Proteinase-mediated insect resistance to Bacillus thuringiensis toxins.

Two Bacillus thuringiensis (Bt)-resistant strains of the Indianmeal moth, Plodia interpunctella, lack a major gut proteinase that activates Bt protoxins. The absence of this enzyme is genetically linked to larval survival on Bt-treated diets. When considered with previous data supporting the existence of receptor-mediated insect resistance to Bt, these results provide evidence that insect adaptation to these toxins occurs through multiple physiological mechanisms, which complicate efforts to prevent or manage resistance to Bt toxins in insect control programs.

Insecticidal proteins of the bacterium Bacillus thuringiensis (Bt) 1 are effective for controlling many insect pest species, but insect resistance threatens the long term effectiveness of these toxins (1). With the introduction of transgenic plants expressing Bt toxins in the field (2), insects will likely be under increased selection pressure for resistance. To develop effective toxin resistance management strategies, a full understanding of the physiological and genetic mechanisms by which insects become resistant to these insecticidal proteins is needed.
More than 100 insecticidal crystal protein genes from Bt subspecies have been described. 2 These genes encode protoxins, referred to as Cry proteins, that have different specificity for lepidopteran, coleopteran, or dipteran insects. The Cry1 subclass consists of lepidopteran-active Bt protoxins with apparent molecular masses of approximately 130 kDa, which are solubilized and processed (activated) by gut enzymes to approximately 65-kDa toxins (4). Toxins interact with receptors in the guts of susceptible insects resulting in pore formation in midgut cell membranes, ionic imbalance, and consequent septicemia in the insect. Alterations in insect gut physiology or biochemistry could disrupt this sequential process and result in toxin resistance.
Several mechanisms of insect resistance to Bt toxins have been proposed (5). One involves changes in the binding of toxins to gut receptors. In a Bt subspecies kurstaki-resistant strain of the Indianmeal moth, Plodia interpunctella, reduced binding of Cry1Ab toxin to larval brush border membrane vesicles was associated with increased resistance to the toxin (6). Decreases in toxin binding have also been reported in resistant strains of the diamondback moth, Plutella xylostella (7)(8)(9)(10)(11)(12), tobacco budworm, Heliothis virescens (13,14), and beet armyworm, Spodoptera exigua (15). However, reduced toxin binding is not always associated with resistance to Bt (16,17), and therefore alternate mechanisms of resistance must exist.
A second mechanism of resistance may involve gut proteinases that interact with Bt toxins. Enzymes from a strain of H. virescens resistant to Bt subspecies kurstaki (HD-73) were reported to process the protoxin more slowly and to degrade toxin faster than enzymes from a susceptible strain (18). In Spodoptera littoralis, an increase in the specific activity of gut proteinases from fifth instar larvae was associated with a loss of sensitivity to Cry1C, possibly due to an increase in the degradation of toxin (19).
Previously, we reported that a strain of P. interpunctella resistant to Bt subspecies entomocidus has low soluble gut proteinase and Bt protoxin-hydrolyzing activities when compared with the parent-susceptible strain and a strain resistant to Bt subspecies kurstaki (20,21). The slower protoxin hydrolysis observed with gut extracts from the entomocidus-resistant strain was an indication that proteinase-mediated mechanisms are involved in resistance to Bt. In this report, we characterize the proteolytic enzyme activity in Bt toxin-resistant insects and demonstrate a genetic linkage between the absence of a major gut proteinase and decreased susceptibility to the toxin.

EXPERIMENTAL PROCEDURES
Insect Dissection-Late fourth instar larvae were chilled, and the posterior and anterior ends were removed. Guts were excised, immediately submersed in ice-cold 200 mM Tris, pH 8.0, 20 mM CaCl 2 (buffer A), aliquoted 1 gut per 25 l of buffer, and frozen at Ϫ20°C for up to 2 weeks until assayed.
Proteinase Activity Assays-For microplate proteinase assays, samples were thawed and spun at 15,000 ϫ g for 2 min, and the supernatant containing soluble gut enzymes was used in assays. Samples were diluted 1:100 in buffer A, and 50 l were added to a microplate well. N-␣-Benzoyl-L-arginine p-nitroanilide (BApNA, Sigma, 100 mg/ml in dimethyl sulfoxide) was diluted 1:100 in buffer A, and 50 l were added to each well to initiate the reaction (final BApNA concentration was 1.15 mM). After a 30-s incubation at 37°C, absorbance was monitored at 405 nm at 15-s intervals over a 5-min period. The change in absorbance per min was calculated by the software KinetiCalc3 (Biotek), and the data were converted to micromoles/min/mg of protein in each gut extract. Protein concentration was determined by the method of Bradford (22) using bovine serum albumin as the standard in a microplate assay (Pierce).
For proteinase activity blots, gut extracts from individual larvae or from five pooled individuals were subjected to sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE) using 10 -20% Tricine gels (Novex, 0.4 gut eq/lane). The proteinase activity blot procedure was performed as described previously (21).
For zymogram analysis, gut samples were first analyzed for BApNAhydrolyzing activity by microplate analysis. Volumes of samples were then adjusted for equivalent activity (⌬A 405 nm /min/ml ϭ 2), and aliquots were subjected to SDS-PAGE on a 10 -20% Tricine gel (Novex). Zymogram analysis was performed by incubating the gel with a Cry1Ac protoxin substrate solution (see below) for 2 h at 37°C, followed by brief * The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Cry1Ac protoxin substrate solution was obtained from Bt subspecies kurstaki HD-73 grown on glucose/yeast extract/salts medium at 30°C for 2-3 days with agitation until sporulation was complete (23). Spores, crystals, and cellular debris were washed by centrifugation three times in phosphate-buffered saline and lyophilized. The lyophilized preparation was resuspended in 100 mM sodium carbonate, pH 10.0, 0.8 M sodium chloride, 10 mM EDTA, 20 mM dithiothreitol. After centrifuging at 1000 ϫ g for 10 min, the supernatant was diluted 1:1 with deionized water and analyzed for Cry1Ac protoxin concentration by SDS-PAGE, Coomassie Blue staining, and scanning gel densitometry (International Technologies International) using homogenous Cry1Ac protoxin as the standard (data not shown). The concentration of Cry1Ac protoxin in the zymogram substrate solution was determined to be 0.3% and constituted greater than 50% of the total protein in the preparation.
Genetic Matings-Fifteen virgin male adults from Bt-susceptible strain 688-sЈ, in which all individuals displayed the enzyme T1 (see "Results" for explanation of enzyme terminology), were mated with 15 virgin female entomocidus-resistant strain 198-r adults, which lacked T1. Gut extracts from approximately 15-day-old larvae from each strain were analyzed for BApNA hydrolytic patterns by proteinase activity blots. Fifteen female F1 adults were backcrossed to 15 resistant 198-r males, and progeny were analyzed for BApNA hydrolytic patterns. Female F1 were chosen because meiotic recombination does not occur in female Lepidoptera, and tight linkage would be observed between any pair of traits located on the same chromosome. Insects were reared on untreated diet throughout the matings.
Proteinase Activity in Backcross Insects-Ten mg of eggs from the backcrossed progeny were added to an untreated diet, and 15 mg were added to a 125 mg/kg Bt subspecies entomocidus (HD-198)-treated diet. Adults were selected from each group (n ϭ 27 from the untreated group and n ϭ 12 from the treated group) and placed on an untreated diet and allowed to mate. Progeny from each group were reared on an untreated diet, and guts were dissected from late fourth instar larvae. Gut extracts were analyzed for proteolytic patterns using proteinase activity blot analysis.

RESULTS
Because trypsin-like proteinases in P. interpunctella guts activate Bt protoxin (21), we examined trypsin-like enzyme activity in Bt-susceptible and resistant strains of the Indianmeal moth. Using the trypsin diagnostic substrate BApNA in a microplate assay, BApNA-hydrolyzing activities were detected in a Bt toxin-susceptible strain of P. interpunctella, 688-s, and three resistant strains, 133-r, 198-r, and Dpl-r, derived from the susceptible parental strain via selection with spore/crystal preparations of Bt subspecies aizawai (HD-133), entomocidus (HD-198), or kurstaki (HD-1, Dipel®), 3 respectively (24) ( Table  I). Specific activities of enzymes in the aizawai-and entomocidus-resistant strains were less than one-half of those in the kurstaki-resistant strain and in the parent-susceptible strain. Activity from the kurstaki-resistant strain was approximately 30% higher than activity from the parent-susceptible strain.
To determine the number and relative size of BApNA-hydrolyzing enzymes in each strain, larval gut extracts from the P. interpunctella strains were analyzed for their ability to hydrolyze BApNA using activity blots (Fig. 1). Enzymes with relatively high molecular masses (Ͼ250 kDa) were observed in extracts from all four strains. Two major BApNA-hydrolyzing enzymes, designated T1 and T2 (apparent molecular masses ϳ45 and ϳ25 kDa, respectively), were observed in the Bt toxinsusceptible strain (688-s) and also in the Dipel-resistant strain (Dpl-r). The enzyme corresponding to T1, however, was not present in gut extracts from the strains resistant to either subspecies entomocidus (198-r) or subspecies aizawai (133-r). A minor trypsin-like enzyme, T3, was observed in all of these strains, which had an apparent molecular mass of approximately 50 kDa.
To determine whether these trypsin-like enzymes hydrolyze protoxin, an experiment was designed to compare the relative hydrolysis of protoxin by gut extracts from Bt-susceptible and -resistant P. interpunctella strains. Gut proteinases from the four P. interpunctella strains were separated electrophoretically by SDS-PAGE, and the gel was incubated in a solution of Bt subspecies kurstaki (HD-73) spores and crystals containing Cry1Ac protoxin as the major protein (Fig. 2). Dark zones in the zymogram demonstrated activity of enzymes from the gut extracts, which are capable of hydrolyzing proteins in the spore/ crystal protein preparation. Major hydrolyzing activity corresponded to enzymes T1 and T2 in the susceptible and kurstakiresistant strains but only T2 in the aizawai-and entomocidusresistant strains. T1 and T2 have similar levels of activity. However, T2 appears to have a higher level of activity because it comigrates with another protoxin-hydrolyzing enzyme identified as one with chymotrypsin-like specificity (data not shown). No hydrolysis of protoxin by the higher molecular mass enzymes (i.e. the Ͼ250-kDa BApNA-hydrolyzing enzymes) was observed.
Until now, BApNA-hydrolyzing enzymes had only been examined in extracts from pooled insects (Fig. 1). When gut extracts from individual susceptible larvae were used in enzyme activity blots, we observed that the strain consisted of a heterogeneous population of insects with respect to the expression of trypsin-like proteinases. Some insects expressed T1 and T2, whereas others expressed only T2 (data not shown). Therefore, single pair lines of susceptible strain 688-s were established to ensure genetic uniformity for subsequent linkage tests, and progeny were analyzed using proteinase activity 3 Dipel® is a registered trademark of Abbott Laboratories.  blots. Progeny from one of these "isofemale" lines, designated as 688-sЈ, were analyzed for four generations, and it was determined that all individuals in this population expressed both T1 and T2 trypsin-like enzymes (a representative blot is shown in Fig. 3, 688-sЈ).
To determine if the T1 allele cosegregates with toxin resistance in P. interpunctella, bulked segregant analysis was used. Insects from the homogeneous susceptible strain 688-sЈ were mated with those from the entomocidus-resistant strain 198-r, and female F1 progeny were subsequently backcrossed to males from the resistant strain. Individual larvae from the parental colonies, the F1 generation, and the backcross were analyzed by proteinase activity blots for BApNA-hydrolyzing enzymes (representative blots are shown in Fig. 3). Based on the analysis of 40 single gut extracts from each strain, the susceptible and resistant strains appeared to be homozygous for the presence and absence, respectively, of the T1 allele. Both parental strains were fixed for T2, since all individuals displayed the T2 enzyme. In the F1 generation, 70 individuals were examined and all exhibited T1 enzyme activity. However, the activity of T1 in the F1 larvae was lower than that in the susceptible strain, consistent with the hypothesis that the F1 were heterozygous for the T1 allele. Of the 70 larvae analyzed for BApNA hydrolytic activity in the backcross, 29 (41%) lacked the T1 enzyme, whereas 41 (59%) expressed both T1 and T2, which is close to the expected 1:1 segregation ratio ( 2 ϭ 2.86, degrees of freedom ϭ 1, p ϭ 0.09).
Insects from this backcross population were then analyzed for the ability to survive on toxin-treated diets. Fifty eggs were placed on 0, 125, 250, and 500 mg of Bt subspecies entomocidus HD-198 per kg of diet, and mortality was determined. A discriminating dose of 125 mg/kg was found to kill all susceptible and heterozygous resistant larvae (data not shown). Mortalities at the three Bt toxin doses, corrected for the control mortality, were 59, 78, and 97%, respectively (25). At the 125 mg/kg dose, 41% of the backcross insects survived, the same proportion of the backcross that lacked the T1 enzyme when reared on untreated diet.
The frequency of the T1 enzyme in descendants of backcross progeny survivors reared on untreated versus toxin-treated diets was determined by proteinase activity blots (Fig. 4). In the untreated group, 20 out of 50 insects (40%) lacked the T1 trypsin-like proteinase, whereas the remainder had both T1 and T2. To eliminate differences in enzyme activity due to dietary toxin influences, adults from backcross progeny surviving on an untreated or HD-198-treated diet (125 mg/kg of diet) were intercrossed en masse and placed on untreated diet to lay eggs. Resulting larvae were analyzed for proteinase patterns. In the progeny from untreated diet, 30 out of 50 displayed both T1 and T2 enzymes. All of the progeny from the survivors on the Bt-treated diet (50 out of 50), however, lacked the major trypsin-like proteinase T1.

DISCUSSION
Results from a microplate assay of BApNA hydrolysis by P. interpunctella gut extracts demonstrated that activity was reduced in the aizawai-and entomocidus-resistant larvae when compared with kurstaki-resistant or susceptible larvae. We have also observed slower protoxin hydrolysis with enzymes from the aizawai-resistant strain, similar to results obtained with the entomocidus-resistant strain (20,21). 4 Therefore, resistance may be a result of reduced proteolytic activity in the aizawai-and entomocidus-resistant strains, but it is probably unrelated to proteinase activity in the kurstaki-resistant strain. The kurstaki-resistant strain is probably resistant due 4  to a receptor adaptation, like another kurstaki-resistant strain of P. interpunctella (5).
Results from a proteinase activity blot assay using BApNA as the substrate revealed that aizawai-and entomocidus-resistant larvae lack a major BApNA-hydrolyzing enzyme, T1. The absence of T1 activity in these strains was also evident in zymogram analysis of Bt toxin-hydrolyzing activity. Since T1 is a protoxin-hydrolyzing enzyme, the lack of an active T1 in the aizawai-and entomocidus-resistant strains would result in less toxin being generated in the gut. We propose that the absence of this T1 enzyme leads to reduced activation of protoxin and, therefore, provides a survival advantage when insects feed on diets containing Bt toxins.
Matings of susceptible insects that have both T1 and T2 enzymes with resistant insects that have only the T1 enzyme resulted in progeny with comparable T2 activity but only a relatively low T1 activity. A backcross of these insects to the resistant strain resulted in progeny consisting of a mixture of phenotypes, with individuals either displaying both T1 and T2 or only T2. All backcross larvae that survived a discriminating dose of Bt toxin lacked the T1 enzyme. Therefore, when insects do not have an active T1, they are able to survive a dose of toxin that is lethal to insects that are homozygous or heterozygous for T1. These data demonstrate a genetic linkage between resistance and the absence of T1 and are strong evidence for a proteinase-mediated mechanism of resistance to Bt.
Alleles of structural genes that have no product detectable by electrophoresis are called "null" alleles and are usually recessive (26). In other null alleles, the absence of the active enzyme is due to a mutation that results in an inactive protein or prevents its synthesis in some way. Esterase null alleles are the most commonly described. The number of copies of a gene EST-2, coding for esterase B in mosquitoes, is more than 250 times greater in insecticide-resistant mosquitoes than in sensitive strains (3). In that case, the null allele is normal for sensitive insects. In P. interpunctella, however, the null allele is a survival advantage for the Bt-resistant insects. This is the first description of a null allele for a trypsin-like proteinase coding gene of which we are aware.
In conclusion, we have demonstrated that some Bt toxinresistant strains of P. interpunctella have lower BApNA-hydrolyzing and protoxin-activating abilities than a susceptible or another resistant strain. These differences are due to the lack of a major gut trypsin-like proteinase in the resistant strains.
Results of a genetic analysis demonstrated that insect resistance to Bt toxins segregated with the loss of this major trypsinlike proteinase. The absence of the trypsin-like enzyme apparently results in reduced levels of toxin in the gut, allowing the insects to avoid exposure to high levels of toxin.
This research has led to the understanding that both the genetic variability in the insect population and the composition of the toxin preparation can influence resistance mechanisms. Selection for reduced proteinase activity apparently is possible with only certain Bt preparations. However, these preparations will not select for a proteinase-deficient phenotype if the genotype is not already present in the population. 5 It is not clear how Bt formulations differ such that they elicit different resistance responses in insects. Understanding the biochemical bases for resistance development in insect populations exposed to different Bt formulations will provide for more effective toxin selection, utilization, and durability in integrated pest management programs that utilize Bt toxins.