larvae were longer at higher concentrations of both proteins. These data provide important information about the behavioral response of key cotton pests to the B. thuringiensis proteins found in commercially available transgenic cotton. This information will be important to develop accurate scouting and management procedures for Bollgard and Bollgard II cotton.     

Comparison between in vivo and in vitro cutaneous penetration of fenvalerate in tobacco budworm (Lepidoptera: Noctuidae)

In vivo and in vitro penetration of fenvalerate were compared in the 3-d-old fifth-instar tobacco budworm, Heliothis virescens (F). In the in vitro studies, a piece of cuticle was excised and mounted on a diffusion cell, and the amount of radioactivity present in the cuticle and medium was determined at various time intervals. At 24 h, approximately 7.4% of the dose was located in the cuticle, whereas only 1.6% actually penetrated. In the in vivo studies, approximately 7.4% of the radioactivity was recovered from the body after cuticular wash.     

Effect of tobacco budworm (Lepidoptera: Noctuidae) infestation level on budworm-resistant and -susceptible varieties of flue-cured tobacco in North Carolina

Field experiments were conducted from 1972 to 1978 and from 1998 to 1999 to evaluate tobacco budworm, Heliothis virescens (F.) (Lepidoptera: Noctuidae), larval feeding on flue-cured tobacco, Nicotiana tabacum (L.), yield in eastern North Carolina. In the earlier studies, using variety Coker 319, treatment plots were evaluated when either 0 or 100% of plants in a plot were infested with H. virescens larvae. Treatment differences based on actual yield loss (kilograms per hectare) were compared with estimations of yield loss based on leaf consumption and leaf loss. Results indicate actual yield loss when 100% of plants were infested was less than the corresponding estimates of yield loss. In the later experiments, two tobacco budworm-resistant lines, 'CU 263' and 'CU 370', were compared with a commercial susceptible variety, K 326, when 0, 10, 20, or 40% of plants were infested (1998) and 0, 10, 40, 75, or 100% of plants were infested (1999). Although significant increases in leaf equivalents consumed were associated with infestations exceeding the recommended threshold, differences were not detected for yield (kilograms per hectare), quality (dollars per kilogram), and value (dollars per hectare) within each tobacco line. Additionally, there was not a significant correlation between value and infestations level for any of the tobacco lines. These results provide economic support for tolerance of a higher treatment threshold. Although K 326 sustained more leaf equivalent loss than CU 263 and CU 370, the value of K 326 harvested was higher than that of CU 263 and CU 370. To justify use of resistant varieties, the combination of pest pressure and the benefit of host plant resistance must be greater than the capacity of a susceptible variety to produce competitive yields, despite sustaining significantly higher loss.     

F2 screen for resistance to a Bacillus thuringiensis-maize hybrid in the sugarcane borer (Lepidoptera: Crambidae)

A novel F2 screening technique was developed for detecting resistance in sugarcane borer, Diatraea saccharalis (F.), to transgenic Bacillus thuringiensis (Bt)-maize expressing the Cry1Ab insecticidal protein. The F2 screening method involved (i) collecting larvae from maize fields; (ii) establishing two-parent families; (iii) screening F2 neonates for survival on Bt-maize leaf tissues; and (iv) confirming resistance on commercial Bt-maize plants. With the F2 screening method, 213 iso-line families of D. saccharalis were established from field collections in northeast Louisiana, USA and were screened for Bt resistance. One family was confirmed to carry a major Bt resistance allele(s). In a laboratory bioassay, larval mortality of the Bt-resistant D. saccharalis on Bt-maize leaf tissues was significantly lower than that of a Bt-susceptible strain. This Bt-resistant D. saccharalis population is the first corn stalk borer species that has completed larval development on commercial Bt-maize. The F2 screening protocol developed in this study could be modified for detecting Bt resistance alleles in other similar corn stalk borers, such as the European corn borer, Ostrinia nubilalis (Hübner), and the southwestern corn borer, D. grandiosella Dyar.     

Bollgard cotton and resistance of tobacco budworm (Lepidoptera: Noctuidae) to conventional insecticides in southern Tamaulipas, Mexico

Insecticide susceptibility in tobacco budworm, Heliothis virescens (F.) (Lepidoptera: Noctuidae), was determined for 8 yr (1991-2001) with larvae sampled from cotton in southern Tamaulipas, Mexico. Before 1996, when Bollgard cotton expressing the Cry1A(c) delta-endotoxin was introduced into the region, two important patterns were documented. The first was economically significant increases in resistance to certain insecticide groups. The second was occurrence of virtually complete control failures in the field during 1994 and 1995. The largest resistance changes were recorded for the type II pyrethroids cypermethrin and deltamethrin. These products are the most widely used products in the region. Resistance ratios for these products increased up to > 100-fold from 1991 to 1995. After 1996, the resistance levels declined. These findings did not occur with other products of scant use (e.g., permethrin, profenofos, and endosulfan) or low tobacco budworm efficacy coupled to a high use pattern (e.g., methyl parathion). This clear trend toward reversal of resistance to type II pyrethroids can be understood, in part, with respect to two factors: 1) the high adoption rate of transgenic cotton in the region, from 31.2% in the beginning (1996) to approximately 90% in 1998; this has considerably curbed the use of synthetic insecticides, with the attending loss of selection pressure on this pest; and 2) the potential immigration to the region of susceptible tobacco budworms from cultivated and wild suitable hosts as well as from transgenic cotton might have influenced the pest population as a whole. The influence of transgenic cotton on southern Tamaulipas can be more clearly seen by the drastic reduction of insecticide use to control this important pest. Now tobacco budworms in this region are susceptible to type II pyrethroids. Two effective and fundamentally different pest management tools are now available to cotton growers in southern Tamaulipas: transgenic cotton, coupled with careful use of pyrethroids, offers the possibility of sustainable and profitable cotton production.     

Effects of gamma radiation on different developmental stages of the oriental tobacco budworm,Helicoverpa assulta (Lepidoptera: Noctuidae)

Ionizing radiation is increasingly used as an alternative to post‐harvest crop fumigation by methyl bromide. We studied the effects of gamma irradiation on Helicoverpa assulta (Lepidoptera: Noctuidae) at different stages of development to determine the minimal dose for the prevention of normal emergence of adults. We selected five doses of gamma rays (100, 200, 300, 400 and 500 Gy) based on preliminary experiments and irradiated eggs, larvae, pupae and adults. A dose of 100 Gy to eggs allowed 21.83% of larvae to pupate, but these all died during the pupal stage. A dose of 100 Gy to last‐instar larvae caused larval or pupal death, or the emergence of abnormal adults; no normal adults developed. Irradiation of pupae with doses of 300 Gy and above resulted either in their death or emergence of abnormal adults; however, after 100 or 200 Gy, normal adults emerged and F₁ eggs were produced, but no eggs hatched. Following irradiation of adults, eggs were produced at all doses, although the numbers were significantly decreased compared to untreated controls (P < 0.05; 69.45–125.50 vs. 475.05 eggs per female); however, none of the eggs hatched. As prevention of normal emergence is a key outcome for measuring the effectiveness of radiation, then the 100 Gy dose was effective for irradiation of eggs and larvae, and 300 Gy for pupae.     

Functional analysis of the SGNP I in the pupal diapause of the oriental tobacco budworm, Helicoverpa assulta (Lepidoptera: Noctuidae)

Helicoverpa assulta suboesophageal ganglion neuropeptide I (Has-SGNP I) is a 24-amino acids peptide amide, which shows 62.5% similarity with the diapause hormone of Bombyx mori (Bom-DH). It has been demonstrated that embryonic diapause is induced by DH in B. mori. Injection of synthetic amidated Has-SGNP I terminated pupal diapause in a dose-dependent manner. Therefore, Has-SGNP I might be referred to a "diapause termination hormone" in H. assulta (Has-DTH). The maximal dose of Has-DTH for diapause termination was 1.0 microg and the half-maximal dose 0.4 microg. The time required for diapause termination of Has-DTH was 2-3 days longer than that of 20-hydroxyecdysone. During the pupal stage, DTH mRNA content in the SGs of nondiapausing pupae was always higher than in diapausing pupae using the combined method of quantitative RT-PCR and Southern blot. DTH gene also expressed at a low level while diapausing pupae were chilled at 4 degrees C, but increased rapidly and largely after being transferred to 25 degrees C. Using a competitive ELISA, Has-DTH-like immunoreactivity in the haemolymph showed the same pattern as that of Has-DTH gene expression. Those results indicated that Has-DTH gene expression was related to diapause development and could be activated by low temperature. Has-DTH might be useful to elucidate the mechanism of diapause termination in pupal diapause species.     

Use of mathematical models to estimate characteristics of pyrethroid resistance in tobacco budworm and bollworm (Lepidoptera: Noctuidae) field populations

Genetic models have been used to examine the evolution of insecticide resistance in pest species subject to data and assumptions regarding genetic, biological, and operational parameters. We used time-series data on pyrethroid tolerance and simple genetic models to estimate underlying genetic and biological parameters associated with resistance evolution in tobacco budworm, Heliothis virescens (F.), and bollworm, Helicoverpa zea (Boddie), Louisiana field populations. Assuming pyrethroid resistance is conferred by one gene at one locus in both species, inheritance of pyrethroid resistance was partially dominant in the tobacco budworm and partially recessive in the bollworm. Relative fitness estimates indicated that fitness costs associated with resistance selected against resistance alleles in the absence of selection pressure in the tobacco budworm, but not in the bollworm. In addition, relative fitness estimates obtained using the indirect method outlined in this study were similar in magnitude to estimates obtained using traditional direct approaches.     

Subcellular localization of the fatty acyl reductase involved in pheromone biosynthesis in the tobacco budworm,Heliothis virescens (Noctuidae: Lepidoptera)

Sex pheromone components are produced in specialized glands of female moths via well-characterized biosynthetic pathways, where a Fatty Acyl Reductase (FAR) is often essential for producing the specific ratio of the different pheromone components. The subcellular localization and membrane topology of FARs is important for understanding how pheromones are synthesized and exported to the exterior for release. We investigated the subcellular localization of HvFAR from the noctuid moth Heliothis virescens by producing recombinant fusion proteins with green fluorescent protein (GFP) in yeast. A C-terminally tagged construct was localized to the endoplasmic reticulum (ER) and retained full reductive activity on a broad range of saturated and unsaturated fatty acyl precursors. In contrast, an N-terminally-tagged construct was poorly expressed in the cytoplasm and was not enzymatically active, indicating that HvFAR requires a free N-terminal for both proper targeting and catalytic activity. A series of truncations of the N-and C-termini of HvFAR was conducted based on in silico-predicted hydrophobic domains and transmembrane regions. The N-terminally truncated protein was found in the cytoplasm and did not retain activity, emphasizing the importance of the N-terminal for FAR function. In addition, the orientation in the membrane of the C-terminus-tagged HvFAR-GFP construct was analyzed using a fluorescence protease protection (FPP) assay, implying that the C-terminal of HvFAR is orientated towards the cytoplasm. These results, together with previous data on the localization of desaturases, confirm the importance of the ER as a subcellular site of pheromone production.     

Using COI gene sequence to barcode two morphologically alike species: the cotton bollworm and the oriental tobacco budworm (Lepidoptera: Noctuidae)

Due to limited morphological difference, the two closely related sister species, the cotton bollworm, Helicoverpa armigera (Hübner) and the oriental tobacco budworm, H. assulta (Guenée) (Lepidoptera: Noctuidae), are very difficult to distinguish, especially at the larvae stage. Recently, DNA sequence has been widely used as a bio-barcode for species identification. In this study, we attempted to distinguish H. armigera and H. assulta using the mitochondrial cytochrome C oxidase subunit I gene (COI) gene sequence as the barcode. We determined a 658 bp segment of the COI gene for 28 individuals of H. armigera, 8 individuals of H. assulta, and 10 individuals of Mamestra brassicae (as the outgroup) in Yunnan Province, southwest of P. R. China, together with one H. assulta and two H. armigera reported sequences from GenBank. Twenty-three haplotypes were identified in all 49 samples. As expected, network analysis of the haplotypes of the three species presented a clustering pattern consistent with the respective species status. Haplotypes of the same species differed from each other by no more than three nucleotide substitutions. However, each haplotype of H. armigera differed from that of H. assulta by at least 22 nucleotide substitutions. Both species differed from M. brassicae by more than 50 nucleotide substitutions. 17 unique diagnostic nucleotides were also used to discriminate the two species. The finding of large COI sequence differences between H. armigera and H. assulta suggested that it could be used to distinguish the two morphologically alike species and be employed for quick species identification during pest control.     

Cloning eleven midgut trypsin cDNAs and evaluating the interaction of proteinase inhibitors with Cry1Ac against the tobacco budworm, Heliothis virescens (F.) (Lepidoptera: Noctuidae)

Midgut trypsins are associated with Bt protoxin activation and toxin degradation. Proteinase inhibitors have potential insecticidal toxicity against a wide range of insect species. This study was conducted to evaluate the interaction of proteinase inhibitors with Bt toxin and to examine midgut trypsin gene profile of Heliothis virescens. A sublethal dose (15ppb) of Cry1Ac, 0.75% soybean trypsin inhibitor, and 0.1% and 0.2% N-α-tosyl-L-lysine chloromethyl ketone significantly suppressed midgut proteinase activities, and resulted in reductions in larval and pupal size and mass. The treatment with inhibitor+Bt suppressed approximately 65% more larval body mass and 21% more enzymatic activities than the inhibitor-only or Bt-only. Eleven trypsin-like cDNAs were sequenced from the midgut of H. virescens. All trypsins contained three catalytic center residues (H(73), D(153), and S(231)), substrate specificity determinant residues (D(225), G(250), and G(261)), and six cysteines for disulfide bridges. These putative trypsins were separated into three distinct groups, indicating the diverse proteinases evolved in this polyphagous insect. These results indicated that the insecticidal activity of proteinase inhibitors may be used to enhance Bt toxicity and delay resistance development.     

Effects of X‐ray irradiation on development and reproduction of the oriental tobacco budworm,Helicoverpa assulta (Lepidoptera: Noctuidae)

All stages of the life cycle of Helicoverpa assulta were irradiated with X‐rays to determine the inhibitory dose for development and reproduction to serve as a quarantine treatment. The 100‐Gy dose was effective for irradiation of eggs and larvae, and the 200‐Gy dose was effective for pupae and mixed‐sex adults. When either adult males or females were irradiated, however, 500 Gy was required to prevent the F1 eggs from hatching, and thus single‐sex adults required much higher doses of X‐ray irradiation. To gather confirmatory data applicable to phytosanitary quarantine regulations, pupae—the immature stage most resistant to X‐ray irradiation—were placed inside paprika in boxes for exportation and were irradiated with 300 Gy as a small‐scale confirmatory test. The dose given to 1,007 individual pupae resulted in 12.62 % survival, and 1.79 % of pupae emerged as normal adults; however, these adults produced only a few eggs that did not hatch, suggesting that a minimum dose of 300 Gy of X‐ray irradiation will provide quarantine security for immature H. assulta in paprika exports.     

Rapid antibody-based field test to distinguish between Helicoverpa armigera (Lepidoptera: Noctuidae) and Helicoverpa punctigera (Lepidoptera: Noctuidae)

Helicoverpa armigera (Hübner) and Helicoverpa punctigera (Wallengren) are the two most important insect pests of cotton production in Australia and require application of insecticides to control them. H. armigera has developed resistance to several insecticides but H. punctigera has not. Cost-effective management of insecticide resistance requires that growers be able to determine the proportion of H. armigera eggs or young larvae present on their crop before applying insecticides. This is impossible visually. We generated two monoclonal antibodies that reacted with the insect protein "lipophorin" and were capable of discriminating individuals of the two species at all life-stages. The antibodies were incorporated into a rapid test kit that was tested under field conditions over two growing seasons. Results obtained with the kit agreed closely with those obtained by rearing larvae through to second instar.     

Genetic similarity and distribution of cry genes of Bacillus thuringiensis pathogenic for Spodoptera exigua (Lepidoptera: Noctuidae) and Agrotis segetum (Lepidoptera: Noctuidae)

Crystals of Bacillus thuringiensis isolates, obtained from dead caterpillars, had potential toxicity against insects in the orders Diptera, Coleoptera, Hemiptera and Lepidoptera. Strain MPU B32b had the highest number and variety of cry genes and seemed to be the most interesting for further research on development as a novel biopesticide.     

Feeding behavior of bollworm and tobacco budworm (Lepidoptera: Noctuidae) larvae in mixed stands of nontransgenic and transgenic cotton expressing an insecticidal protein

Feeding behavior of third-instar bollworm, Helicoverpa zea (Boddie), and tobacco budworm, Heliothis virescens (F.), was observed in pure and mixed stands of nontransgenic and transgenic cotton (BTK), Gossypium hirsutum L., expressing an insecticidal protein CryIA(c) from a bacterium, Bacillus thuringiensis Berliner subsp. kurstaki. Five plant stands composed of BTK and non-BTK plants were evaluated; two pure stands and three mixed stands. Percentage ratios of BTK to non-BTK plants in the stands were 100:0, 75:25, 50:50, 25:75 and 0:100, respectively. In all stands with BTK plants, fewer bollworm and tobacco budworm larvae were found on BTK plants than non-BTK plants 24 h after infestation with third instars. At 48 h, significantly fewer tobacco budworm larvae, but not fewer bollworm larvae, were found on BTK plants. However, the number of larvae of either insect did not increase on non-BTK plants compared with the initial infestation density of three larvae per plant. The number of obacco budworm injured flower buds, and capsules was lower in all plant stands containing BTK plants compared with the pure stand of non-BTK at 48 h after infestation. Higher numbers of larvae on non-BTK plants were possibly the result of larval intoxication, reduced feeding, and increased plant abandonment and death on BTK plants rather than a classical feeding preference. Unexpectedly, the number of flower buds and capsules injured by bollworm and tobacco budworm when averaged per plant for all plants in a stand, differed little among the 75:25, 50:50 and 25:75 plant mixtures. These data suggest that larvae of both species frequently moved among plants, feeding indiscriminately on BTK and non-BTK plants.