Effects of black-eyed pea trypsin/chymotrypsin inhibitor on proteolytic activity and on development of Anthonomus grandis

The cotton boll weevil Anthonomus grandis (Boheman) is one of the major pests of cotton (Gossypium hirsutum L.) in tropical and sub-tropical areas of the New World. This feeds on cotton floral fruits and buds causing severe crop losses. Digestion in the boll weevil is facilitated by high levels of serine proteinases, which are responsible for the almost all proteolytic activity. Aiming to reduce the proteolytic activity, the inhibitory effects of black-eyed pea trypsin/chymotrypsin inhibitor (BTCI), towards trypsin and chymotrypsin from bovine pancreas and from midguts of A. grandis larvae and adult insects were analyzed. BTCI, purified from Vigna unguiculata (L.) seeds, was highly active against different trypsin-like proteinases studied and moderately active against the digestive chymotrypsin of adult insects. Nevertheless, no inhibitory activity was observed against chymotrypsin from A. grandis larval guts. To test the BTCI efficiency in vivo, neonate larvae were reared on artificial diet containing BTCI at 10, 50 and 100 microM. A reduction of larval weight of up to approximately 54% at the highest BTCI concentration was observed. At this concentration, the insect mortality was 65%. This work constitutes the first observation of a Bowman-Birk type inhibitor active in vitro and in vivo toward the cotton boll weevil A. grandis. The results of bioassays strongly suggest that BTCI may have potential as a transgene protein for use in engineered crop plants modified for heightened resistance to the cotton boll weevil.     

Molecular cloning of a cysteine proteinase cDNA from the cotton boll weevil Anthonomus grandis (Coleoptera: Curculionidae)

The cotton boll weevil (Anthonomus grandis) causes severe cotton crop losses in North and South America. This report describes the presence of cysteine proteinase activity in the cotton boll weevil. Cysteine proteinase inhibitors from different sources were assayed against total A. grandis proteinases but, unexpectedly, no inhibitor tested was particularly effective. In order to screen for active inhibitors against the boll weevil, a cysteine proteinase cDNA (Agcys1) was isolated from A. grandis larvae using degenerate primers and rapid amplification of cDNA ends (RACE) techniques. Sequence analysis showed significant homologies with other insect cysteine proteinases. Northern blot analysis indicated that the mRNA encoding the proteinase was transcribed mainly in the gut of larvae. No mRNA was detected in neonatal larvae, pupae, or in the gut of the adult insect, suggesting that Agcys1 is an important cysteine proteinase for larvae digestion. The isolated gene will facilitate the search for highly active inhibitors towards boll weevil larvae that may provide a new opportunity to control this important insect pest.     

Toxicity to cotton boll weevil Anthonomus grandis of a trypsin inhibitor from chickpea seeds

Cotton (Gossypium hirsutum L.) is an important agricultural commodity, which is attacked by several pests such as the cotton boll weevil Anthonomus grandis. Adult A. grandis feed on fruits and leaf petioles, reducing drastically the crop production. The predominance of boll weevil digestive serine proteinases has motivated inhibitor screenings in order to discover new ones with the capability to reduce the digestion process. The present study describes a novel proteinase inhibitor from chickpea seeds (Cicer arietinum L.) and its effects against A. grandis. This inhibitor, named CaTI, was purified by using affinity Red-Sepharose Cl-6B chromatography, followed by reversed-phase HPLC (Vydac C18-TP). SDS-PAGE and MALDI-TOF analyses, showed a unique monomeric protein with a mass of 12,877 Da. Purified CaTI showed significant inhibitory activity against larval cotton boll weevil serine proteinases (78%) and against bovine pancreatic trypsin (73%), when analyzed by fluorimetric assays. Although the molecular mass of CaTI corresponded to alpha-amylase/trypsin bifunctional inhibitors masses, no inhibitory activity against insect and mammalian alpha-amylases was observed. In order to observe CaTI in vivo effects, an inhibitor rich fraction was added to an artificial diet at different concentrations. At 1.5% (w/w), CaTI caused severe development delay, several deformities and a mortality rate of approximately 45%. These results suggested that CaTI could be useful in the production of transgenic cotton plants with enhanced resistance toward cotton boll weevil.     

Purification of a 6.5 kDa protease inhibitor from Amazon Inga umbratica seeds effective against serine proteases of the boll weevil Anthonomus grandis

A 6.5 kDa serine protease inhibitor was purified by anion-exchange chromatography from the crude extract of the Inga umbratica seeds, containing inhibitor isoforms ranging from 6.3 to 6.7 kDa and protease inhibitors of approximately 19 kDa. The purified protein was characterized as a potent inhibitor against trypsin and chymotrypsin and it was named I. umbratica trypsin and chymotrypsin inhibitor (IUTCI). MALDI-TOF spectra of the IUTCI, in the presence of DTT, showed six disulfide bonds content, suggesting that this inhibitor belongs to Bowman-Birk family. The circular dichroism spectroscopy indicates that IUTCI is predominantly formed by unordered and beta-sheet secondary structure. It was also characterized, by fluorescence spectroscopy, as a stable protein at range of pH from 5.0 to 7.0. Moreover, this inhibitor at concentration of 75 microM presented a remarkable inhibitory activity (60%) against digestive serine proteases from boll weevil Anthonomus grandis, an important economical cotton pest.     

Evaluation of Catolaccus grandis (Burks) (Hym., Pteromalidae) as a biological control agent against cotton boll weevil

Abstract: An evaluation of augmentative releases carried out at the Agreste site of the state of Paraiba, Brazil, provided significant insight into the ecology and potential impact of Catolaccus grandis (Burks) against the boll weevil, Anthonomus grandis Boheman in that cotton agroecosystem. The rate of increase in density of C. grandis was higher than the boll weevil. Catolaccus grandis showed ability to effectively search and reproduce within the release environment and revealed pronounced host and habitat preferences. Parasitism by C. grandis was largely confined to third instar weevil larvae, the majority of which (86.9%) occurred in abcised cotton squares. Catolaccus grandis inflicted significant mortality on third instar weevil larvae in the plot resulting in a significant level of suppression. The net effect was a higher boll weevil mortality in the release plot in comparison with the control. The use of augmentative releases of C. grandis has a very high potential for supplementing and enhancing available technology for suppressing boll weevil populations in the Agreste Paraiba.     

Expression and Chloroplast Targeting of Cholesterol Oxidase in Transgenic Tobacco Plants

Cholesterol oxidase represents a novel type of insecticidal protein with potent activity against the cotton boll weevil (Anthonomus grandis grandis Boheman). We transformed tobacco (Nicotiana tabacum) plants with the cholesterol oxidase choM gene and expressed cytosolic and chloroplast-targeted versions of the ChoM protein. Transgenic leaf tissues expressing cholesterol oxidase exerted insecticidal activity against boll weevil larvae. Our results indicate that cholesterol oxidase can metabolize phytosterols in vivo when produced cytosolically or when targeted to chloroplasts. The transgenic plants exhibiting cytosolic expression accumulated low levels of saturated sterols known as stanols, and displayed severe developmental aberrations. In contrast, the transgenic plants expressing chloroplast-targeted cholesterol oxidase maintained a greater accumulation of stanols, and appeared phenotypically and developmentally normal. These results are discussed within the context of plant sterol distribution and metabolism.     

Larval development and proteolytic activity of Anticarsia gemmatalis Hübner (Lepidoptera: Noctuidae) exposed to different soybean protease inhibitors

Anticarsia gemmatalis represents a relevant factor for lowering soybean and other legume crop productivities. Protease inhibitors affect protein degradation and reduce the availability of amino acids, impairing the development and survival of insect pests. To evaluate the possible use of proteinaceous protease inhibitors in the management of this pest, the activities of midgut proteases and the growth and development of A. gemmatalis larvae exposed to soybean Bowman–Birk trypsin-chymotrypsin inhibitor (SBBI) and soybean Kunitz trypsin inhibitor (SKTI) were determined. The survival curves obtained using Kaplan–Meier estimators indicated that SKTI and SBBI stimulated larval survival. However, the development of A. gemmatalis was delayed, and prepupal weight decreased in the presence of both inhibitors. The results showed that SKTI and SBBI inhibited the trypsin-like and total proteolytic activities of larvae on the 12th day after eclosion. On the 15th day after eclosion, larvae exposed to SKTI increased the activities of trypsin and total proteases. Although SKTI and SBBI did not affect the survival of the insect, they had effects on midgut proteases in a stage wherein A. gemmatalis fed voraciously, increased the larval cycle, and decreased prepupal weight. These findings provide baseline information about the potential of proteinaceous protease inhibitors to manage the velvetbean caterpillar, avoiding chemical pesticides.     

Susceptibility of Anthonomus grandis (cotton boll weevil) and Spodoptera frugiperda (fall armyworm) to a cry1ia-type toxin from a Brazilian Bacillus thuringiensis strain

Different isolates of the soil bacterium Bacillus thuringiensis produce multiple crystal (Cry) proteins toxic to a variety of insects, nematodes and protozoans. These insecticidal Cry toxins are known to be active against specific insect orders, being harmless to mammals, birds, amphibians, and reptiles. Due to these characteristics, genes encoding several Cry toxins have been engineered in order to be expressed by a variety of crop plants to control insectpests. The cotton boll weevil, Anthonomus grandis, and the fall armyworm, Spodoptera frugiperda, are the major economically devastating pests of cotton crop in Brazil, causing severe losses, mainly due to their endophytic habit, which results in damages to the cotton boll and floral bud structures. A cry1Ia-type gene, designated cry1Ia12, was isolated and cloned from the Bt S811 strain. Nucleotide sequencing of the cry1Ia12 gene revealed an open reading frame of 2160 bp, encoding a protein of 719 amino acid residues in length, with a predicted molecular mass of 81 kDa. The amino acid sequence of Cry1Ia12 is 99% identical to the known Cry1Ia proteins and differs from them only in one or two amino acid residues positioned along the three domains involved in the insecticidal activity of the toxin. The recombinant Cry1Ia12 protein, corresponding to the cry1Ia12 gene expressed in Escherichia coli cells, showed moderate toxicity towards first instar larvae of both cotton boll weevil and fall armyworm. The highest concentration of the recombinant Cry1Ia12 tested to achieve the maximum toxicities against cotton boll weevil larvae and fall armyworm larvae were 230 microg/mL and 5 microg/mL, respectively. The herein demonstrated insecticidal activity of the recombinant Cry1Ia12 toxin against cotton boll weevil and fall armyworm larvae opens promising perspectives for the genetic engineering of cotton crop resistant to both these devastating pests in Brazil.     

Relationships of abscised cotton fruit to boll weevil (Coleoptera: Curculionidae) feeding, oviposition, and development

Abscised cotton, Gossypium hirsutum L., fruit in field plots planted at different times were examined to assess adult boll weevil, Anthonomus grandis grandis Boheman (Coleoptera: Curculionidae), use of squares and bolls during 2002 and 2003 in the Lower Rio Grande Valley of Texas. Although boll abscission is not necessarily related to infestation, generally more bolls abscised than squares and abundances of fallen bolls were not related to the planting date treatments. During 2003, fallen squares were most abundant in the late-planted treatment. Although large squares (5.5-8-mm-diameter) on the plant are preferred for boll weevil oviposition, diameter of abscised squares is not a reliable measurement because of shrinkage resulting from desiccation and larval feeding. Fallen feeding-punctured squares and bolls were most abundant in late plantings but differences between fallen feeding-punctured squares versus fallen feeding-punctured bolls were found in only one treatment in 2003. During the same year, fallen oviposition-punctured squares were more numerous in the late-planted treatment than in the earlier treatments. Treatment effects were not found on numbers of oviposition-punctured bolls, but fallen oviposition-punctured squares were more common than bolls in the late-planted treatment compared with earlier treatments each year. Dead weevil eggs, larvae, and pupae inside fallen fruit were few and planting date treatment effects were not detected. Living third instars and pupae were more abundant in fallen squares of the late-planted treatment than in the earlier treatments and bolls of all three treatments. This study shows that fallen squares in late-planted cotton contribute more to adult boll weevil populations than bolls, or squares of earlier plantings.     

Effects of routine late-season field operations on numbers of boll weevils (Coleoptera: Curculionidae) captured in large-capacity pheromone traps

Flat and cylindrical adhesive boll weevil, Anthonomus grandis grandis Boheman (Coleoptera: Curculionidae), pheromone traps captured significantly more (P < or = 0.05) boll weevils than the Hercon (Hercon Environmental, Emigsville, PA) trap during the late cotton-growing season, and larger adhesive areas were associated with higher captures; a flat plywood board collected the most boll weevils because it had the largest surface area. The flat board trap, chosen for measuring large late-season adult boll weevil populations common to the Lower Rio Grande Valley of Texas in 2000 and 2001, collected more (P < or = 0.05) weevils when deployed in proximity to natural and cultivated perennial vegetation, and mean numbers of captured boll weevils were higher (P < or = 0.0001) on the leeward sides of the board traps than on the windward sides. The board trap had an estimated potential capacity of approximately 27,800 boll weevils, and the large capacity of the board trap allowed for more accurate measurements of large adult boll weevil populations than the more limited Hercon trap. Measurement of adult boll weevil numbers after the routine field operations of defoliation, harvest, shredding, and stalk-pulling, demonstrated that large populations of boll weevils persist in cotton fields even after the cotton crop has been destroyed. Increases (P < or = 0.05) in the percentage variation of trapped boll weevils relative to the numbers collected just before each field operation were observed after defoliation, harvest, shredding, and stalk-pulling, but the percentage variations followed a quadratic pattern with significant correlation (P < 0.0001; 0.59 < adjusted r2 < 0.73). Numbers of adult boll weevils caught on board traps deployed at 15.24-m intervals on windward and leeward edges of cotton fields suggested that boll weevil populations in flight after field disturbances might be affected by large-capacity trapping.     

Potential for escape of live boll weevils (Coleoptera: Curculionidae) into cottonseed, motes, and cleaned lint at the cotton gin

Reintroductions of the boll weevil, Anthonomus grandis grandis Boheman, into areas of the United States where it has been eradicated or suppressed are very expensive to mitigate. There is concern that a cotton gin in an eradication zone may serve as a site of boll weevil reintroductions when processing cotton harvested in a neighboring infested zone. Similarly, there is a question whether weevil-free areas can safely import gin products, such as cottonseed and baled lint, from infested areas without risking an introduction. Many countries require fumigation of imported U.S. cotton bales to protect against boll weevil introductions, costing the U.S. cotton industry millions of dollars annually. In previously reported experiments, we quantified the potential for boll weevils to survive passage through precleaning machinery in the gin. In this study, we quantified survival potential of boll weevils passing through the gin stand and segregating into the cottonseed, mote, or lint fractions. We also examined boll weevil survival when passed with ginned lint through a lint cleaner. We present a flow chart of experimentally determined survival potentials of boll weevils passing through the various subprocesses of the gin, from which one can calculate the risk of a live boll weevil reaching any point in the process. Our data show that there is virtually no chance of a boll weevil being segregated alive into the cottonseed or of one surviving in the lint to approach the bale press. Therefore, quarantine or fumigation of cottonseed and cotton bales to guard against boll weevil introductions is unnecessary.     

Ecology and phenology of the boll weevil (Coleoptera: Curculionidae) on an unusual wild host, Hibiscus pernambucensis, in southeastern Mexico.

The phenology and ecology of Hibiscus pernambucensis Arruda and its interaction and importance in maintaining populations of the boll weevil, Anthonomus grandis Boheman, were studied over a period of 3 yr in the Soconusco Region of the state of Chiapas, Mexico. H. pernambucensis is a small tree of Neotropical distribution, restricted to lowland areas, and generally associated with halophytic vegetation. This species is found exclusively along the shores of brackish estuaries, in or near mangrove swamps in southeastern Mexico. In this region, H. pernambucensis has a highly seasonal flowering pattern in which the greatest bud production occurs shortly after the start of the rainy season in May and the highest fruit production occurs in July and August. Boll weevil larvae were found in buds of H. pernambucensis during all months but February and densities of buds and weevils were highest from May through September. The percentage of buds infested with boll weevil larvae rarely exceeded 30%. Because plant densities and reproductive output of H. pernambucensis is relatively low and, consequently, the number of oviposition and larval development sites for boll weevils is limited, the importance of this plant as a source of boll weevils with potential of attacking commercial cotton is minimal in comparison with the quantity produced in cultivated cotton. However, the plant could be important as a reservoir of boll weevils in areas of boll weevil quarantine and eradication programs. The factors and circumstances that may have led to this apparent recent host shift of the boll weevil in this region are discussed.     

Digestive proteolytic activity in larvae of tomato moth,Lacanobia oleracea; effects of plant protease inhibitors in vitro and in vivo

Three distinct digestive protease activities, with strongly alkaline pH optima, were identified in the gut of tomato moth (Lacanobia oleracea) larvae, and characterised using specific synthetic substrates and inhibitors. These were; a trypsin-like activity, a chymotrypsin-like activity specific for substrates and inhibitors containing more than one amino acid residue, and an elastase-like activity, accounting for 40%, 30% and 20% of overall proteolysis respectively. The protease activities differed in their sensitivities to inhibition by different plant protein protease inhibitors (PIs), as estimated by I(50) values. Soya bean Kunitz trypsin inhibitor (SKTI) was the only plant PI tested to inhibit all three digestive protease activities at concentrations <40 &mgr;g/ml (approx. 5x10(-6)M). Incorporation of SKTI into a potato leaf-based artificial diet at 2% of total protein, decreased larval survival and growth (by approx. 33% and 40% respectively after 21 days) and retarded development (by approx. 2 days). However, when SKTI was expressed in transgenic potato plants at approx. 0.5% of total protein, only marginal effects on L. oleracea larvae were observed, which decreased with time. Whilst the presence of SKTI in artificial diet increased endogenous larval trypsin-like activity by up to four-fold, no effects on this activity were observed in larvae feeding on transgenic plants.     

The diamondback moth, Plutella xylostella, specifically inactivates Mustard Trypsin Inhibitor 2 (MTI2) to overcome host plant defence

The mustard trypsin inhibitor family has so far only been described among cruciferous species which represent the host plants for the specialist diamondback moth (DBM), Plutella xylostella. The performance of a Dutch and Chinese strain of DBM was assessed on transgenic Arabidopsis expressing Mustard Trypsin Inhibitor 2 (MTI2) at a level of 84 microg/g fresh weight equivalent to 12 microM. No significant differences in larval mortality or development were found relative to the control. Trypsin activity in gut extracts from larvae feeding on either control or transgenic plants were titrated with MTI2 and SKTI (Soybean Kunitz Trypsin Inhibitor) to assess the basis of the insensitivity to MTI2. The specific trypsin activity per gut of larvae reared on MTI2 plants was not significantly higher compared to the control, and ca. 80% of trypsin activity could be inhibited by both inhibitors in both treatments, suggesting no specific induction of PI-insensitive activity in response to MTI2 in the diet. On the basis of the apparent equilibrium dissociation constant of Plutella trypsins for MTI2 (80 nM), the gut trypsin concentration (4.8 microM), and the MTI2 concentration in the leaves (12 microM) it was calculated that 99% of the gut trypsin activity sensitive to MTI2 should be inhibited in vivo, unless MTI2 was degraded. Indeed, we found that a pre-incubation of MTI2 and SKTI with gut proteases for 3 h resulted in complete loss of inhibitory activity of MTI2, but not of SKTI, at the concentration ratios found in planta. This process was enzymatic as it was inactivated by heat. Gut extracts of larvae reared on control or MTI2 leaves were equally well capable of this degradation indicating that the inactivating enzymes are constitutively expressed. In conclusion, it appears that the insensitivity of the diamondback moth to MTI2 can be sufficiently explained by the specific degradation of MTI2, thereby protecting itself against this protease inhibitor which is part of the defense of cruciferous plant species.     

Partial characterization of the proteolytic enzymes in the gut of the banana weevil, Cosmopolites sordidus, and effects of soybean Kunitz trypsin inhibitor on larval performance

The proteolytic enzymes in the gut of the banana weevil, Cosmopolites sordidus (Germar) (Coleoptera: Curculionidae), have been characterized. Both larvae and adults rely on a complex proteolytic system based on at least cathepsin D‐, cathepsin B‐, trypsin‐, chymotrypsin‐, leucine aminopeptidase‐, carboxypeptidase A‐, and carboxypeptidase B‐like activities. All endoproteolytic activities were higher in the anterior section of the gut, whereas the exopeptidases were evenly distributed in the anterior and middle sections, and almost no activity was detected in the posterior section. Gelatin‐containing gels confirmed the spatial organization of the proteolytic digestive process. According to this proteolytic profile, the STI (soybean Kunitz trypsin inhibitor) was tested in vivo to establish its potential as a resistance factor against C. sordidus. Newly hatched larvae fed on diets containing 0.2% (w/w) STI experience lower survival rates and display significant reductions in larval growth. Biochemical analysis carried out on guts of larvae reared on STI‐treated diet showed a reduction of trypsin‐like activity compared to that from larvae fed on control diet. This decrease was compensated with an induction of cathepsin B, whereas cathepsin D, chymotrypsin, and leucine aminopeptidase were not affected. These results are discussed as a basis for selecting appropriate inhibitors to obtain transgenic banana and plantain plants with enhanced resistance to this pest.     

Obtaining a Transgenic Upland Cotton Harboring Two Insecticidal Genes

pBinLK carried two insecticidal genes, pea lectin (P-Lec)gene and soybean Kunitz trypsin inhibitor (SKTI) gene, were successfully transferred into 4 upland cotton ( Gossypium hirsutum L. ) cultivars, "Xinluzao-1 ", "Xinluzhong-2", "Jihe-321" and "Liao-9" via Agrobacterium-mediated transformation. Hypocotyl segments from aseptic seedlings were used as receipient. After co-cultivation of hypocotyl segments with A. tumefac/ens (Smith et Townsend) Conn, kanamycin-resistant calli were screened, and somatic embryos and regenerated plants were obtained through various media. Transgenic cotton plants harboring two insecticidal genes were confirmed by NPT-Ⅱ ELISA, PCR and PCR Southern. rllae results of bioassay demonstrated that the transgenic plants showed significant resistance to the larvae of cotton bollwonn (Heliothis armigera Hubner).     

Reducing boll weevil populations by clipping terminal buds and removing abscised fruiting bodies

Cotton pests damaging fruiting bodies (squares and young bolls) are difficult to control and their damage results in direct yield loss. Small growers, with low technological inputs, represent a large portion of cotton growers worldwide comprising more than 76 countries; they rely mainly on cultural practices to counteract pest attack in their crops. Boll weevil, Anthonomus grandis Boheman (Coleoptera: Curculionidae), oviposition involves puncturing cotton squares and young bolls, causing abscission. We examined the impact on boll weevil population of collecting abscised cotton fruiting bodies and clipping plant terminals at 50% boll maturation in the field during two cotton‐growing seasons and under field cage conditions. Greatest numbers of damaged squares occurred ca. 117 days after planting and clipped plants resulted in reduction of abscised structures and adult boll weevils compared with non‐clipped plants, irrespective of cotton variety. Damaged young bolls were found ca. 128 days after planting in 2009 and 2011, but clipping had no effect. Numbers of boll weevils found in plants of the varieties BRS 201 and BRS Rubi (both in 2009) and BRS Rubi (in 2011) were, respectively, 13‐, 17‐, and 20‐fold greater when clipping plus collecting abscised fruiting bodies were not practiced. Furthermore, the average percentage of the boll weevil parasitoid Bracon vulgaris Ashmead (Hymenoptera: Braconidae) emerging from abscised and collected structures was similar between clipped and non‐clipped plant terminals in both seasons. Clipping plant terminals did not result in yield reduction and reduced adult boll weevil production. Collecting abscised reproductive structures, clipping plant terminals, and using both practices together reduced boll weevil populations by as much as 63, 57, and 79%, respectively, in cage trials. Thus, these practices cause significant impact on boll weevil populations and are feasible of adoption, especially for smallholder cotton growers.     

Biochemical characterization of midgut digestive proteases from Mamestra brassicae (cabbage moth; Lepidoptera: Noctuidae) and effect of soybean Kunitz inhibitor (SKTI) in feeding assays

Proteolytic activities in soluble protein extracts from Mamestra brassicae (cabbage moth) larval midgut were analysed using specific peptide substrates and proteinase inhibitors. Serine proteinases were the major activities detected, with chymotrypsin-like and trypsin-like activities being responsible for approximately 62% and 19% of the total proteolytic activity towards a non-specific protein substrate. Only small amounts of elastase-like activities could be detected. The serine proteinases were active across the pH range 7-12.5, with both trypsin-like and chymotrypsin-like activities maximal at pH 11.5. The digestive proteinases were stable to the alkaline environment of the lepidopteran gut over the timescale of passage of food through the gut, with 50% of trypsin and 40% of chymotrypsin activity remaining after 6h at pH 12, 37 degrees C. Soybean Kunitz trypsin inhibitor (SKTI) ingestion by the larvae had a growth-inhibitory effect, and induced inhibitor-insensitive trypsin-like activity. Qualitative and quantitative changes in proteinase activity bands after gel electrophoresis of gut extracts were evident in SKTI-fed larvae when compared with controls, with increases in levels of most bands, appearance of new bands, and a decrease in the major proteinase band present in extracts from control insects.