Spruce budworm elastase precipitates Bacillus thuringiensis delta-endotoxin by specifically recognizing the C-terminal region

A gut juice protein from Choristoneura fumiferana (spruce budworm) larvae that precipitates certain delta-endotoxins shows a unique specificity for the C-terminal amino acid sequence. Using homolog scanning mutants, we have identified a contiguous region of the Cry1Aa toxin which interacts with the 75-kDa toxin precipitating protein (TPP-75)' resulting in precipitation. The contiguous region from Cry1Aa can be transferred to Cry1Ac and results in an identical precipitation reaction. The precipitation reaction occurs rapidly and is unique in that the ratio of precipitating protein to toxin is low (estimated at 0.01), unlike antibody-antigen reactions which exhibit mole ratios close to 1. TPP-75 has been characterized as an elastase-like serine protease. We have taken advantage of this serine protease character and incorporated a radiolabel using an irreversible inhibitor. The radiolabel has allowed us to show the coincidence of the catalytically-inhibited TPP-75 with the toxin in a blotting assay and to follow the degradation of TPP-75 during storage. TPP-75 represents the first evidence that gut juice proteins may selectively attenuate the activity of delta-endotoxins, prior to binding to putative receptors on susceptible cells. TPP-75 should be evaluated as a possible resistance mechanism for those larvae that do not exhibit a receptor-based resistance.     

Analytical investigations of the delta-endotoxin of Bacillus thuriginiensis (author's transl)

Biochemical properties of the crystalline delta-endotoxin of Bacillus thuringiensis, such as stability and solubility in different solvents, were investigated. The dissolved compounds were characterized by gel-electrophoresis and gel-filtration. Covalent and non-covalent bonds are responsible for the crystallisation of the protein molecules. The solubilisation of crystals with non-enzymatic solvents led to high molecular weight products (MW greater than or equal to 800,000) and to components with a molecular weight less than or equal to 10,000. Only the high molecular weight compounds showed toxic activity. The enzymatic degradation of the protein crystals yielded components with molecular weights of greater than or equal to 800,000, 250,000, 100,000, and less than or equal to 10,000. Again, the fraction less than or equal to 10,000 showed no toxic activity. Digestion of the crystals with proteases isolated from the gut juice of Pieris brassicae resulted in a highly toxic fraction with a molecular weight of 100,000. This component, which is resistant to further degradation, appears to be the toxic unit of the protein crystal.     

The major excretory/secretory protease from Lucilia cuprina larvae is also a gut digestive protease

The larvae of the fly Lucilia cuprina excrete or secrete a chymotrypsia (LCTb) onto the skin of sheep to facilitate the establishment of the larval infestation. A combination of immunoblotting and RT-PCR approaches has established that this protease is also a gut digestive protease. LCTb is synthesized primarily in the cardia, a small highly specialized organ located at the anterior end of the midgut and by midgut cells. There is also some expression by the hindgut but no expression by salivary glands. Excretion of LCTb with waste products or regurgitation of the gut contents of the larvae may explain how this protease is transferred from the larval gut onto ovine skin. LCTb is first expressed in eggs and constitutively expressed throughout each larval instar, but is not expressed in pupae or adult flies. It is concluded that LCTb could be involved in the establishment of larvae on sheep skin as well as acting as a general gut digestive enzyme.     

Pain. A prelude

The regulatory basis for differences among species in the developmental rate at which successive life stages are reached ("heterochrony") is a subject of much controversy among vertebrate and invertebrate developmental biologists. The heterochrony in development of different insect species is characterized in part by the intercalcation between the embryo and adult of a (varied) number of heteromorphic larval instars. These heteromorphic larval instars exhibit changes of body form from one larval instar to the next, prior to the final metamorphic molt to the pupal form. The intractability of larval heteromorphosis to experimental dissection is due in part to the lack of suitable experimental probes that can test the nature of the coupling of each heterochronically expressed instar-specific program. The epistatic basis of expression of heteromorphic developmental programs was assessed by two-dimensional electrophoretic analysis of hemolymph proteins during the normal and experimentally manipulated feeding stages of the 3rd, 4th, and 5th (final) instar larvae, and during the prepupal stage, of Trichoplusia ni. Of the several hundred protein spots tracked, some were identified that were uniquely detected during a single stage, while others were observed during combinations of certain stages. The nature of coupling of sequential heterochronic expression of these proteins during successive instars or stages was tested by use of a parasite (Chelonus sp.) that injects regulatory material into the host embryo that later causes the subsequent precocious expression of the final instar larval program. Following the expression of a normal 3rd instar pattern, such larvae were observed to omit expression of the 4th instar program, including omission of the proteins heteromorphically specific to that instar, and instead then express an essentially normal final instar pattern. Thus, normal expression of the final instar feeding stage pattern was not invariantly coupled to prior expression of the penultimate instar-specific proteins or pattern. Also, expression of the full program of the final instar feeding stage was epistatic to teh penultimate instar program, i.e., the protein pattern unique to the penultimate larval instar was not co-expressed with the precociously expressed final instar pattern. Larvae developmentally redirected in this manner failed to fully express the final instar prepupal stage pattern of protein expression, due at least in part to failed expression of prepupal ecdysteroids, but this was shown not to arise from omission of any of the first 4 larval instars per se. The nature of the redirections in host development caused by this parasite finally provides means of probing the coupling of successive expression on heteromorphic programs during larval development.     

The prevalence of Gasterophilus intestinalis in horses in northern England and Wales

The stomachs of 448 horses from northern England and Wales were examined for Gasterophilus larvae, and 237 (52.7%) were found to be infected with G. intestinalis. Larvae were present in stomachs examined during each month of the year except August. Second instar larvae occurred from September through February and third instars were present from November through July. Adult fly activity began in August as indicated by the presence of eggs on horses. The life-cycle of G. intestinalis in northern England and Wales is outlined from the data presented. The mean instar burdens were 15.7 second and 38.0 third instars, and more than 75% of the infections consisted of up to 50 larvae. Prevalence of infection and mean larval burdens declined with increasing age of host. Only one of 258 duodena examined was infected with G. nasalis and this horse originated from the south coast of England, outside of the catchment area of the other horses examined.     

Domain III substitution in Bacillus thuringiensis delta-endotoxin CryIA(b) results in superior toxicity for Spodoptera exigua and altered membrane protein recognition

To test our hypothesis that substitution of domain III of Bacillus thuringiensis delta-endotoxin (Cry) proteins might improve toxicity to pest insects, e.g., Spodoptera exigua, in vivo recombination was used to produce a number of cryIA(b)-cryIC hybrid genes. A rapid screening assay was subsequently exploited to select hybrid genes encoding soluble protoxins. Screening of 120 recombinants yielded two different hybrid genes encoding soluble proteins with domains I and II of CryIA(b) and domain III of CryIC. These proteins differed by only one amino acid residue. Both hybrid protoxins gave a protease-resistant toxin upon in vitro activation by trypsin. Bioassays showed that one of these CryIA(b)-CryIC hybrid proteins (H04) was highly toxic to S. exigua compared with the parental CryIA(b) protein and significantly more toxic than CryIC. In semiquantitative binding studies with biotin-labelled toxins and intact brush border membrane vesicles of S. exigua, this domain III substitution appeared not to affect binding-site specificity. However, binding to a 200-kDa protein by CryIA(b) in preparations of solubilized and blotted brush border membrane vesicle proteins was completely abolished by the domain III substitution. A reciprocal hybrid containing domains I and II of CryIC and domain III of CryIA(b) did bind to the 200-kDa protein, confirming that domain III of CryIA(b) was essential for this reaction. These results show that domain III of CryIC protein plays an important role in the level of toxicity to S. exigua, that substitution of domain III may be a powerful tool to increase the repertoire of available active toxins for pest insects, and that domain III is involved in binding to gut epithelium membrane proteins of S. exigua.     

Site-directed mutations in the third domain of Bacillus thuringiensis delta-endotoxin CryIAa affect its ability to increase the permeability of Bombyx mori midgut brush border membrane vesicles

A series of mutant Bacillus thuringiensis CryIAa delta-endotoxin proteins was prepared by replacing the first, second, and last arginine residues of the conserved third-domain sequence, R-521 YRVRIR-527, with other amino acids. The stable mutant proteins were bioassayed against Bombyx mori larvae and found to all be approximately half as active as wild-type CryIAa. The toxins were also tested by means of a light-scattering assay for their ability to increase permeability of larval B. mori midgut brush border membrane vesicles. Three of the mutant toxins were as active as the wild-type toxin in the vesicle permeability assay.     

Prothoracicotropic hormone in Manduca sexta: localization by a larval assay

1. A new method for the assay of insect prothoracicotropic hormone (PTTH) is described, using fourth instar larvae of Manduca sexta. Larvae neck-ligated at a critical time to prevent release of PTTH from the head fail to undergo the next larval moult. Such ligated larvae moult to fifth instar larvae or larval-pupal intermediates after injection of brain homogenates from Manduca larvae, pupae or pharate adults. The degree of response is proportional to the concentration of brain homogenate injected. 2. The source of PTTH in the pupal brain is the dorsal region of the protocerebrum containing the lateral neurosecretory cells. Microhomogennates of single pieces of brain showed activity with this method. 3. PTTH activity in partially purified extracts is water soluable, stable to boiling for 10 min, and is destroyed by Pronase or trypsin.     

Control of mucin molecular forms expression by salivary protease: differences with caries

1. A protease activity capable of degradation of the high mol. wt salivary mucus glycoprotein to a low mol. wt glycoprotein form was identified in human submandibular gland secretion. 2. The protease exhibited optimum activity at pH 7.0-7.4, and gave on SDS-PAGE under reducing conditions two major protein bands of 48 and 53 kDa. The enzyme showed susceptibility to PMSF, alpha 1antitrypsin, and egg white and soybean inhibitors, a characteristic typical to serine proteases. 3. The activity of the protease towards the high mol. wt mucus glycoprotein was found to be 3.8-fold higher in submandibular gland secretion of caries-resistant individuals than that of caries-susceptible. Furthermore, the enzyme from both groups displayed greater activity against the mucus glycoprotein of caries-resistant subjects. 4. Since the low mol. wt salivary mucus glycoprotein form is more efficient in bacterial clearance than the high mol. wt mucin, the enhanced expression of this indigenous salivary protease activity towards mucin may be the determining factor in the resistance to caries.     

M100907 and clozapine, but not haloperidol or raclopride, prevent phencyclidine-induced blockade of NMDA responses in pyramidal neurons of the rat medial prefrontal cortical slice

Antiserum to leucokinin I, a neuropeptide originally isolated from the cockroach Leucophaea maderae, was used for immunocytochemical labeling of neurons in the brain and ventral ganglia of the moth Spodoptera litura during postembryonic development. In the ventral ganglia, leucokinin-like immunoreactivity begins to occur in the abdominal ganglion A3 to A7 of first instar larva. One to two weakly labeled pairs of bilateral LK-LI cell bodies are located in the subesophageal ganglion of fourth to sixth instar larvae and in the abdominal ganglia A1 to A7 of second to sixth instar larvae. The abdominal ganglion A1 of fourth to sixth instar larvae and A8 of sixth instar larva each contain one weakly labeled pair of median LK-LI cell bodies. Two strongly labeled pairs of bilateral LK-LI neurons are found in A3 to A7 of third to sixth instar larvae. Abdominal ganglia A1 to A8 of prepupa, pupa and adult contain one to three weakly labeled pairs of bilateral LK-LI neurons. Two strongly labeled pairs of bilateral LK-LI neurons in each of the abdominal ganglia of larva, prepupa, pupa and adult send axons to the neuropil, and then each axon bifurcates into two axonal branches. Theses axonal branches from two bundles. From each of the two pairs of neurons an axon exits through the posterior ventral nerve (N2) which runs to the transverse nerve of the next posterior segment. In larval brains, 2-16 pairs of bilateral LK-LI cell bodies can be found together with LK-LI processes in the central neuropil. The larval brains show large changes in the number of LK-LI neurons throughout postembryonic development. The number of LK-LI cell bodies are reduced in number from sixth instar larval brain. Therefore, prepupal, pupal and adult brains contain a smaller number of LK-LI cell bodies. Two pairs of LK-LI median neurosecretory cells located immediately beside the pars intercerebralis in larval brains increase to three pairs in the 7-day-old pupal brain. In the adult, however, LK-LI median neurosecretory cells decrease to one pair.     

Immunotherapy against murine leukemia

Three distinct digestive protease systems were induced in larvae of the herbivorous pest, Colorado potato beetle (CPB; Leptinotarsa decemlineata Say), and used as a model to assess the ability of the proregion of papaya proteinase IV (PPIV; glycyl endopeptidase, EC 3.4.22.25) to act as an inhibitor of insect digestive cysteine proteinases. As shown by gelatin/PAGE and complementary inhibition assays, a recombinant form of the proregion produced in Escherichia coli inhibited a fraction of the insect proteases also inhibited by the well-characterized inhibitor of cysteine proteinases, oryzacystatin I (OCI). In contrast with OCI, the inhibitory potency of the proregion was affected by an increase of the temperature, suggesting a certain alteration of its structural integrity by the insect non-target proteases. This apparent susceptibility to proteolysis was confirmed by SDS-PAGE, after challenging the proregion with the different insect extracts. As seen on gel, selective inhibition of the insect aspartate proteinase, cathepsin D, with the inhibitor pepstatin A preserved the activity of the proregion against cysteine proteinases by preventing its hydrolysis. Taken together, these observations suggest the potential of plant protease proregions as regulators of cysteine proteinases in biotechnological systems, and show the ability of protease inhibitors to preserve the integrity of 'companion' defense-related proteins from the action of insensitive proteases in target pests.     

Survival, developmental and morphogenic deficiencies of Parasarcophaga argyrostoma (Diptera: Sarcophagidae) induced by the biosynthesis inhibitor, chlorfluazuron (IKI-7899)

To evaluate the activity of benzoylphenyl urea chitin biosynthesis inhibitor chlorfluazuron (IKI-7899) against Parasarcophaga argyrostoma, seven doses were topically applied (once) onto early third (last) instar larvae, puparia, or newly formed pupa: 150, 100, 50, 10, 1.0, 0.5, and 0.25 microgram/insect. After topical treatment of last instar larvae, the highest mortality was caused by both higher doses and the lowest mortality was caused by the lowest dose. The lethal activity of IKI-7899 as pronouncedly decreased as the treatment was lately carried out (at the puparial time). IKI-7899 failed to cause cumulative mortality because no pupal or adult mortalities were observed, irrespective of the time of treatment. Treated larvae suffered the action of IKI-7899 because they had decreased weight gain. Except the lowest dose, the weight gain of larvae inversely correlated with the dose-levels. IKI-7899 prolonged not only the larval duration but also the pupal duration after topical treatment of last instar larvae with doses 50-0.25 micrograms/larva. With no exception, all doses topically applied onto puparia or newly formed pupae enhanced pupae to live longer. Topical application onto last instar larvae resulted in different degrees of reduction of pupation rate, but IKI-7899 could not affect the pupal morphogenesis after larval treatment except by its highest dose which led to 8.33% pupal deformities and 7.69% larval-pupal intermediates. The dose 100 micrograms/larva topically applied onto last instar larvae detained 7.69% of what known as "permanent larvae" which suffered the action of the compound along 16 days and eventually perished without any external feature of puparium formation. A metamorphic effect of IKI-7899 pronouncedly appeared in the adult stage. Three higher doses completely arrested the adult flies. Topical application of the compound onto prepupae did not greatly reduce the pupation rate especially at the doses 50, 10 and 1.0 micrograms/puparium. The dose 50 micrograms/puparium was only the dose halting the pupal moulting program because 7.14% of permanent prepupae remained about 12 days and then died. In respect to adult emergence, the highest dose led to zero rate and the lowest dose allowed to all pupae to emerge without malformation.     

Efficient synthesis of mosquitocidal toxins in Asticcacaulis excentricus demonstrates potential of gram-negative bacteria in mosquito control

The control of mosquitoes with chemical insecticides pollutes the environment and leads to resistance in mosquito populations. Bacterial control of mosquito larvae with Bacillus sphaericus and Bacillus thuringiensis subsp. israelensis, which produce protein toxins, has proved useful, safe, and nonpolluting. These bacteria do, however, suffer from disadvantages, including rapid setting, UV sensitivity, and lack of persistance of spores, proteolysis of toxins, narrow host range, and high production costs. Here we show that the Gram-negative bacterium Asticcacaulis excentricus is a promising host for delivering toxins to mosquito larvae. Plasmid-transformed A. excentricus cells expressing the binary toxin of B. sphaericus exhibited toxicity to Culex and Anopheles mosquito larvae similar to that of the high-toxicity strains of B. sphaericus which produce several toxins. A. excentricus has potential advantages as a larvicide compared with the bacilli, especially persistance in the larval feeding zone, resistance to UV light, lack of toxin-degrading proteases, and low production costs.     

Cytokine dysregulation in the post-Q-fever fatigue syndrome

High levels of protease inhibitors are induced in potato leaves by wounding. These inhibitors, when ingested by Colorado potato beetle (Leptinotarsa decemlineata Say) larvae, induce expression of specific proteolytic activities in the gut. Induced protease activities cannot be inhibited by potato inhibitors and thus enable the insects to overcome this defence mechanism of potato plants. The induced aminopeptidase and endoproteolytic activities both have the characteristics of cysteine proteases. Twenty-one protein inhibitors of different structural types have been examined for their ability to inhibit these activities in vitro. Members of the cystatin superfamily were found to be poor inhibitors of the induced endoproteolytic activities, except for the third domain of human kininogen, which was a fairly strong inhibitor (75% inhibition). The strongest inhibition (85%) of induced endoproteolytic activity was obtained using structurally different thyroglobulin type-1 domain-like inhibitors--equistatin and MHC class II-associated p41 invariant fragment. Experiments performed using three synthetic substrates for endoproteases gave similar results and indicate the existence of at least different endoproteolytic enzymes resistant to potato inhibitors. The induced aminopeptidase activity can be inhibited only by stefin family of inhibitors in cystatin superfamily. In in vivo experiments, Colorado potato beetle larvae fed on equistatin-coated potato leaves were strongly retarded in their growth and almost 50% died after 4 days. This demonstrated the potential of equistatin to protect crops from insect attack.     

Effects of insect diapause and parasitization of a braconid, Bracon brevicornis Wesm. on the haemolymph of its host Sesamia cretica Led

Eight types of haemocytes were defined in the 6th instar larvae of the greater sugar cane borer Sesamia cretica Led, as follows prohaemocytes, plasmatocytes, granular cells, spindle cells, adipohaemocytes, oenocytoids, spherule cells and cystocytes. Total haemocyte counts (THCs) and differential counts (DHCs) were estimated for diapausing and non-diapausing larvae. During diapause, there was a significant reduction in the numbers of all haemocyte types. Upon termination of diapause, the haemocyte level increased. This reduction in the haemocyte level observed during diapause may be due to the decreased in the metabolic activity. Also, a decrease in the total haemocyte counts appeared after the parasitization of 6th instar S. cretica larvae by the 2nd larval instar of braconid ectoparasioid Bracon brevicornis Wesm. (Hymenoptera: Braconidae) as compared to the unparasitized ones. However, the DHCs in parasitized larvae showed an increase in the number of phagocytic cells (plasmatocytes, granular cells and spindle cells) and a remarkable decrease in the prohaemocyte counts was detected as compared to that of the unparasitized ones. Moreover, teratocytes appeared in the blood smears of paraitized S. cretica by B. brevicornis. They were round to oval, opaque and stain dark with Giemsa stain and ranged between 5-30 mu in diameter.     

Submandibular salivary proteases: lack of a role in anti-HIV activity

Whole human saliva contains a number of proteolytic enzymes, mostly derived from white blood cells and bacteria in the oral cavity. However, less information is available regarding proteases produced by salivary glands and present in salivary secretions. In the present study, we have analyzed submandibular saliva, collected without contaminating cells, and identified multiple proteolytic activities. These have been characterized in terms of their susceptibility to a series of protease inhibitors. The submandibular saliva proteases were shown to be sensitive to both serine and acidic protease inhibitors. We also used protease inhibitors to determine if salivary proteolytic activity was involved in the inhibition of HIV infectivity seen when the virus is incubated with human saliva. This anti-HIV activity has been reported to occur in whole saliva and in ductal saliva obtained from both the parotid and submandibular glands, with highest levels of activity present in the latter fluid. Protease inhibitors, at concentrations sufficient to block salivary proteolytic activity in an in vitro infectivity assay, did not block the anti-HIV effects of saliva, suggesting that the salivary proteases are not responsible for the inhibition of HIV-1 infectivity.     

Effects of Mesocyclops longisetus (Copepoda:Cyclopidae) on mosquitoes that inhabit tires: influence of litter type, quality, and quantity

A 59-week study was conducted to evaluate the impact of adult Mesocyclops longisetus populations on larval mosquito species inhabiting tires. Greater than 90% reduction of number of 1st and 2nd instars was recorded by 4 wk with 90% reduction of number of 3rd and 4th instars after 7 wk. Reduced control was noted with the onset of cooler winter water temperature. Overall. a 52% reduction in the number of 1st and 2nd instars was achieved, and a 57% reduction was noted in number of 3rd- and 4th-instar mosquito larvae. Cooler temperatures resulted in a decline of adult Mesocyclops, which resulted in reduced larval control. Significantly greater numbers of Mesocyclops adults were collected in tires with either new litter or heavy amounts of litter regardless of litter type. Lastly, litter type, either oak leaves or pine needles, did not influence mosquito reduction or abundance of Mesocyclops populations.     

Osteoprotegerin mRNA is expressed in primary human osteoblast-like cells: down-regulation by glucocorticoids

Oryzacystatins (OCs) are protease inhibitors (PIs) that inhibit Colorado potato beetle (CPB) digestive proteases, and transgenic potato plants containing these PIs are currently under test. However, OCs could interfere with the digestive system of beneficial insects. Protease activity and susceptibility to class-specific protease inhibitors were studied in protein extracts of Perillus bioculatus, a stinkbug predator that has shown potential for biological control of the CPB. At physiological pH, the analysis of protease activity showed that up to 90% of P. bioculatus protease activity is of the cysteine type. All active life stages of the predator were tested, and electrophoretic characterization detected no major qualitative variation in protease pattern between stages. Protease activity in extracts of P. bioculatus nymphs was significantly reduced, up to 70%, by the two recombinant cystatins from rice (OCI and OCII), and by stefin A, a PI encoded by a human gene. These results clearly indicate that cysteine PIs are active not only against the CPB digestive protease complex, but also against proteases of one of its most important natural predators.     

Midgut carboxypeptidase from Helicoverpa armigera (Lepidoptera: Noctuidae) larvae: enzyme characterisation, cDNA cloning and expression

Using synthetic substrates we have characterised carboxypeptidase activity in gut extracts from Helicoverpa armigera larvae. Carboxypeptidase A activity predominates, with only low levels of carboxypeptidase B activity present. Maximum carboxypeptidase A activity occurs over a broad pH range and is inhibited by phenanthroline and potato carboxypeptidase inhibitor. A cDNA clone encoding carboxypeptidase (the first such sequence from a lepidopteran insect) was isolated from a larval gut library. The sequence predicts a secreted polypeptide of Mr 46.6 k with homology to metallocarboxypeptidases from mammalian and invertebrate species. The presence of a serine residue at the active site suggests carboxypeptidase A activity. To further characterise the gene product, the complete cDNA sequence was expressed in insect cells using the baculovirus system. Culture supernatant from these cells contained carboxypeptidase A activity, with no activity against a carboxypeptidase B substrate; the carboxypeptidase B activity in gut extracts must thus be due to a separate enzyme. In agreement with this conclusion, the expressed carboxypeptidase cDNA is a member of a small multigene family. Chronic ingestion of soybean Kunitz trypsin inhibitor by H. armigera larvae results in increased accumulation of carboxypeptidase mRNA in the midgut cells, and an increase in carboxypeptidase A activity detected in gut extract.