苏芸金杆菌广谱杀虫Tml3—14菌株晶体毒素及毒力特性

以新筛选的对鞘翅目,鳞翊目、双翅目均具毒性的B.t.Tml3-14菌株为材料,用HD-1、3370-1为参考菌株,比较了伴孢晶体蛋白的多肽组成,以及经三种昆虫肠道酶降解产生的抗酶多肽组分。SDS-PAGE分析表明：Tml3-14晶体蛋白含1 38kD、l 32kD主要成分和65kD次要我分。其晶体分别由三种昆虫肠道酶消化产生的毒性多肽,经生物测定,杀家蚕的毒性成分为68.5kD、59kD多肽;杀斜纹夜蛾毒性成分为71kD,67kD和59.6kD多肽;杀马铃薯瓢虫毒性成分为69kD、65kD多肽。它与HD01、3370-1晶体均有明显差异。     

Some notes on the ecology of a GermanBranchipus schaefferi population (Crustacea: Anostraca)

Habitat preference, seasonal occurrence, starvation resistance, hatching eggs ofBranchipus schaefferi, and effects of predation onB. schaefferi were studied.Branchipus was only present in turbid, unvegetated ponds and absent in ponds which contain higher aquatic vegetation and theSpirogyra sp. The first individuals ofB. schaefferi appeared in April when water temperature was 10 °C and the last adults in November at a water temperature of 3.5 °C. Up to 6 reproducing generations were observed during this period. Abundance ofB. schaefferi was higher in temporary ponds than in permanent ponds. Sex ratio was close to unity for most of the year. Body size ofB. schaefferi males and females was significantly positively correlated with pond volume. Without foodB. schaefferi could survive for 1.5 to 2 days at 20 °C and 4 to 5 days at 10 °C. Hatching success of eggs decreased when eggs were dried for 7 months. Freezing of eggs had no effect on hatching success. From] the predators tested,Chaoborus sp. larvae clearly selected smallB. schaefferi; one consumed approximately 6Branchipus d–1 at a density of 6 to 12 prey 1–1. The other predators, dragonfly larvae, and larvae and adults ofTriturus alpestris selected alternative prey types, for exampleTubifex sp. and ostracods.     

Biochemical Characterization of HL-7 and HL-10 Peptides Identified from Scorpion Venom of <Emphasis Type="Italic">Hemiscorpius lepturus</Emphasis>

In this study, the scorpion venom of H. lepturus was fractionized by reversed phase high-pressure liquid chromatography (RP-HPLC). The sequences of two peptide fractions were identified by tandem mass spectrometry and named as HL-10 (AFPYYGHHLG) and HL-7 (YLYELAR), respectively. Antioxidant activity and cellular effect of synthetic peptides on A549 cell line were investigated. Results showed that the two peptides had high activities in radical scavenging and inhibition of lipid peroxidation in a concentration-dependent manner. The HL-10 and HL-7 peptides demonstrated cytotoxicity on A549 without any hemolytic effect. By increasing of peptide concentrations induced significantly (P?≤?0.01) activities of catalase and glutathione peroxidase. Our results showed that HL-7 peptide had higher antioxidant potency, whereas the HL-10 peptide revealed a great cytotoxicity on A549 cell line by MTT assay. Our results suggested that the peptides of H. lepturus, possessed free radicals scavenging likewise increased antioxidant enzyme activities in A549 cells.     

Characterization of a Gloverin-Like Antimicrobial Peptide Isolated from Muga Silkworm, <Emphasis Type="Italic">Antheraea assamensis</Emphasis>

Antimicrobial peptides (AMPs) are produced in all living organisms including insects in a non-specific manner, and act as innate immune defense arsenal against the invading pathogens. Muga silkworm (Antheraea assamensis) larvae were injected with Candida albicans and AMPs were isolated from the hemolymph after extracting with methanol, acetic acid and water mixture (90:1:9) and evaluated for antimicrobial activity against fungal and bacterial pathogens. Further purification was done through successive semipreparative and analytical reversed phase HPLC using C-18 column. The obtained fractions were collected, lyophilized and tested for antimicrobial activity. Among the HPLC fractions, one showed highest activity with MIC value of 64 µg/ml against Gram-negative bacteria, Escherichia coli and Enterobacter cloacae. Purity of this isolated peptide was confirmed by SDS-PAGE and TLC, and its molecular mass was determined as 9.052 kDa by MALDI-TOF mass spectrometry. From the mass fingerprinting analysis of this peptide after trypsin digestion a peptide fragment with molecular mass of 2622.7 Da was obtained. De novo sequencing of this peptide fragment following MS/MS analysis identified few amino acid residues as “KSGGGGWGS” with a total score of 46.9 with gloverin peptide of A. mylitta. The peptide inhibited biofilm formation of the Gram-negative bacterial pathogens. SEM study revealed that peptide disrupted bacterial cell wall to leach out intracellular materials and may be the major target for its antimicrobial activity.     

Nondevelopment ofCotesia kariyai(Hymenoptera: Braconidae) in Entomopoxvirus-Infected Larvae ofPseudaletia separata(Lepidoptera: Noctuidae)

Interaction between an entomopoxvirus (PsEPV) and a gregarious braconid endoparasitoid,Cotesia kariyai,inPseudaletia separatalarvae showed that infection of larvae with PsEPV was deleterious to the development and survival ofC. kariyai.The survival and development ofC. kariyaiin PsEPV-infectedP. separatalarvae depended on the length of time between parasitization and viral infection. No parasitoid larvae emerged from PsEPV-infected hosts when host larvae were exposed simultaneously to parasitization and PsEPV inoculation whereas more than 80% of the hosts produced parasitoids when PsEPV was administered 5 days postparasitization.C. kariyailarvae in PsEPV-infected hosts showed a retarded development, shrank, and died about 8 days after viral exposure. Virion-free plasma from PsEPV-infectedP. separatalarvae was toxic to the parasitoid larvae even up to a dilution level of 32 when it was injected intrahemocoelically into the host larvae. Development of parasitoids in hosts that were simultaneously parasitized and injected with the virion-free plasm never progressed beyond the egg stage. The parasitizedP. separatalarvae injected with the virion-free plasma did not pupate and died within 30 days after injection.     

Venom peptides from solitary hunting wasps induce feeding disorder in lepidopteran larvae

The cell lytic activity and toxicity against lepidopteran larvae of 13 venom peptides (4 OdVPs and 9 EpVPs) from two solitary hunting wasps, Orancistrocerus drewseni and Eumenes pomiformis, were examined with mastoparan as a reference peptide. Of the 13 peptides, 7 were predicted to have α-helical structures that exhibit the typical character of amphipathic α-helical antimicrobial peptides. The remaining peptides exhibited coil structures; among these, EpVP5 possesses two Cys residues that form an internal disulfide bridge. All the helical peptides including mastoparan showed antimicrobial and insect cell lytic activities, whereas only two of them were hemolytic against human erythrocytes. The helical peptides induced a feeding disorder when injected into the vicinity of the head and thorax of Spodoptera exigua larvae, perhaps because their non-specific neurotoxic or myotoxic action induced cell lysis. At low concentrations, however, these helical peptides increased cell permeability without inducing cell lysis. These findings suggest that the helical venom peptides may function as non-specific neurotoxins or myotoxins and venom-spreading factors at low concentrations, as well as preservatives for long-term storage of the prey via antimicrobial, particularly antifungal, activities.     

Toxicity of the extracellular products ofVibrio damsela isolated from diseased fish

In this work we analyzed the pathogenic in vivo and in vitro activities for both fish and mammals of extracellular products (ECP) of several isolates ofVibrio damsela implicated in disease problems in marine culture. The ECP from all the strains were strongly lethal for fish (LD₅₀ ranging from 0.06 to 3.7 g protein/g fish) and mice (LD₅₀ ranging from 0.02 to 0.43 g protein/g mouse), causing death between 4 and 72 h after inoculation. These ECP samples possessed low proteolytic activity without production of caseinase, gelatinase, or elastase. However, most of them showed remarkable phospholipase and hemolytic activity for sheep, human, and turbot red blood cells. In addition, all the ECP samples were cytotoxic for fish and homoiothermic cell lines. The levels of enzymic and cytotoxic activities were clearly associated with the degree of virulence for fish. Moreover, the enzymic patterns of both live cells and ECP evaluated with the API-ZYM system were very similar among the strains, indicative that most of the activities are associated with exoenzymes.The in vivo and in vitro biological activities were considerably reduced after heat treatment (100°C for 10 min), but not totally lost in the highly virulent strains. Although we have demonstrated that the toxicity of the ECP is not directly associated with their lipopolysaccharides (LPS) content, these compounds could confer some heat-stabilizing effect to the toxic fractions.     

Comparative study of the hemolysins ofTreponema hyodysenteriae andTreponema innocens

The nucleic acid-bound hemolysins from two strains ofTreponema hyodysenteriae (-hemolytic) and two strains ofT. innocens (weakly -hemolytic) were purified and characterized. All four hemolysins shared similar molecular weights, stability to oxygen, and pH and heat lability. The hemolysins were inactivated by pronase and lipase and inhibited by trypan blue. Although lacking detectable proteolytic or phospholipase activity,T. hyodysenteriae andT. innocens hemolysins differ in their sensitivity to phospholipids; the former was insensitive to cardiolipin, which completely inhibited the latter. Furthermore, both groups of hemolysins differed in their hemolytic spectra and in their specific activities on rabbit erythrocytes. The results showed that the observed hemolytic patterns ofT. hyodysenteriae andT. innocens on blood agar were due to different hemolysins.     

Sodium polyacrylamide polyacrylate, a gelling agent in diets for cabbage looper, omnivorous looper and western avocado leafroller

Ingredient substitutions for agar or carrageenan in artificial diets for lepidopterous larvae were evaluated. A sodium polyacrylamide polyacrylate gelling agent, Water-Lock G-400, was compared with agar for solidifying diets of the cabbage looper, Trichoplusia ni (Hübner), omnivorous looper, Sabulodes aegrotata (Gueneé), and the western avocado leafroller, Amorbia cuneana Walsingham. Water-Lock G-400 is considerably less expensive than agar or carrageenan, it does not require heating to gel, and it is made from a dependable supply of agricultural starch. Sabulodes aegrotata pupae from larvae reared on agar were significantly heavier than those reared on Water-Lock G-400. However, the remainder of the variables observed (larval survival, developmental times, fecundity, sex ratio and pupal weights for A. cuneana and T. ni) were all independent of diet. Decreasing the total amount of ingredients in a Water-Lock-G-400 diet produced fewer and smaller T. ni pupae than did an agar diet when the larvae were reared using a mass production protocol. However, these lower values were still above acceptable levels for mass production and reduced the cost of diet production by 65%.Further ingredient substitutions impaired larval development (i.e., replacing pinto beans with soy protein or combinations of soy protein and -cellulose), while others did not (i.e., excluding pinto beans and reducing water content and replacing pinto beans with -cellulose or corn cob grits). Moisture control by adding corn cob grits to diets increased the yield of pupae, while removing water from diets increased survivorship and the yield of pupae.     

Multicellular-vesicle-promoting polypeptide from Trichoplusia ni: Tissue distribution and N-terminal sequence

An N-terminal amino acid sequence of a 16.9 kDa hemolymph polypeptide, “Vesicle Promoting Factor” (VPF) from Trichoplusia ni, revealed a high sequence homology (70%) with Manduca sexta apolipophorin-III. A polyclonal antibody developed against VPF, however, was not immunoreactive with either purified M. sexta or T. ni apolipophorin-III. Immunoblots of tissue homogenates of T. ni indicated that VPF was present in imaginal wing discs, central nervous system (CNS), silk glands, midgut and hemocytes from fifth instar larvae, and also in the IAL-TND1 cell line which can grow as either fluid-filled multicellular vesicles or multicellular aggregates. VPF was also detected immunologically in the hemolymph of adults of T. ni, and in hemolymph of adults and larvae of Galleria mellonella and Heliothis virescens. Testes, midgut, hemocytes, and wing discs, but not Malpighian tubules, of T. ni released VPF into tissue culture medium during a 3 h incubation period. © 1995 Wiley-Liss, Inc.

1 This article is a US Government work and, as such, is in the public domain in the United States of America.

     

DNA homology between the crystal toxin genes of several mosquito pathogenicBacillus thuringiensis strains

With the recombinant pVB131 plasmid, which encodes the mosquitocidal 130 kilodalton peptide ofBacillus thuringiensis var.israelensis as a probe, DNA homology between crystal toxin genes of several dipteran-toxic strains was tested. Results from this study indicate that, while the crystal toxin genes ofB. thuringiensis var.kyushuensis and var.morrisoni isolate PG-14 share homology to the crystal toxin gene of var.israelensis, the -endotoxin genes of other dipteran-active strains tested (i.e., var.colmeri and var.kurstaki) do not exhibit any homology. The crystal toxin genes of vars.kyushuensis andmorrisoni isolate PG-14 were found to be located on plasmids of 60 and 94 megadaltons, respectively.     

Characterization of Six Highly Mosquitocidal Bacillus thuringiensis Strains That Do Not Belong to H-14 Serotype

Four strains belonging to Bacillus thuringiensis serovars thompsoni, malaysiensis, canadensis, jegathesan and two auto-agglutinating B.t. strains were identified as being highly toxic to the mosquito larvae of the species Aedes aegypti, Anopheles stephensi, and Culex pipiens. Their larvicidal and hemolytic activities were determined and compared with those of strains known to be highly mosquitocidal and/or cytolytic from serovars of B.t. israelensis, morrisoni, darmstadiensis, medellin, kyushuensis, and fukuokaensis. The electrophoretic protein profiles of purified crystals and immunological relationships with B.t.i. polypeptides were studied. Five out of the six new strains showed the same larvicidal and hemolytic activities and the same crystal proteins and toxin genes as B.t.i. One strain, B.t. jegathesan 367, presented a novel pattern of larvicidal activity and a protein profile different from those of other strains.     

The 23-kilodalton CytB protein is solely responsible for mosquito larvicidal activity ofBacillus thuringiensis serovarkyushuensis

To identify the actual mosquito larvicidal toxin(s) inBacillus thuringiensis serovarkyushuensis, we conducted a comparative toxicity study by a combination of ion-exchange chromatographies and SDS-PAGE. Among the proteins contained in solubilized and protease-treated inclusions, only the 23-kDa protein, a cytolysin referable to the CytB protein, exhibited mosquitocidal activity. The LC₅₀ value of this protein was 51.4 g/ml for the third-instar larvae of the mosquito,Aedes aegypti. The fractions of other inclusion proteins showed no mosquito larvicidal activity at protein concentrations up to 1000 g/ml.     

Pre-ingestive and post-ingestive effects of soya bean extracts and rutin on Trichoplusia ni growth

Total ethanolic crude extracts from soya bean (Glycine max L. Merrill) PI 227687 and `Davis', three purified fractions (A, B and C) from PI 227687, and pure rutin were tested for their effect on the growth of Trichoplusia ni (Hübner) (Lepidoptera: Noctuidae) larvae, from egg hatching to pupation. Analysis of covariance (ANCOVA), followed by bicoordinate utilisation plots, was used to remove the effect of feeding time from consumption and weight of pupae and to separate pre- ingestive and post- ingestive effects of different diets on T. ni growth. Survival, pupal weight, and feeding time of T. ni were all negatively affected by PI 227687 total extracts. Pupal weight and consumption remained affected by diet, after removing the effect of feeding time by ANCOVA. Larvae fed on the `Davis' extract diet grew larger and consumed more rapidly a larger amount of food than those fed on PI 227687 and control diets. T. ni pupal weight was strongly influenced by consumption. In general, those insects that consumed more food were heavier but larvae fed on diet containing PI 227687 extract grew less than the others, irrespective of the amount of ingested and digested food. The feeding time was adversely affected by PI 227687 fraction A, composed of two flavonol glycosides (rutin and quercetin 3-O-glucosylgalactoside) and of an isoflavone glycoside (genistin). Survival and pupal weight were not affected, although the consumption and assimilation of ingested food was lower when larvae were fed on fractions A and C. The toxicity of the higher rutin concentration (2%) was similar to that observed when the insects were fed on unfractioned PI 227687, except that effects on food consumption were not observed. Results indicated that the feeding deterrence of PI 227687 must be caused by other compounds from fractions A and C. Moreover, rutin, acting as an antibiotic, appears to account for the adverse effects of PI 227687 on the physiology of T. ni.     

Influence of tungsten on metabolic patterns in Pyrococcus furiosus,a hyperthermophilic archaeon

Pyrococcus furiosus is a strictly anaerobic heterotroph that grows optimally around 100 °C. It can be cultured in an artificial seawater-based medium with either peptides or maltose as the carbon source. Significant stimulation of cell yields were observed when trace levels of tungsten (as tungstate) were added to an energy-limited chemostat culture of P. furiosus when maltose is present, but not when peptides were the sole carbon source. The addition of tungsten also led to dramatic increases in the specific activities within cell-free extracts of aldehyde ferredoxin oxidoreductase (AOR), which is a tungsten-iron-sulfur protein. Moreover, the addition of tungsten to cells growing in maltose/peptide medium dramatically reduced the specific activity of intracellular proteases, suggesting a preference for the utilization of maltose over peptides as the carbon and energy source in the presence of tungsten.Non-standard abbreviations EPPS N-[2-Hydroxyethyl]-piperazine-N-[3-propane-sulfonic acid] - VFA volatile fatty acids - LNA 1-Lys-p-nitroaniline - MeOSAPTNA MeO-Suc-Arg-Pro-Tyr-p-nitroaniline - AOR aldehyde ferredoxin oxidoreductase     

Screening Antimicrobial Activities of Basic Protein Fractions from Dry and Germinated Wheat Seeds

Two small cationic peptide fractions (5 kDa) were isolated from dry and germinated seeds of wheat, named WAP and GWAP, respectively. The antifungal and antibacterial activities of the peptides were analyzed using disk diffusion and turbidity measurement assays. The peptides in vitro exhibited effective antifungal activity against four plant pathogenic fungi at minimum concentration of 15 g(protein) cm^–3. Their antimicrobial activity was negatively affected by the presence of 5 mM CaCl₂. The peptides were less effective against Gram-negative bacterium Erwinia carotovora subsp. carotovora, but they demonstrated inhibitory activity against Gram-positive bacterium Clavibacter michiganensis subsp. sepedonicus. The antimicrobial activity of GWAP was more effective than WAP.     

The endocrine basis for developmentally stationary prepupae in larvae ofTrichoplusia ni pseudoparasitized byChelonus insularis

Summary Larvae of the cabbage looper,Trichoplusia ni, precociously initiate metamorphosis in the penultimate instar when parasitized byChelonus insularis. Some larvae developing from stung eggs precociously spin cocoons, but upon dissection contain no live or obvious parasites. Such pseudoparasitized larvae greatly slow down in development as prepupae, due to a suppressed ecdysteroid titer which in turn may be caused by a suppressed juvenile hormone titer.Abbreviations JH juvenile hormone - JHA juvenile hormone analog - JHE juvenile hormone esterase     

Molecular epidemiology ofCladosporium carrionii

The effects of cuticle from larvae ofTrichoplusia ni andHelicoverpa (=Heliothis)zea on expression of proteases and chitinases by germinating conidia ofNomuraea rileyi in submerged cultures were studied. Increasing the concentration ofT. ni orH. zea cuticle resulted in a 13- and 15-fold increase in protease activity, respectively. Endochitinase and N-acetylglucosaminidase activity on theT. ni andH. zea substrates increased as cuticular concentrations increased to 2.5%, then stabilized or decreased thereafter. The simultaneous expression of both proteases and chitinases suggests that they are controlled by a multiple-regulatory system.This article reports the results of research only. Mention of a proprietary product in this paper does not constitute a recommendation for use by US Department of Agriculture.     

Chromatographic fractionation of Aspergillus endotoxins

Summary Crude preparations of the endotoxins extracted from the mycelia ofAspergillus fumigatus andAspergillus flavus, after preliminary concentration by ammonium sulfate, have been fractionated by column chromatography on DEAE-cellulose. Although the biological activity of the chromatographed preparations was not limited to a single fraction, examination of the most active fractions by starch gel electrophoresis showed no bands common to the two nephrotoxins.The highest hemolytic and toxic activities of the fumigatus toxin were found in different fractions of the chromatographed material, and starch gel electrophoresis showed no bands common to these two fractions.The molecular weight of the flavus toxin has been estimated to be in the range of 32,000 to 34,000 as judged by the results of ultracentrifugation and microelectrodialysis in starch gels of the most toxic fractions.Both of the toxins have been shown to contain small amounts of hexosamine and larger amounts of non-amino sugars.Presented in part at the 46th Annual Meeting of the American Society of Biological Chemists, Atlantic City, New Jersey, April, 1962.     

Hemocyte responses to implanted tissues inDrosophila melanogaster larvae

Summary At 26° C temperature-sensitivetu(1) Sz ^ts larvae ofDrosophila melanogaster develop melanotic tumors consisting of aberrant caudal adipose tissue encapsulated by precociously differentiated hemocytes (lamellocytes). Whentu-Sz ^ts larvae are grown at 18° C, lamellocytes are present but the caudal fat body surfaces remain normal and melanotic tumors do not develop (Rizki and Rizki, preceding paper). In this paper we demonstrate that the lamellocytes intu-Sz ^ts larvae at 18° C encapsulate implants of mechanically-damaged fat bodies and adipose cells devoid of basement membrane, while leaving host fat bodies or implanted fat bodies with intact basement membrane unencapsulated. Therefore, low temperature blocks melanotic tumor formation by normalizing the surfaces of the prospective tumor-forming sites intu-Sz ^ts.The discriminatory ability oftu-Sz ^ts lamellocytes was examined by challenging them with undamaged heterospecific tissues. Tissues from sibling species ofD. melanogaster were not encapsulated whereas tissues fromDrosophila species outside theD. melanogaster species subgroup were. Ultrastructural examination of encapsulated heterospecific tissues showed intact basement membrane, so we propose that distinction between self and not self by lamellocytes depends upon the molecular architecture of the basement membrane. In similar series of experiments usingD. virilis donor tissues inOre-R wild type larval hosts, fat bodies remained unencapsulated and imaginal disks metamorphosed. These studies suggest that continued presence of lamellocytes in the larval host is a prerequisite for encapsulation.