Effects of a mustard trypsin inhibitor expressed in different plants on three lepidopteran pests

The effects of mustard trypsin inhibitor MTI-2 expressed at different levels in transgenic tobacco, arabidopsis and oilseed rape lines have been evaluated against three different lepidopteran insect pests. 1. Plutella xylostella (L.) larvae were the most sensitive to the ingestion of MTI-2. The inhibitor expressed at high levels in arabidopsis plants caused rapid and complete mortality. High mortality and significantly delayed larval development were also detectable in oilseed rape expressing MTI-2 at lower levels. 2. Mamestra brassicae (L.) larvae were sensitive only at high MTI-2 expression level, as obtained in transgenic tobacco and arabidopsis, whereas no effects were observed for larvae fed on plants showing relatively low expression levels such as those of oilseed rape lines. 3. Feeding bioassays with Spodoptera littoralis (Boisduval) larvae were carried out using the same oilseed rape lines, showing that at these low expression levels no mortality was observed although a delay in larval development did occur. The levels of insect gut proteolytic activities of the larvae still alive at the end of a 7 day feeding bioassay were usually higher than in the controls, but no new proteinases were expressed in any case. The combined results described in this paper demonstrate altogether the relevance of a case-by-case analysis [target insects and proteinase inhibitor (PI) level of expression in planta] in a PI-based strategy for plant protection.     

The mustard trypsin inhibitor 2 affects the fertility of Spodoptera littoralis larvae fed on transgenic plants

The effects of mustard trypsin inhibitor MTI-2 expressed at different levels in transgenic tobacco lines have been evaluated by feeding the lepidopteran Spodoptera littoralis throughout its larval life. Specific conditions were selected to study the long-term effects of feeding larvae on transgenic plants expressing the inhibitor at various levels. The data obtained led to the establishment of three relevant parameters to be considered during the experimentation: (i) the PI content of the plant lines to be used; (ii) the developmental stage of larvae sensitive to that PI content; (iii) the ratio of MTI-2/proteases sufficient to inhibit gut proteases. The experimental data obtained from feeding S. littoralis larvae using these conditions led to two main results. First, when L2 S. littoralis larvae were fed on high MTI-2 expressing tobacco plants, no effects on larval development were detected but there was a significantly reduced fertility. When the same larvae were fed on low expressing MTI-2 tobacco plants, only a less marked lowering of fertility was observed. Second, after the first generation, no differences in protease activity were observed in insects derived from larvae fed on high or low MTI-2 expressing tobacco lines, suggesting that genetic traits observed in previous studies were not inherited.     

Impact of oilseed rape expressing the insecticidal serine protease inhibitor, mustard trypsin inhibitor-2 on the beneficial predator Pterostichus madidus

Abstract Insect-resistant transgenic plants have been suggested to have deleterious effects on beneficial predators feeding on crop pests, through transmission of the transgene product by the pest to the predator. To test this hypothesis, effects of oilseed rape expressing the serine protease inhibitor, mustard trypsin inhibitor -2 (MTI-2), on the predatory ground beetle Pterostichus madidus were investigated, using diamondback moth, Plutella xylostella as the intermediary pest species. As expected, oilseed rape expressing MTI-2 had a deleterious effect on the development and survival of the pest. However, incomplete pest mortality resulted in survivors being available to predators at the next trophic level, and inhibition studies confirmed the presence of biologically active transgene product in pest larvae. Characterization of proteolytic digestive enzymes of P. madidus demonstrated that adults utilize serine proteases with trypsin-like and chymotrypsin-like specificities; the former activity was completely inhibited by MTI-2 in vitro. When P. madidus consumed prey reared on MTI-2 expressing plants over the reproductive period in their life cycle, no significant effects upon survival were observed as a result of exposure to the inhibitor. However, there was a short-term significant inhibition of weight gain in female beetles fed unlimited prey containing MTI-2, with a concomitant reduction of prey consumption. Biochemical analyses showed that the inhibitory effects of MTI-2 delivered via prey on gut proteolysis in the carabid decreased with time of exposure, possibly resulting from up-regulation of inhibitor-insensitive proteases. Of ecological significance, consumption of MTI-2 dosed prey had no detrimental effects on reproductive fitness of adult P. madidus.     

Insecticidal Potential of Defense Metabolites from Ocimum kilimandscharicum against Helicoverpa armigera

Genus Ocimum contains a reservoir of diverse secondary metabolites, which are known for their defense and medicinal value. However, the defense-related metabolites from this genus have not been studied in depth. To gain deeper insight into inducible defense metabolites, we examined the overall biochemical and metabolic changes in Ocimum kilimandscharicum that occurred in response to the feeding of Helicoverpa armigera larvae. Metabolic analysis revealed that the primary and secondary metabolism of local and systemic tissues in O. kilimandscharicum was severely affected following larval infestation. Moreover, levels of specific secondary metabolites like camphor, limonene and β-caryophyllene (known to be involved in defense) significantly increased in leaves upon insect attack. Choice assays conducted by exposing H. armigera larvae on O. kilimandscharicum and tomato leaves, demonstrated that O. kilimandscharicum significantly deters larval feeding. Further, when larvae were fed on O. kilimandscharicum leaves, average body weight decreased and mortality of the larvae increased. Larvae fed on artificial diet supplemented with O. kilimandscharicum leaf extract, camphor, limonene and β-caryophyllene showed growth retardation, increased mortality rates and pupal deformities. Digestive enzymes of H. armigera - namely, amylase, protease and lipase- showed variable patterns after feeding on O. kilimandscharicum, which implies striving of the larvae to attain required nutrition for growth, development and metamorphosis. Evidently, selected metabolites from O. kilimandscharicum possess significant insecticidal activity.     

Adaptation of Helicoverpa armigera (Lepidoptera: Noctuidae) to a proteinase inhibitor expressed in transgenic tobacco

A giant taro proteinase inhibitor (GTPI) cDNA was expressed in transgenic tobacco using three different gene constructs. The highest expression level obtained was ca. 0.3% of total soluble protein when the cDNA was driven by the Arabidopsis rbcS ats1 promoter. Repeated feeding trials with Helicoverpa armigera larvae fed on clonally derived T₀ and T₁ plants expressing GTPI demonstrated that, relative to those fed on control plants, some growth inhibition (22–40%) occurs, but there was no increase in larval mortality. Proteinase activities of larvae fed on GTPI-expressing tobacco or GTPI-containing diet were examined to monitor the spectrum of digestive proteinases in the midgut. Total proteinase activity was reduced by 13%, but GTPI-insensitive proteinase activity was increased by up to 17%. Trypsin was inhibited by 58%, but chymotrypsin and elastase were increased by 26% and 16% respectively. These results point to an adaptive mechanism in this insect that elevates the levels of other classes of proteinases to compensate for the trypsin activity inhibited by dietary proteinase inhibitors.     

Organellar and Cytosolic Localization of Four Phosphoribosyl Diphosphate Synthase Isozymes in Spinach

We report on the efficacy of proteinase inhibitors (PIs) from three host plants (chickpea [Cicer arietinum], pigeonpea [Cajanus cajan], and cotton [Gossypium arboreum]) and three non-host (groundnut [Arachis hypogea], winged bean [Psophocarpus tetragonolobus], and potato [Solanum tuberosum]) in retarding the growth of Helicoverpa armigera larvae, a devastating pest of important crop plants. Enzyme assays and electrophoretic analysis of interaction of H. armigera gut proteinases (HGPs) with PIs revealed that non-host PIs inhibited HGP activity efficiently whereas host PIs were ineffective. In the electrophoretic assay, trypsin inhibitor activity bands were detected in all of the host and non-host plants, but HGP inhibitor activity bands were present only in non-host plants (except cotton in the host plant group). H. armigera larvae reared on a diet containing non-host PIs showed growth retardation, a reduction in total and trypsin-like proteinase activity, and the production of inhibitor-insensitive proteinases. Electrophoretic analysis of PI-induced HGP showed differential regulation of proteinase isoforms. Interestingly, HGP activity induced in response to dietary potato PI-II was inhibited by winged bean PIs. The optimized combination of potato PI-II and winged bean PIs identified in the present study and their proposed successive use has potential in developing H. armigera-resistant transgenic plants.     

Bioevaluation of Subabul (Leucaena leucocephala) proteinase inhibitors on Helicoverpa armigera

Helicoverpa armigera, a highly polyphagous pest, has a broad host spectrum, causes significant levels of yield loss in many agriculturally important crops. Serine primarily responsible for most of the proteolytic activity in the larval gut of lepidopteron insects. Neonate larvae were reared on artificial diet and chickpea seeds smeared with Subabul Trypsin Inhibitor. Larvae fed with artificial diet showed reduction in larval weight up to 21% (HSTI) and 43% (LSTI). However, larvae fed on seeds showed significant reduction in weight, 52.4% (HSTI) and 60.3% (LSTI), suggesting that the diet also plays a vital role on the effectiveness of the inhibitors on larval growth and development. HSTI and LSTI inhibited the gut proteinases from larvae fed on artificial diet significantly (41.40% and 64.36%) compared to the gut proteinases (27.80% and 38.90%) from larvae fed on chickpea seeds. Seeds smeared with 10,000 TIU resulted in complete mortality of larvae while there was no mortality observed in artificial diet. The results reveal that LSTI is a stronger inhibitor of insect gut proteinases and for larvae fed with poor nutrition in the natural ecosystems, low level expression of inhibitor would be enough to affect the growth and development. Handling editor: Chen-Zhu Wang     

Growth stimulation of beetle larvae reared on a transgenic oilseed rape expressing a cysteine proteinase inhibitor

The resistance of a transgenic line of oilseed rape expressing constitutively the cysteine proteinase inhibitor oryzacystatin I (OCI) was assessed against Psylliodes chrysocephala L. (Coleoptera: Chrysomelidae). The levels of OCI expression in the transformed line averaged 0.2% and 0.05% of total soluble protein in leaves and petioles respectively. In vitro analyses showed that P. chrysocephala larvae use both cysteine and serine proteinases for protein digestion, and that all the cysteine proteolytic activity is OCI-sensitive. However, bioassays showed that adults fed identically on leaf discs from control or transformed plants. When larvae were reared on transgenic plants expressing OCI, they showed an increase in weight gain compared to those reared on control plants. Furthermore, those larvae from transgenic plants exhibited a 2-fold increase in both cysteine and serine proteolytic activity as a reponse to the presence of OCI. The plasticity of insect digestive physiology and feeding behaviour are discussed, as well as the relevance of engineering a genotype expressing both types of proteinase inhibitors.     

Crocidolomia pavonana larval foraging: behavior and feeding site preferences on cabbage, Brassica oleracea

Experiments were conducted to determine feeding site preferences of Crocidolomia pavonana (Fabricius) (Lepidoptera: Crambidae) larvae within cabbage plants, Brassica oleracea L. var. capitata cv. Warrior (Brassicaceae), and to determine whether induced plant responses to herbivory affect the behavior of larvae. In the first experiment, intra-plant damage and larval distribution were recorded to account for the spreading pattern of damage and larval feeding behavior on a plant; larvae initially fed on the base of leaves and moved progressively to the bud, leaf tips were avoided. In the second experiment, larval performance (the duration of the first instar, survival to the second instar, and weight of second instars) was assessed when larvae fed on the bud, the base, and the tip of the youngest fully expanded leaf on a plant. Crocidolomia pavonana larvae performed best when they fed on bud leaf tissue and most poorly when they fed on leaf tissue at the base of leaves. In the third experiment, expression of induced resistance was tested on each of the three plant parts using a first-instar bioassay. Negative impacts on larval growth and development were not detected when larvae fed on the bud or base tissue when plants were damaged prior to the assay. However, negative effects were detected in larvae feeding on tip leaf tissue when the base of the leaf was damaged prior to the assay or if the bud tissue was damaged simultaneously with the assay. The findings indicate that resource heterogeneity for C. pavonana within-cabbage plants is determined by both the initial quality of food at a location and by subsequent induced changes as a result of larval feeding; both contribute to the feeding pattern observed in these gregarious larvae.     

Susceptibility of Tuta absoluta (Meyrick) (Lep., Gelechiidae) reared on two species of Lycopersicon to Bacillus thuringiensis var. kurstaki

The mortality of Tuta absoluta (Meyrick) larvae that were fed on leaves of Lycopersicon hirsutum f. glabratum (PI 134417, insect resistant) and Lycopersicon esculentum (cultivar Santa Clara, susceptible) treated with Bacillus thuringiensis var. kurstaki (Btk) was evaluated. Feeding on untreated PI 134417 was detrimental to the survival of T. absoluta larvae. When Btk was applied to the two Lycopersicon plants, mortality occurred in all T. absoluta instars. Application of Btk on tomato leaves had synergistic or additive effects with the resistant genotype on larval survival. This effect was dependent on the instar at which the larvae were fed Btk-treated leaves. Delayed Btk application may cause higher insect mortality if the insects become more susceptible to the pathogen after a longer period of feeding on the resistant crop.     

Antibiosis in Soybean Genotypes and the Resistance Levels to Spodoptera eridania (Cramer) (Lepidoptera: Noctuidae)

The southern armyworm (SAW) Spodoptera eridania (Cramer) is one of the most common armyworm species defoliating soybeans. Preliminary screening trials have indicated that some soybean genotypes exhibit resistance to SAW. Therefore, in this study, we evaluated the development of SAW larvae fed on ten soybean genotypes in order to identify genotypes with antibiosis-type resistance. Neonate SAW larvae were daily fed with young leaves collected from plants at the vegetative growth stages V4–V5. Larval development and survival were recorded. Genotypes PI 227687 and PI 227682 delayed larval, pupal, and larva-adult development and yielded larvae with the lowest weight and survival and pupae with the lowest weight. Genotypes IAC 100 and DM 339 also negatively affected larval and pupal development and larval survival but at a lower level. Based on our results, the soybean lines PI 227687 and PI 227682 could be used as sources of genes for soybean breeding programs aiming to develop high yield, SAW-resistant cultivars. Moreover, further trials must be carried out under field conditions to validate if the commercial cultivars IAC 100 and DM 339, which expressed moderate levels of antibiosis-type resistance in the laboratory, are effective in suppressing SAW larvae populations.     

Physiological adaptation explains the insensitivity of Baris coerulescens to transgenic oilseed rape expressing oryzacystatin I

Larvae of Baris coerulescens Scop. (Coleoptera: Curculionidæ) exhibit a complex array of gut proteinase activities comprising cysteine and serine proteinases. The major cysteine proteinase activity, showing an optimum at pH 6.0, corresponds to at least 4 different proteinases. On the contrary, the minor serine proteinase activity, with an optimum at pH 9.0, seems to be due essentially to a single proteinase. The cysteine proteinase inhibitor oryzacystatin I (OC-I) inhibits completely the cysteine proteinase activity in vitro. However, larval growth and survival were not significantly different on control and transgenic oilseed rape plants expressing high levels of active OC-I. In larvae grown on transgenic plants, cysteine proteinase activity was dramatically decreased, whereas serine proteinase activity was increased by more than 2-fold, when compared to larvae raised on control plants. For both activities, no new proteinase was detected in insects fed plants expressing OC-I. These results suggest that partial compensation of the inhibition of cysteine proteinase activity by the increase in serine proteinase activity allowed the larvae to overcome the effects of OC-I consumption. This case illustrates problems that could arise when trying to achieve high levels of protection for plants against Coleopteran pests possessing a complex digestive proteinase pool.     

Improved tolerance against <Emphasis Type="Italic">Helicoverpa armigera</Emphasis> in transgenic tomato over-expressing multi-domain proteinase inhibitor gene from <Emphasis Type="Italic">Capsicum annuum</Emphasis>

Plant proteinase inhibitors (PIs) are plant defense proteins and considered as potential candidates for engineering plant resistances against herbivores. Capsicum annuum proteinase inhibitor (CanPI7) is a multi-domain potato type II inhibitor (Pin-II) containing four inhibitory repeat domains (IRD), which target major classes of digestive enzymes in the gut of Helicoverpa armigera larvae. Stable integration and expression of the transgene in T1 transgenic generation, were confirmed by established molecular techniques. Protein extract of transgenic tomato lines showed increased inhibitory activity against H. armigera gut proteinases, supporting those domains of CanPI7 protein to be effective and active. When T1 generation plants were analyzed, they exhibited antibiosis effect against first instar larvae of H. armigera. Further, larvae fed on transgenic tomato leaves showed delayed growth relative to larvae fed on control plants, but did not change mortality rates significantly. Thus, better crop protection can be achieved in transgenic tomato by overexpression of multi-domain proteinase inhibitor CanPI7 gene against H. armigera larvae.     

Complexity in specificities and expression of Helicoverpa armigera gut proteinases explains polyphagous nature of the insect pest

Helicoverpa armigera is a devastating pest of cotton and other important crop plants all over the world. A detailed biochemical investigation of H. armigera gut proteinases is essential for planning effective proteinase inhibitor (PI)-based strategies to counter the insect infestation. In this study, we report the complexity of gut proteinase composition of H. armigera fed on four different host plants, viz. chickpea, pigeonpea, cotton and okra, and during larval development. H. armigera fed on chickpea showed more than 2.5- to 3-fold proteinase activity than those fed on the other host plants. H. armigera gut proteinase composition revealed the predominance of serine proteinase activity; however, the larvae fed on pigeonpea revealed the presence of metalloproteases and low levels of aspartic and cysteine proteases as well. Gut proteinase activity increased during larval development with the highest activity seen in the fifth instar larvae which, however, declined sharply in the sixth instar. Over 90% of the gut proteinase activity of the fifth instar larvae was of the serine proteinase type, however, the second instar larvae showed the presence of proteinases of other mechanistic classes like metalloproteases, aspartic and cysteine proteases along with serine proteinase activity as evident by inhibition studies. Analysis of fecal matter of larvae showed significant increase in proteinase activity when fed on an artificial diet with or without non-host PIs than larvae fed on a natural diet. The diversity in the proteinase activity observed in H. armigera gut and the flexibility in their expression during developmental stages and depending upon the diet provides a base for selection of proper PIs for insect resistance in transgenic crop plants.     

Indirect interaction between a fungal plant pathogen and a herbivorous beetle of the weed<Emphasis Type="Italic"> Cirsium arvense</Emphasis>

Interactions between plants and their natural enemies are well studied, but investigations on the indirect interactions between plant enemies that simultaneously exploit a host plant are rare. Yet these plant-mediated interactions are important because they may affect not only the impact of plant antagonists on plant survival but may also influence the performance of the other plant exploiters. This study focused on the indirect effects of a systemic infection of creeping thistle, [irsium arvense (L.) Scop., with the necrotrophic fungus Phoma destructiva (Plowr.) on the phytophagous leaf beetle Cassida rubiginosa Müller, by examining egg deposition, food plant choice, and larval and pupal performance of the beetle. Thus, the results give a broader view than most other studies of plant-mediated effects of a pathogen on a phytophagous insect. Since both the beetle and the fungus are considered as agents for the biological control of C. arvense, the results are also of interest for applied ecology. Potted plants of C. arvense were inoculated with a conidiospore suspension of P. destructiva to cause a systemic infection of the plants. In a cage experiment, ovipositing females of C. rubiginosa showed a significant preference for healthy thistles. In dual-choice tests, adults of C. rubiginosa preferred leaf discs from healthy thistles over those from Phoma-infected thistles. The beetles also consumed significantly more leaf tissue from healthy than from infected plants. Development time from freshly hatched larvae until pupation was significantly longer for larvae fed on infected leaves. The weight of last-instar larvae and pupae was lower, and larval and pupal mortality was higher when larvae had been fed with infected compared to healthy leaves. Thus, the combined use of both potential biological control agents may be of lowered efficiency because (1) C. rubiginosa avoided infected thistles for both egg deposition and adult feeding and (2) Phoma infection negatively affected larval development and increased larval and pupal mortality of the beetle.     

Co‐expression of the proteinase inhibitors oryzacystatin I and oryzacystatin II in transgenic potato alters Colorado potato beetle larval development

Colorado potato beetle (CPB; Leptinotarsa decemlineata Say, Coleoptera: Chrysomelidae) has shown a remarkable adaptability to a variety of control measures. Although oryzacystatin I and II (OCI and OCII) have potential in controlling pests that use cysteine proteinases for food digestion, expression of a single OC gene in potato exhibited a minimal or no effect on CPB fitness traits. The aim of this study was to examine the effect of coexpressed OCI and OCII in potato (Solanum tuberosum L.) cultivars Desiree, Draga?evka and Jelica on CPB larvae. Growth parameters, consumption rates and food utilization, as well as activity of proteases of CPB larvae were assayed. Second and third instar larvae fed on transformed leaves molted earlier and had higher relative growth and consumption rates than larvae fed on nontransformed leaves, while efficiency of food utilization was unaffected. In contrast, fourth instar maximum weight gain and amount of leaves consumed were about 20% lower for the larvae fed on transgenic potato. Analysis of total protease activity of third instar larvae revealed reduction in overall proteolytic activity measured by azocasein hydrolysis, accompanied with inhibition of cysteine proteinase activity 24 h after ingestion of potato leaves expressing OCI and OCII. However, after long‐term feeding on transformed leaves proteolytic activities of larvae became similar to the controls. Although feeding on OCI/OCII leaves did not affect larval survival, coexpression of OC genes reduced the development time and thus significantly decreased plant damage caused by CPB larvae.     

Larval gregariousness and neonate establishment of the eucalypt-feeding beetle Chrysophtharta agricola (Coleoptera: Chrysomelidae: Paropsini)

The selective advantage offered to individuals living within groups may relate to natural enemy defence, but in leaf feeding insects may also relate to overcoming plant defences, especially with respect to feeding establishment. We conducted a series of experiments focusing on neonate larval survival, examining the effect of group size and leaf age on the survival of a eucalypt-feeding beetle, Chrysophtharta agricola , which formed groups of up to 43 larvae on the foliage of Eucalyptus nitens in the field. In the laboratory, in the absence of natural enemies, we found that initial density, leaf age and damage to the leaf margin significantly affected larval survival. Survival of solitary first-instar larvae on young foliage was around 80% whereas on older foliage it was around 11%. Prior damage to the leaf margin significantly increased survival on older leaves to around 61%. Initial larval density also affected survival, although mortality was always significantly higher on older leaves. On older leaves the larval group size above which mortality increased no further was over two-fold that on young leaves. Observations of group feeding behaviour at each instar showed that the majority of larvae (75.7%) were aligned facing away from the feeding site and that only around 7.5%, or just 1–2 larvae per group, fed at any one time. Feeding larvae chewed the leaf edge by straddling the leaf margin. Measurements of leaf margins showed that older leaves had significantly thicker leaf margins and 'thickness' ratios (leaf margin to leaf lamina proper). In the field, approximately 85% of all larvae occurred on the first two expanded leaf pairs, and larval mortality was highest between eclosion and establishment of the first instar. However, beetles apparently did not adjust clutch size according to leaf age.     

Development of transgenic sorghum for insect resistance against the spotted stem borer (Chilo partellus)

Transgenic sorghum plants expressing a synthetic cry1Ac gene from Bacillus thuringiensis (Bt) under the control of a wound-inducible promoter from the maize protease inhibitor gene (mpiC1) were produced via particle bombardment of shoot apices. Plants were regenerated from the transformed shoot apices via direct somatic embryogenesis with an intermittent three-step selection strategy using the herbicide Basta. Molecular characterisation based on polymerase chain reaction and Southern blot analysis revealed multiple insertions of the cry1Ac gene in five plants from three independent transformation events. Inheritance and expression of the Bt gene was confirmed in T₁ plants. Enzyme-linked immunosorbant assay indicated that Cry1Ac protein accumulated at levels of 1–8 ng per gram of fresh tissue in leaves that were mechanically wounded. Transgenic sorghum plants were evaluated for resistance against the spotted stem borer (Chilo partellus Swinhoe) in insect bioassays, which indicated partial resistance to damage by the neonate larvae of the spotted stem borer. Reduction in leaf damage 5 days after infestation was up to 60%; larval mortality was 40%, with the surviving larvae showing a 36% reduction in weight over those fed on control plants. Despite the low levels of expression of Bt -endotoxin under the control of the wound-inducible promoter, the transgenic plants showed partial tolerance against first instar larvae of the spotted stem borer.     

Leaf toughness changes the effectiveness of larval aggregation in the butterfly Byasa alcinous bradanus (Lepidoptera: Papilionidae)

Two subspecies of the papilionid butterfly Byasa alcinous , B. a. bradanus and B. a. alcinous , have varying degrees of larval aggregation. Early instar larvae of ssp. bradanus always occur in aggregations. To determine the functions of larval aggregation in this subspecies, we examined the effects of leaf toughness on larval performance when caterpillars were reared alone and in aggregations. Newly hatched larvae were reared either individually or in groups of 10 and were fed either tough or tender leaves of Aristolochia debilis . When fed tough leaves, more gregarious larvae survived the first instar. This difference between solitary and aggregated larvae did not occur when caterpillars were fed soft leaves. The effects of aggregation on larval weight and duration were not significant between leaf-toughness treatments. Larval aggregation of B. a. bradanus improves larval survivorship in early instars that use host plants with tough leaves.     

Avidin expressed in transgenic tobacco leaves confers resistance to two noctuid pests,Helicoverpa armigera and Spodoptera litura

Fertile transgenic tobacco plants with leaves expressing avidin in the vacuole have been produced and shown to halt growth and cause mortality in larvae of two noctuid lepidopterans, Helicoverpa armigera and Spodoptera litura. Late first instar H. armigera larvae and neonate (<12-h-old) S. litura larvae placed on leaves excised from T₀ tobacco expressing avidin at 3.1–4.6M (moles/kg of fresh leaf tissue) had very poor growth over their first 8 days on the leaves, significant numbers had died by days 11 or 12 and all were dead by day 22 (H. armigera) or day 25 (S. litura). Similar results were obtained when late first instar H. armigera larvae were placed on leaves from T₁ plants expressing avidin at six different average concentrations, ranging from 3.7 to 17.3M. Two larvae on the lowest expressing leaves survived to pupation, but there was total mortality among the other groups and no relationship between avidin concentration and the effects on the larvae. Synergistic effects between avidin-expressing tobacco plants and a purified Bt toxin, Cry1Ba, were demonstrated. Late instar H. armigera larvae fed with leaves from T₂ plants expressing avidin at average concentrations of either <5.3 or >12.9M, and painted with Cry1Ba protein at a rate equivalent to an expression level of 0.5% of total leaf protein, died significantly faster than larvae given either of the two treatments alone. Larvae fed with avidin-expressing leaves painted with the protease inhibitor, aprotinin, at a rate equivalent to 1% of total leaf protein had mortality similar to those given avidin-leaves alone. There was no evidence of antagonism between these two proteins.