The pharmacological flexibility of the insect voltage gated sodium channel: toxicity of AaIT to knockdown resistant (kdr) flies.

AaIT is an insect selective neurotoxic polypeptide shown to affect insect neuronal sodium conductance by binding to excitable sodium channels. In the present study the paralytic potency of AaIT to wild type and various mutant strains of houseflies (Musca domestica) and fruitflies (Drosophila melanogaster) was examined and it has been shown that: On the basis of body weight when compared to published data on Sarcophaga falculata blowflies, the Musca and Drosophila flies reveal at least two orders of magnitude decreased susceptibility to the AaIT. When compared to wild type flies the toxicity of AaIT is greatly altered in knockdown resistant fly strains which are mutated in their para gene encoding the voltage gated sodium channel. Several strains, with genetically mapped para mutations conferring pyrethroid resistance, exhibited opposing response to AaIT. The para ts2 Drosophila strain, with a point of mutation in domain I of the para gene conferring a 6-fold resistance to deltamethrin also showed about 15-fold tolerance to AaIT. On the other hand the Musca kdr and super-kdr flies, with a single or a double point mutation, respectively in domain II of the para gene, are about 9- and 14-fold more susceptible to AaIT, respectively. The above data are interpreted in terms of the pharmacological diversity and flexibility ("allosteric coupling") of voltage gated sodium channels and their implications for the management of pesticide resistance are discussed.     

Proteolytic breakdown of the Neb-trypsin modulating oostatic factor (Neb-TMOF) in the hemolymph of different insects and its gut epithelial transport

The degradation of the unblocked hexapeptide, trypsin modulating oostatic factor of the flesh fly Neobellieria (Sarcophaga) bullata (Neb-TMOF) was studied in vitro in the hemolymph of the lepidopteran Spodoptera frugiperda, the orthopteran Schistocerca gregaria and the dictyopteran Leucophaea maderae. The half-life in the different species varied from approximately 3min in L. maderae to approximately 25min in S. gregaria. Purification of the degradation products and ESI-Qq-oa-Tof mass spectrometry revealed the fragments Asn-Pro-Thr-Asn, Leu-His and Asn-Pro, which were the same in the hemolymph of all species. Except in Leucophaea, Neb-TMOF was cleaved in dipeptides starting from the C-terminus and the reaction could be, at least partially, inhibited by captopril. These observations suggest that a dipeptidase, which has very similar enzymatic properties as mammalian angiotensin converting enzyme (ACE) and which circulates in the hemolymph, apparently is involved in the breakdown of Neb-TMOF and might be a common but not a universal enzyme in insect hemolymph.The introduction of Neb-TMOF into the gut of S. gregaria with the help of a capillary tube (intubation) demonstrated that the intact peptide is able to cross the gut epithelium and to appear in the hemolymph compartment. Since [3H]-inulin, which is too large to cross cell membranes, was found to penetrate the gut walls at a measurable rate, the paracellular pathway might be also permeable to smaller peptides. There was indeed a clear correlation between the molecular weight of inulin, Neb-TMOF, and inositol and the rate of penetration of these compounds through the gut epithelium to the hemolymph. These are promising findings in view of a potential use of such peptides for insect control purposes.     

AaIT: from neurotoxin to insecticide

AaIT is a single chain neurotoxic polypeptide derived from the venom of the Buthid scorpion Androctonus australis Hector, composed of 70 amino acids cross-linked by four disulfide bridges. Its strict selectivity for insects has been documented by toxicity, electrophysiological and ligand receptor binding assays. These last have shown that various insect neuronal membranes possess a single class of non-interacting AaIT binding sites of high affinity (K(D) = 1-3(n)M) and low capacity (0.5-2.0 pmol/mg prot.). The fast excitatory paralysis induced by AaIT is a result of a presynaptic effect, namely the induction of a repetitive firing in the terminal branches of the insect's motor nerves resulting in a massive and uncoordinated stimulation of the respective skeletal muscles. The neuronal repetitive activity is attributed to an exclusive and specific perturbation of sodium conductance as a consequence of toxin binding to external loops of the insect voltage-dependent sodium channel and modification of its gating mechanism. From a strictly agrotechnical point of view AaIT involvement in plant protection has taken the following two complementary forms: firstly, as a factor for the genetic engineering of insect infective baculoviruses resulting in potent and selective bio-insecticides. The efficacy of the AaIT-expressing, recombinant baculovirus is attributed mainly to its ability to continuously provide and translocate the gene of the expressed toxin to the insect central nervous system; secondly, based on the pharmacological flexibility of the voltage-gated sodium channel, as a device for insecticide resistance management. Channel mutations conferring resistance to a given class of insecticidal agents (such as the KDR phenomenon) may greatly increase susceptibility to the AaIT expressing bioinsecticides. Thus the AaIT is a pharmacological tool for the study of insect neuronal excitability and chemical ecology and the development of new approaches to insect control.     

Penetrability of orally toxic protein from cobra venom through the gut of a blowfly

The oral toxicity of a radioiodinated toxic polypeptide isolated from a cobra snake venom as assayed by Sarcophaga falculata blowflies coupled with assays on competitive displacement have indicated that: (a) During 3–4 h 8% of the orally active toxin is able to pass through the digestive system of the fly; (b) the orally active toxin after passing the gut binds to body tissues. The strong affinity of the toxin to tissue membranes explains its absence in the insect's hemolymph following oral applications as well as injection.The removal of traces of phospholipase A, which is extremely toxic, by injection of the orally active toxin has significantly lowered its injection toxicity without affecting its oral toxicity, thus indicating the absence of any interaction with phospholipases in oral toxicity. This conclusion was supported by additional experimentation.     

The tolerance of lepidopterous larvae to an insect selective neurotoxin

The interaction of the fast-neurotoxic and insect selective polypeptide derived from scorpion venom (AaIT) with lepidopterous larvae tissues was studied through assays of toxicity, chromatography, binding and light microscopical autoradiography. The native and/or radioiodinated toxin was shown to:

• 1.(1) Induce a delayed, slow, progressive paralysis (within 24–48 h) of Spodoptera larvae by relatively high doses (paralytic unit = 2.4 μg/100 mg) corresponding to about only 10% of the total toxicity of the crude venom. Larvae of six species representing five families of Lepidoptera responded similarly to the toxin.
• 2.(2) Resist an in vitro incubation in the insect's hemolymph.
• 3.(3) Lose 80% of its toxicity in the insect's body within 24 h, accompanied by a progressive process of degradation and elimination by the excretory system.
• 4.(4) Specifically bind to a single class of non-interacting binding sites of high affinity and low capacity (0.2 pmol/mg protein, similar to tritiated saxitoxin) in an in vitro, homogenate derived, neuronal preparation.
• 5.(5) Specifically bind with high affinity to desheathed but otherwise intact nerves.
• 6.(6) Be devoid of accessibility to peripheral-terminal branches of Spodoptera motor nerves in situ—strongly contrasting those of the toxin susceptible Periplaneta nerves.

It may be thus concluded that the tolerance of the lepidopterous larvae to AaIT can be substantially attributed to pharmacokinetic aspects of toxin accessibility barriers and degradation processes.     

Recombinant scorpion insectotoxin AaIT kills specifically insect cells but not human cells

The nucleotide sequence deduced from the amino acid sequence of the scorpion insectotoxin AaIT was chemically synthesized and was expressed in Escherichia coli. The authenticity of this in vitro expressed peptide was confirmed by N-terminal peptide sequencing. Two groups of bioassays, artificial diet incorporation assay and contact insecticidal effect assay, were carried out separately to verify the toxicity of this recombinant toxin. At the end of a 24 h experimental period, more than 60% of the testing diamondback moth (Plutella xylostella) larvae were killed in both groups with LCs0 value of 18.4 uM and 0.70 μM respectively. Cytotoxicity assay using cultured Sf9 insect cells and MCF-7 human cells demonstrated that the toxin AaIT had specific toxicity against insect cells but not human cells. Only 0.13 μM recombinant toxin was needed to kill 50% of cultured insect cells while as much as 1.3μM toxin had absolutely no effect on human cells. Insect cells produced obvious intrusions from their plasma membrane before broken up. We infer that toxin AaIT bind to a putative sodium channel in these insect cells and open the channel persistently, which would result in Na influx and finally cause destruction of insect cells.     

An excitatory and a depressant insect toxin from scorpion venom both affect sodium conductance and possess a common binding site

Two insect selective toxins were purified by gel-permeation and ion-exchange chromatographies from the venom of the scorpion, Leiurus quinquestriatus quinquestriatus, and their chemical and pharmacological properties were studied. The first toxin (LqqIT1) induces a fast excitatory contraction paralysis of fly larvae and is about 40 times more toxic than the crude venom. It is a polypeptide composed of 71 amino acids, including 8 half-cystines and devoid of methionine and tryptophan, with an estimated molecular weight of 8189 and a pI value of 8.5. The second toxin (LqqIT2) induces a slow depressant, flaccid paralysis of fly larvae. It is composed of 72 amino acids, including 8 half-cystines, is devoid of proline methionine and histidine, and has an estimated molecular weight of 7990 and a pI value of 8.3. The contrasting symptomatology of these toxins is interpreted in terms of their effects on an isolated axonal preparation of the cockroach in current and voltage clamp conditions. LqqIT1 (0.5-4 microM) induced repetitive firing of the axon which was attributable to two changes in the sodium conductance, a small increase in the peak conductance and a slowing of its turning off. LqqIT2 (1-8 microM) caused a blockage of the evoked action potentials, attributable to both a strong depolarization of the axonal membrane and a progressive suppression of the sodium current. Neither toxin affected potassium conductance. The two toxins differ mainly in their opposite effects on the activatable sodium permeability. In binding assays to a preparation of insect synaptosomal membrane vesicles, the two toxins were shown to competitively displace the radioiodinated excitatory insect toxin derived from the venom of the scorpion, Androctonus australis [( 125I]AaIT), which strongly resembles, in its chemistry and action, the LqqIT1 toxin. The present two toxins have demonstrated a strong affinity closely resembling the AaIT, with KD values of 0.4, 1.9, and 1.0 nM for LqqIT1, LqqIT2, and AaIT, respectively. These data suggest the possibility that the excitatory and depressant insect toxins share a common binding site associated with sodium channels in insect neuronal membranes.     

Rapid purification and molecular modeling of AaIT peptides from venom of Androctonus australis

As recombinant viruses expressing scorpion toxins are moving closer toward the market, it is important to obtain large amounts of pure toxin for biochemical characterization and the evaluation of biological activity in nontarget organisms. In the past, we purified a large amount of Androctonus australis anti-insect toxin (AaIT) present in the venom of A. australis with an analytical reversed-phase column by repeated runs of crude sample. We now report 20 times improved efficiency and speed of the purification by employing a preparative reversed-phase column. In just two consecutive HPLC steps, almost 1 mg of AaIT was obtained from 70 mg crude venom. Furthermore, additional AaIT was obtained from side fractions in a second HPLC run. Recently discovered insect selective toxin, AaIT5, was isolated simultaneously from the same venom batch. It shows different biological toxicity symptoms than the known excitatory and depressant insect toxins. AaIT5 gave 100% mortality with a dose of less than 1.3 μg against fourth-instar tobacco budworms Heliothis virescens 24 h after injection. During the purification process, we implemented mass spectrometry in addition to bioassays to monitor the presence of AaIT and AaIT5 in the HPLC fractions. Mass spectrometric screening can unambiguously follow the purification process and can greatly facilitate and expedite the downstream purification of AaIT and AaIT5 eliminating the number of bioassays required. Further, electrospray ionization was compared with matrix-assisted desorption/ionization and evaluated as a method of choice for mass spectrometric characterization of fractions from the venom purification for it provided higher mass accuracy and relative quantitation capability. Molecular models were built for AaIT5, excitatory toxin AaIT4, and depressant toxin LqhIT2. Three-dimensional structure of AaIT5 was compared with structures of the other two toxins, suggesting that AaIT5 is similar to depressant toxins. Arch. Insect Biochem. Physiol. 38:53–65, 1998. © 1998 Wiley-Liss, Inc.     

Differential protection of Cry1Fa toxin against Spodoptera frugiperda larval gut proteases by cadherin orthologs correlates with increased synergism

The Cry proteins produced by Bacillus thuringiensis (Bt) are the most widely used biopesticides effective against a range of crop pests and disease vectors. Like chemical pesticides, development of resistance is the primary threat to the long-term efficacy of Bt toxins. Recently discovered cadherin-based Bt Cry synergists showed the potential to augment resistance management by improving efficacy of Cry toxins. However, the mode of action of Bt Cry synergists is thus far unclear. Here we elucidate the mechanism of cadherin-based Cry toxin synergism utilizing two cadherin peptides, Spodoptera frugiperda Cad (SfCad) and Manduca sexta Cad (MsCad), which differentially enhance Cry1Fa toxicity to Spodoptera frugiperda neonates. We show that differential SfCad- and MsCad-mediated protection of Cry1Fa toxin in the Spodoptera frugiperda midgut correlates with differential Cry1Fa toxicity enhancement. Both peptides exhibited high affinity for Cry1Fa toxin and an increased rate of Cry1Fa-induced pore formation in S. frugiperda. However, only SfCad bound the S. frugiperda brush border membrane vesicle and more effectively prolonged the stability of Cry1Fa toxin in the gut, explaining higher Cry1Fa enhancement by this peptide. This study shows that cadherin fragments may enhance B. thuringiensis toxicity by at least two different mechanisms or a combination thereof: (i) protection of Cry toxin from protease degradation in the insect midgut and (ii) enhancement of pore-forming ability of Cry toxin.     

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.     

In vitro release of digestive enzymes by FMRF amide related neuropeptides and analogues in the lepidopteran insect Opisina arenosella (Walk.)

The insect neuropeptides FMRF amide, leucomyosupressin (LMS) and neuropeptide analogues leucosulfakinins (FLSK and LSK II Ser (SO(3)H)), perisulfakinin (PSK), proleucosulfakinin (PLSK), 14A[phi1]WP-I, 542phi1, and 378A[5b]WP-I were assayed for their effects on the release of amylase and protease from the midgut tissue of larvae of Opisina arenosella. In the bioassay, empty midgut tubes ligated at both ends using hair were incubated with insect saline containing neuropeptides/analogues in a bioassay apparatus at 37 degrees C for 30 min. After incubation the contents of the midgut preparations were analyzed for amylase and protease activity. In control experiments, the midgut preparations were incubated in insect saline without neuropeptides. The results of the study reveal that for stimulating amylase release from midgut tissue, the peptides require an FXRF amide (X may be methionine or leucine) sequence at the C-terminal. The presence of HMRF amide at C-terminal of peptides may inhibit the release of amylase. Meanwhile, peptides with both FMRF and HMRF amide sequence at the C-terminal are found to be effective in stimulating protease release. The tetrapeptide segment at the C-terminal probably represent the active core of the neuropeptide.     

Localization of receptor sites for insect-selective toxins on sodium channels by site-directed antibodies.

Site-directed antibodies corresponding to conserved putative extracellular segments of sodium channels, coupled with binding studies of radiolabeled insect-selective scorpion neurotoxins, were employed to clarify the relationship between the toxins' receptor sites and the insect sodium channel. (1) The depressant insect toxin LqhIT2 was shown to possess two noninteracting binding sites in locust neuronal membranes: a high-affinity (KD1 = 0.9 +/- 0.6 nM) and low-capacity (Bmax1 = 0.1 +/- 0.07 pmol/mg) binding site as well as a low-affinity (KD2 = 185 +/- 13 nM) and high-capacity (Bmax2 = 10.0 +/- 0.6 pmol/mg) binding site. (2) The high-affinity site serves as a target for binding competition by the excitatory insect toxin AaIT. (3) The binding of LqhIT2 was significantly inhibited in a dose-dependent manner by each of four site-directed antibodies. The binding inhibition resulted from reduction in the number of binding sites. (4) The antibody-mediated inhibition of [125I]AaIT binding differs from that of LqhIT2: three out of the four antibodies which inhibited LqhIT2 binding only partially affected AaIT binding. Two antibodies, one corresponding to extracellular and one to intracellular segments of the channel, did not affect the binding of either toxin. These data suggest that the receptors to the depressant and excitatory insect toxins (a) comprise an integral part of the insect sodium channel, (b) are formed by segments of external loops in domains I, III, and IV of the sodium channel, and (c) are localized in close proximity but are not identical in spite of the competitive interaction between these toxins.     

Differences in the midgut proteolytic activity of two Heliothis virescens strains,one susceptible and one resistant to Bacillus thuringiensis toxins

The development of resistance to Bacillus thuringiensis toxic proteins is a growing concern because it could threaten both conventional and gene transfer use of this environmentally safe biological insecticide. The most common mechanism of resistance involves changes in binding affinity of toxin receptors in the insect midgut membrane. This has not been the case in Heliothis virescens. We have investigated changes in midgut proteolytic activity as a possibility to explain the resistance observed in this insect species. We have developed an improvement of known methods to demonstrate proteolytic activity in crude extracts. Using this method we have found differences in the proteolytic activity profile of midgut extracts of a susceptible and a resistant H. virescens strain. We also have studied the in vitro processing of CrylA(b) toxin and protoxin by midgut contents of both strains. SDS-PAGE of the in vitro degradation products showed differences between the strains. The resistant strain degrades protoxin more slowly and processes the active toxin more quickly than the susceptible strain. © 1996 Wiley-Liss, Inc.     

Transport of peroxidase through the midgut epithelium of Glossina M. Morsitans (diptera, glossinidae)

The peritrophic membrane (pm) of teneral female tsetse flies, Glossina morsitans morsitans, did not extend to the full length of the midgut 1-12 hr after emergence. The ingested blood did not reach the posterior part of the midgut (p-part), and the crop still contained food 12 hr after feeding. In these flies, the p-part contained the remains of the larval gut, the meconium, and bacteria. Ferritin molecules fed to tsetse females together with human serum were only found in the endoperitrophic space of the gut. This electron-dense tracer did not penetrate and cross the pm. On the other hand, ingested peroxidase passed the pm, and was transported through intercellular clefts, the basal labyrinth and the basal lamina to the hemolymph. This uptake was observed in the anterior part and to a smaller extent in the middle part of the midgut within 2 hr after feeding. Peroxidase was incorporated from the hemolymph into fat body cells, where it was found 2 hr and later after feeding. Pinocytosis of the tracer molecules, as an additional intracellular pathway to the intercellular route of transport, could not be demonstrated.     

Transgenic plants expressing the AaIT/GNA fusion protein show increased resistance and toxicity to both chewing and sucking pests

The adoption of pest‐resistant transgenic plants to reduce yield losses and decrease pesticide use has been successful. To achieve the goal of controlling both chewing and sucking pests in a given transgenic plant, we generated transgenic tobacco, Arabidopsis, and rice plants expressing the fusion protein, AaIT/GNA, in which an insecticidal scorpion venom neurotoxin (Androctonus australis toxin, AaIT) is fused to snowdrop lectin (Galanthus nivalis agglutinin, GNA). Compared with transgenic tobacco and Arabidopsis plants expressing AaIT or GNA, transgenic plants expressing AaIT/GNA exhibited increased resistance and toxicity to one chewing pest, the cotton bollworm, Helicoverpa armigera. Transgenic tobacco and rice plants expressing AaIT/GNA showed increased resistance and toxicity to two sucking pests, the whitefly, Bemisia tabaci, and the rice brown planthopper, Nilaparvata lugens, respectively. Moreover, in the field, transgenic rice plants expressing AaIT/GNA exhibited a significant improvement in grain yield when infested with N. lugens. This study shows that expressing the AaIT/GNA fusion protein in transgenic plants can be a useful approach for controlling pests, particularly sucking pests which are not susceptible to the toxin in Bt crops.     

Mode of action of Bacillus thuringiensis δ-endotoxin: General characteristics of intoxicated Bombyx larvae

The general pathology induced by δ-endotoxin in terms of larval behavior and hemolymph chemistry has been widely studied in the so-called Type I insect, Bombyx mori. The succession of symptoms is divided into four arbitrary stages: Stage 0, appearance and locomotion normal, no feeding; Stage 1, slightly sluggish; Stage 2, extremely sluggish; and Stage 3, complete paralysis. The action of δ-endotoxin is highly specific to the midgut since contractile movement of both foregut and hindgut continues long after all locomotor activity and heartbeat have stopped. Immediately after the silkworm stops feeding and blood pH sharply rises, there is an associated abrupt rise in the K⁺ concentration of hemolymph. Thereafter, the rise in K⁺ is linear while the rise in pH is not. In vivo measurements have not yielded the same simple linear dependence of pH on K⁺ concentrations that is found in in vitro mixtures of hemolymph and midgut juice. Ligation experiments showed that the same pathological sequence (rise of pH and K⁺ concentration, and general paralysis) follows whether the toxin has unrestricted access to the entire midgut or only part of it (anterior or posterior). From the results of injections of midgut juice or various salt solutions into hemocoel, we came to the conclusions that the blood pH and the symptoms are not necessarily parallel and the intact midgut and Malpighian tubules have strong functions for ion regulation.     

Changes in protease activity and Cry3Aa toxin binding in the Colorado potato beetle: implications for insect resistance to Bacillus thuringiensis toxins

Widespread commercial use of Bacillus thuringiensis Cry toxins to control pest insects has increased the likelihood for development of insect resistance to this entomopathogen. In this study, we investigated protease activity profiles and toxin-binding capacities in the midgut of a strain of Colorado potato beetle (CPB) that has developed resistance to the Cry3Aa toxin of B. thuringiensis subsp. tenebrionis. Histological examination revealed that the structural integrity of the midgut tissue in the toxin-resistant (R) insect was retained whereas the same tissue was devastated by toxin action in the susceptible (S) strain. Function-based activity profiling using zymographic gels showed specific proteolytic bands present in midgut extracts and brush border membrane vesicles (BBMV) of the R strain not apparent in the S strain. Aminopeptidase activity associated with insect midgut was higher in the R strain than in the S strain. Enzymatic processing of toxin did not differ in either strain and, apparently, is not a factor in resistance. BBMV from the R strain bound approximately 60% less toxin than BBMV from the S strain, whereas the kinetics of toxin saturation of BBMV was 30 times less in the R strain than in the S strain. However, homologous competition inhibition binding of (125)I-Cry3Aa to BBMV did not reveal any differences in binding affinity (K(d) approximately 0.1 microM) between the S and R strains. The results indicate that resistance by the CPB to the Cry3Aa toxin correlates with specific alterations in protease activity in the midgut as well as with decreased toxin binding. We believe that these features reflect adaptive responses that render the insect refractory to toxin action, making this insect an ideal model to study host innate responses and adaptive changes brought on by bacterial toxin interaction.     

棉铃虫和甜菜夜蛾中肠丝氨酸蛋白酶活性测定以及活化、降解Cry1Ac毒素分析

昆虫中肠对Bt原毒素活化与对活化毒素降解的变化被认为是害虫对Bt产生的机制之一,研究比较棉铃虫Helicoverpa armigera(Hübner)与甜菜夜蛾Spodoptera exigua(Hübner)的中肠液、BBMV蛋白酶的活性,通过SDS-PAGE分析2种昆虫对原毒素的活化速度与对活化毒素的降解速度。2种昆虫的中肠液蛋白酶活性均显著高于BBMV蛋白酶活性,中肠液与BBMV均能迅速活化原毒素并继续降解活化后的毒素,与中肠液相比,BBMV对原毒素的活化与对活化毒素的降解均慢于中肠液,甜菜夜蛾对毒素的活化与降解又慢于棉铃虫。另外,还测定抑制剂对中肠液蛋白酶活性的抑制作用,结果表明,各抑制剂对棉铃虫和甜菜夜蛾相应酶活性的抑制表现出相同的趋势,TLCK对丝氨酶蛋白酶具较好的抑制作用,而PMSF对胰蛋白酶的抑制作用次之,TPCK对胰凝乳蛋白酶的抑制作用较弱。     

棉铃虫和甜菜夜蛾中肠丝氨酸蛋白酶活性测定以及活化、降解CrylAc毒素分析

昆虫中肠对Bt原毒素活化与对活化毒素降解的变化被认为是害虫对Bt产生的机制之一,研究比较棉铃虫Helicoverpa armigern（Hǔbner）与甜菜夜蛾Spodoptera exigm（Hǔbner）的中肠液、BBMV蛋白酶的活性,通过SDS-PAGE分析2种昆虫对原毒素的活化速度与对活化毒素的降解速度。2种昆虫的中肠液蛋白酶活性均显著高于BBMV蛋白酶活性,中肠液与BBMV均能迅速活化原毒素并继续降解活化后的毒素,与中肠液相比,BBMV对原毒素的活化与对活化毒素的降解均慢于中肠液,甜菜夜蛾对毒素的活化与降解又慢于棉铃虫。另外,还测定抑制剂对中肠液蛋白酶活性的抑制作用,结果表明,各抑制剂对棉铃虫和甜菜夜蛾相应酶活性的抑制表现出相同的趋势,TLCK对丝氨酶蛋白酶具较好的抑制作用,而PMSF对胰蛋白酶的抑制作用次之,TPCK对胰凝乳蛋白酶的抑制作用较弱。     

Isolation, characterization, and amino acid sequence of a ubiquitin-like protein from insect eggs

A protein with a primary structure identical to that of human and bovine ubiquitin has been purified from insect eggs. The isolation, secondary structure, and amino acid sequence of this ubiquitin-like protein are reported. The sequence was determined by automatic Edman degradation of the intact molecule as well as by the manual sequence analysis of the enzymatic cleavage products. The polypeptide has 74 amino acid residues and internal homology regions. Interactions of the protein with peptides results in protective effects against proteolysis. This paper reports for the first time the presence of the ubiquitin molecule in invertebrates.