Trichostrongylus colubriformis: egg lethality due to Bacillus thuringiensis crystal toxin

A toxin from crystals of Bacillus thuringiensis israelensis was lethal in vitro to eggs of the ruminant nematode Trichostrongylus colubriformis, with an LD50 of 1.8 ng/ml. Larval viability declined after a 2-hr exposure to B. t. israelensis and was dependent on the development period of eggs prior to exposure. Alkaline solubilization suggested that the insecticidal delta-endotoxin of B. t. israelensis was not responsible for nematicidal activity. Filtration of the toxin through 0.2- or 0.45-micron-pore filters revealed that the nematicidal activity was retained on the filter. Toxicity for nematode eggs was decreased by the enzyme inhibitor L-1-tosylamide 2-phenylethylchloromethyl ketone (10(-4) M) or ethylenediaminetetraacetic acid (10(-5) M) and phenylmethylsulfonyl fluoride (10(-6) M). Ethylenediaminetetraacetic acid from 10(-9) to 10(-5) M had no effect on the toxicity while phenylmethylsulfonyl fluoride from 10(-9) to 10(-5) M inhibited toxicity. Fourteen mammalian and microbial enzymes had no significant effect on larval viability while 12 sugars and lipids failed to reduce the toxicity. Addition of 5 mM calcium to the eggs' medium decreased the B. t. israelensis toxicity by 20-fold. The calcium-dependent inhibition of toxicity was reversed by ethylenediaminetetraacetic acid (10(-5) M) and lanthanum chloride (100 microM). The ionophore A-23187 decreased the LD50 by 18-fold to 33.5 ng/ml. Addition of 5 mM calcium chloride to the ionophore and toxin yielded an LD50 of 9.2 ng/ml. Treatment of nematode eggs with B. t. israelensis toxin for 2 or 24 hr had no effect on subsequent binding of selected fluoresceinated lectins to the eggshell.     

Exopeptidase activity in eggs of Trichostrongylus colubriformis (Nematoda)

Summary

A supernatant from eggs of the ruminant nematode Trichostrongylus colubriformis contained an enzyme that was similar to leucine aminopeptidase (LAP), based on hydrolysis of the substrate L-leucine β-naphthylamide to β-naphthylamine. A Michaelis-Menten constant (K _m) of 0.155 mM was obtained. Rate of hydrolysis of 16 substrates revealed that L-phenylalanine and L-tyrosine β-naphthylamides were hydrolyzed most readily while seven additional substrates were hydrolyzed at lesser rates. The optimum pH for enzymatic activity was 6.75–7.5. Enzymatic activity was lost by heating the egg supernatant to 60°C for 5 min or freezing at 0°C for 28 days. Addition of millimolar concentrations of the chlorides of zinc, manganese and magnesium to the egg supernatant had no stimulatory effect on enzyme activity while 10 and 100 mM concentrations significantly reduced activity. Ethylenediamine tetraacetic acid at 10^?4 M had no effect on enzymatic activity. Activity was inhibited by 10^?4 M 1,10-phenanthroline, but the inhibition was reversed by zinc chloride at 10^?3 M. Di-isopropylphosphofluoridate at 10^?3 M reduced enzymatic activity moderately. Enzyme activity in egg supernatant increased 2.2-fold from 21 days to 60–90 days of a primary infection in the host while a 3.3-fold increase was found in primary versus secondary infections.     

Alteration of Trichostrongylus colubriformis egg permeability by Bacillus thuringiensis israelensis toxin

A toxin from the bacterium Bacillus thuringiensis israelensis is lethal to nematode eggs. Exposure of eggs of the ruminant nematode Trichostrongylus colubriformis to the toxin significantly increased the eggs' permeability to radiolabeled phenylalanine within 2 hr. Calcium chloride inhibited the toxin-induced change in egg permeability. Iodine staining of eggs that were exposed to the microbial toxin revealed that egg permeability was altered within 5 min and was dependent on the dose of toxin. Addition of 34 mM sucrose, 17 mM sodium chloride, or 17 mM potassium chloride to the eggs' medium increased the toxin's lethality. Exopeptidase activity in eggs of T. colubriformis was reduced significantly after exposure to the B. t. israelensis toxin. Tetrodotoxin, tetraethylammonium chloride, ouabain, 4-acetamido-4'-isothiocyano-stilbene-2,2'disulfonic acid (SITS), 4,4'-diisothiocyano-2,2'disulfonic acid stilbene (DIDS), valinomycin, and sodium vanadate, which affect membrane transport, had no significant effect on the activity of B. t. israelensis toxin for eggs. Likewise, a series of nucleotides and their derivatives had no effect on the toxin's activity. Ovicidal activity of the microbial toxin was increased by 4-aminopyridine (4.4 X), but was decreased by furosemide (97 X), nigericin (263 X), or monensin (125 X). Microscopic measurement of T. colubriformis eggs after treatment with the microbial toxin revealed no significant size change.     

Tubulin and benzimidazole-resistance in Trichostrongylus colubriformis (Nematoda)

Benzimidazole treatment produced greater effects on microtubule-dependent acetylcholinesterase secretion, the presence of microtubules in intestinal cells, and colchicine binding in susceptible compared with benzimidazole-resistant Trichostrongylus colubriformis. In addition, the binding of benzimidazoles was markedly reduced in preparations from the latter strain, indicating that the mechanism of resistance to benzimidazoles in this nematode involves a reduced affinity of tubulin for benzimidazoles.     

Oviposition by Trichostrongylus colubriformis (Nematoda) in vitro

Summary

Oviposition by females of the ruminant nematode Trichostrongylus colubriformis was influenced by in vitro conditions. Release of eggs declined linearly during incubation for 4.5 h. An average of 8.8 eggs was deposited by females during the first hour in vitro. Decreased pH, temperature, and osmolarity caused reduced production of eggs. Storage of females at 4°C had no effect on subsequent oviposition at 37°C. Oviposition was similar under light and dark conditions. Concomitant males or the female's age did not influence production of eggs in vitro. The release of eggs in vitro was significantly decreased by octapamine and GABA while serotonin elevated oviposition. Thus, environmental stimuli from the host may partially modulate helminth fecundity.     

Sex steroid content and metabolism in Trichostrongylus colubriformis (Nematoda)

Testosterone, progesterone and cholesterol were found in mixed sexes of the nematode Trichostrongylus colubriformis from goats, according to thin-layer, gas-liquid and high-performance liquid chromatography. The structure of these steroids was confirmed by proton nuclear magnetic resonance and mass spectroscopy. Melting points of the worms' steroids were similar to authentic standards of the steroids. Estradiol was not detected in worms from either goat sex. Cholesterol was about 0.08% of the worms' dry weight in helminths from either sex of host. Testosterone was 0.02% of the dry weight when worms were taken from male goats, but only 0.005% from female goats. Progesterone was not detected in worms from male goats, but was 0.005% of the dry weight of helminths from female hosts. Incubation of a worm preparation with tritiated steroids showed that progesterone was converted to 17-alpha-hydroxyprogesterone, based on retention during radioactive thin-layer and gas-liquid chromatography, and co-crystallization. Testosterone, cholesterol and 17-beta-estradiol were not metabolized.     

Trichostrongylus colubriformis: isolation and characterization of ovicidal activity from Bacillus thuringiensis israelensis

Bioassay of media fractions from cultivation of Bacillus thuringiensis israelensis revealed that ovicidal activity for eggs of the ruminant nematode Trichostrongylus colubriformis was found in microbial crystals, but was not released into culture medium. The purified delta-endotoxin of B. t. israelensis, composed of two 25 kDa proteins, had no effect on nematode eggs. A fraction that had high ovicidal activity for eggs of T. colubriformis was isolated by high performance liquid chromatography from crystals of B. t. israelensis. Retention of the compound(s) on size exclusion columns indicated a mol wt of 1510 when compared to standards. The LD50 of this fraction for nematode eggs was not altered significantly by 5 mM calcium chloride with and without ethylenediaminetetraacetic acid or the enzyme inhibitor phenylmethylsulfonyl fluoride (10(-6) M). The fraction was susceptible to proteolytic hydrolysis by bacterial protease VI whereas the fungal protease XIII slightly decreased the ovicidal effects of the fraction. The ovicidal activity was stable for 2 days at ambient temperature or 2 months at 0 C, but declined after 7 days at ambient temperature. Little activity was lost after heating to 100 C for 60 min. The ovicidal effects were also pH dependent with increased toxicity at alkaline pH values. The fraction, however, had no effect on larvae of the mosquito Aedes aegypti or mice after intraperitoneal injection.     

Binding of steroidal sex hormones by supernatant from Trichostrongylus colubriformis (Nematoda)

• 1.1. Binding of sex steroids by a supernatant from the ruminant nematode Trichostrongylus colubriformis was determined by isoelectric focusing of radiolabeled hormones.
• 2.2. Progesterone was recovered from the gels at pH 6.3 and 7.7 while 17-β-estradiol was found at pH 5.9 and 6.5. Testosterone was focused at pH 4.3. The binding of hormones was different in the sexes of nematode.
• 3.3. T. colubriformis may contain binding proteins and/or receptors for sex steroids.

     

Anhydrobiosis in the infective juveniles of Trichostrongylus colubriformis (Nematoda: Trichostrongylidae)

The infective juveniles of Trichostrongylus colubriformis can survive exposure to 0% relative humidity after desiccation at higher relative humidities. During rehydration there is a lag phase before recovery. The infective juveniles are thus capable of anhydrobiosis. Removal of the sheath does not affect desiccation survival but does affect the duration of the lag phase. Morphological changes during the lag phase are described. The appearance of birefringence in the muscle cells during the anomalous shrinkage which occurs during the lag phase is considered to reflect physiological changes necessary for the recovery of normal muscle function.     

Changes in Morphology of Trichostrongylus colubriformis Eggs and Juveniles Caused by Bacillus thuringiensis israelensis

Eggs and rhabditiform juveniles of the ruminant parasite Trichostrongylus colubriformis developed normally in Caenorhabditis briggsae Maintenance Medium. A toxin from a crystal-enriched preparation of the bacterium Bacillus thuringiensis israelensis was lethal to nematode eggs and juveniles within 24 hours and to eggs and juveniles after 24 hours of development. Treated eggs had refractive granules and development was arrested, whereas nontreated eggs developed normally. Eggs treated after 24 hours of development contained juveniles that were granulated, had esophageal derangements, and were moribund or dead. The ovicidal toxin from B. t. israelensis may facilitate microbial control of parasitic nematodes.     

Effects of changes in temperature and saturation deficit on the survival of eggs of Trichostrongylus colubriformis (Nematoda: Trichostrongylidae)

and 1972. Effects of changes in temperature and saturation deficit on the survival of eggs of Trichostrongylus colubriformis (Nematoda: Trichostrongylidae) International Journal for Parasitology, 2: 439–447. In conformity with a hypothesis relating to survival of the developing T. colubriformis egg exposed to desiccation, samples of eggs initially at the early blastomere stage of development showed decreased mortality during development with increasing temperature of incubation up to 25°C, for approximately constant rates of evaporation. At 30°C there was a higher percentage mortality for fixed evaporation rate than at 20° or 25°C. It is suggested that at 30°C there may be an abrupt increase in the initial rate of water loss from the developing embryo resulting from a change in the permeability to water of the lipid layer of the egg envelope.

Fully embryonated T. colubriformis eggs were obtained by incubation at 20°C in the presence of a moderate saturation deficit during development. When such eggs were transferred to 30° and 40°C there was no mortality at the higher temperature, providing that the saturation deficit was substantially increased. A hypothesis proposed for survival at high temperature is based on analogy with water loss through the arthropod cuticle and is attributed to a decrease in permeability of the protein-chitin layer of the egg envelope under conditions of high evaporation rate, even though permeability of the lipid layer might be increased by high temperature.     

Effects of Bacillus thuringiensis kurstaki on Malpighian tubule cells of Thaumetopoea pityocampa (Lepidoptera: Thaumetopoeidae) larvae

In this study effects of Bacillus thuringiensis kurstaki (Btk) on Malpighian tubule cells of Thaumetopoea pityocampa (Lepidoptera: Thaumetopoeidae) larvae was investigated by electron microscopy. 3 mg/l Btk was given with food. After Btk administration, the Malpighian tubule cells were investigated and compared with a control group. 3 and 6 hrs after Btk administration swelling in Malpighian tubule cells was observed. Swelling of mitochondria and separation of their cristae was seen after 12 hrs. After 24 hrs dissolution of the basal cytoplasm, swelling and vacuolization of all mitochondria, partial dissolution of the nucleoplasm, and swelling and separation ofmicrovilli was documented. A membrane-body in the nucleus was seen after 48 hrs. The nucleoplasm was completely dissolved after 72 hrs and after 96 hrs large vacuoles appeared in the cytoplasm and shortening of microvilli was observed.     

Simple method for the isolation of the antilepidopteran toxin from Bacillus thuringiensis subsp. kurstaki

A protein with a molecular mass of 66 kDa was isolated by a simple, rapid, and inexpensive method, using 3-N-morpholinopropanesulfonic acid, potassium thiocyanate, and dithiothreitol, from a mixture of spores, parasporal crystals, and cell debris of Bacillus thuringiensis subsp. kurstaki. The protein was active against the third instar larvae of Trichoplusia ni, was soluble in 19 mM Na2CO3, and was characterized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and confirmed as the insecticidal component of the 132-kDa protoxin of B. thuringiensis subsp. kurstaki by an enzyme-linked immunosorbent assay using antibodies prepared against the protoxin.     

Facile preparation and characterization of the toxin from Bacillus thuringiensis var. kurstaki.

We report a simple three-step method of generating a homogeneous toxic fragment (toxin) in high yield from B. thuringiensis var. kurstaki. Purified crystals were digested with trypsin at pH 10.5, followed by (NH4)2SO4 precipitation and dialysis. For the HD73 strain the preparation is toxic to eastern-spruce-budworm (Choristoneura fuminiferana) larvae. It gives a single 66 kDa band on polyacrylamide-gel electrophoresis and a single band with an isoelectric point of 5.5 on an isoelectric-focusing gel. A single isoleucine N-terminus was detected, and the first 20 amino acids were found to be identical with those predicted from the gene nucleotide sequence. A single lysine C-terminus was detected, and the amino acid composition was in excellent agreement with tryptic cleavages at arginine-28 and lysine-623 of the protoxin. Raman spectroscopic analysis gave values of 20% alpha-helix, 35% beta-sheet and 45% unordered structure. The resistance of the toxin to most proteinases and its susceptibility to proteolysis by papain and Pronases indicates a compact multidomain structure.