Interaction of Nosema pyrausta and temperature on Ostrinia nubilalis egg production and hatch

Nosema pyrausta is an obligate pathogen causing reduced fecundity and longevity of Ostrinia nubilalis. This study was conducted to determine the combined effects of N. pyrausta infection and temperature on O. nubilalis egg production and hatch. N. pyrausta-infected and noninfected O. nubilalis were maintained in two different temperature regimes. The first regime allowed females to oviposit under optimum conditions (27 degrees C, 65% RH, 16:8 (L:D)), while females in the second regime were held initially under the same humidity and light conditions, but a constant temperature of 16 degrees C for 1 week after which they were transferred to optimum ovipositional conditions. Studies were performed initially with O. nubilalis populations and later with individual mating pairs. In studies with O. nubilalis populations, the mean number of eggs laid per female under optimum conditions was 660, while N. pyrausta-infected females held initially at 16 degrees C laid 116 eggs per female. In studies with individual mating pairs, N. pyrausta infection reduced egg production per female 53 and 11% in the 16 and 27 degrees C temperature regimes, respectively, compared to noninfected females under optimum conditions. Exposure to 16 degrees C temperatures early in the ovipositional period had a more profound impact on reducing egg production in N. pyrausta-infected than noninfected O. nubilalis.     

Interactions between Nosema pyrausta (Microsporidia: Nosematidae) and Bacillus thuringiensis subsp. kurstaki in the European corn borer (Lepidoptera: Pyralidae).

Larval susceptibility to Bacillus thuringiensis was determined for Nosema pyrausta-infected and uninfected European corn borers, Ostrinia nubilalis (Hübner), in bioassays using a commercial formulation of B. thuringiensis subsp. kurstaki, Dipel ES, incorporated into diet. LC50 values for N. pyrausta-infected larvae were significantly lower (P<0.0001) than for uninfected larvae and declined with increasing levels of infection. LC50 values for a 15-d bioassay using field-colony first instars were 0.006 and 0.027 mg of Dipel ES/kg of diet for larvae moderately infected by N. pyrausta and uninfected larvae, respectively. Nosema pyrausta-infected larvae reared on Dipel ES-amended diets produced 70-fold fewer spores (P<0.0001) than larvae reared on standard diet. For example, 15 d after placement as first instars on standard diet, infected field-colony larvae produced 7.6-8.7 million N. pyrausta spores per larva; similar larvae placed on diet containing 0.09 mg of Dipel ES/kg of diet produced 85-103 thousand spores per larva. Infected larvae also weighed less and failed to mature on Dipel ES-amended diets. Increased susceptibility of N. pyrausta-infected larvae to Dipel ES and reduced N. pyrausta spore production in larvae feeding on diet containing Dipel ES suggest that Bt corn will have a direct adverse effect on the survival and continual impact of N, pyrausta as a regulating factor on European corn borer populations.     

Growth, development, and survival of Nosema pyrausta-infected European corn borers (Lepidoptera: Crambidae) reared on meridic diet and Cry1Ab

Transgenic corn, Zea mays L., hybrids expressing crystal protein endotoxin genes from Bacillus thuringiensis Berliner are an increasingly popular tactic for managing the European corn borer, Ostrinia nubilalis (Hübner), in North America. O. nubilalis populations also are often vulnerable to the ubiquitous entomopathogenic microsporidium Nosema pyrausta (Paillot). We examined the effect of feeding meridic diet incorporated with purified Cry1Ab on growth, development, and survival of Nosema-infected and uninfected neonate O. nubilalis. Infected larvae developed more slowly than uninfected larvae. Increasing the concentration of Cry1Ab in diet reduced larval development, and this effect was amplified by microsporidiosis. Infected larvae weighed significantly less than uninfected larvae. The relationship among Nosema infection, Cry1Ab concentration, and larval weight was fitted to an exponential function. The LC50 of infected larvae was one-third that of uninfected larvae, indicating that infected larvae are more vulnerable to toxin. This work has implications for resistance management of O. nubilalis and demonstrates that it is important to determine whether N. pyrausta is present when testing susceptibility of larvae to transgenic corn hybrids.     

PERMANENT GENETIC RESOURCES: Isolation and characterization of microsatellite loci from the European corn borer, Ostrinia nubilalis (Hübner) (Insecta: Lepidoptera: Crambidae)

Few useful microsatellites are available for population studies of the European corn borer, Ostrinia nubilalis (Hübner). An enrichment strategy was used to develop microsatellite markers for O. nubilalis, and over 500 positive clones were isolated. Seventy-five contained unique microsatellites, 10 of which were polymorphic with discernable polymerase chain reaction products. The 10 loci were surveyed for variability in 72 wild individuals from central Iowa. Five loci showed no deviation from Hardy-Weinberg proportions, and all were successfully cross-amplified in the related Asian corn borer, Ostrinia furnacalis. These loci represent a significant addition to microsatellites appropriate for population studies of O. nubilalis.     

Determination of rate constants for nucleotide dissociation from Na,K-ATPase.

A method for determining individual rate constants for nucleotide binding to and dissociation from membrane bound pig kidney Na,K-ATPase is presented. The method involves determination of the rate of relaxation when Na,K-ATPase in the presence of eosin is mixed with ADP or ATP in a stopped-flow fluorescence apparatus. It is shown that the nucleotide dependence of this rate of relaxation--taken together with measured equilibrium binding values for eosin and ADP--makes possible a reasonably reliable determination of the rate constant for dissociation of nucleotide, i.e., determination of the rate constant k-1 in the following model (where E denotes Na,K-ATPase): [formula: see text] All experiments are carried out at about 4 degrees C in a buffer containing 200 mM sucrose, 10 mM EDTA, 25 mM Tris and 73 mM NaCl (pH 7.4). Values obtained for the rate constants for dissociation are about 6 s-1 for ADP and 2-3 s-1 for ATP.     

DNA persistence length revisited

DNA restriction fragments ranging from 79 to 789 base pairs in length have been characterized by transient electric birefringence (TEB) measurements at various temperatures between 4 and 43 degrees C. The DNA fragments do not contain runs of four or more adenine residues in a row and migrate with normal electrophoretic mobilities in polyacrylamide gels, indicating that they are not intrinsically curved or bent. The low ionic strength buffers used for the measurements contained 1 mM Tris Cl, pH 8.0, EDTA, and variable concentrations of Na(+) or Mg(2+) ions. The rotational relaxation times were obtained by fitting the TEB field-free decay signals with a nonlinear least-squared fitting program; the decay of the birefringence was monoexponential for fragments < or = 241 base pair (bp) in length and multiexponential for larger fragments. The terminal relaxation times, characteristic of the end-over-end rotation of the DNA molecules, were then used to determine the persistence length (p) and hydrodynamic radius (r) of DNA as a function of temperature and ionic strength, using several different hydrodynamic models. The specific values obtained for p and r are model dependent. The wormlike chain model of P. J. Hagerman and B. H. Zimm (Biopolymers 1981, Vol. 20, pp. 1481-1502) combined with the revised Broersma equation (J. Newman et al., Journal of Mol Biol 1997, Vol. 116, pp. 593-606) appears to be the most suitable for describing the flexibility of DNA in low ionic strength solutions. The values of p and r obtained from the global least squares fitting of this equation are independent of DNA length, and the deviations of the individual values from the average are reasonably small. The consensus r value calculated for DNA in various low ionic strength solutions containing 1 mM Tris buffer is 14.7 +/- 0.4 A at 20 degrees C. The consensus p values decrease from 814 approximately 564 A in solutions containing 1 mM Tris buffer plus 0.2-1 mM NaCl and decrease still further to 440 A in solutions containing 0.2 mM Mg(2+) ions. The persistence length exhibits a shallow maximum at 20 degrees C and decreases slowly upon either increasing or decreasing the temperature, regardless of the model used to fit the data. By contrast, the consensus values of the hydrodynamic radius are independent of temperature. The calculated persistence lengths and hydrodynamic radii are compared with other data in the literature.     

Optimal isolation conditions for rainbow trout (Salmo gairdneri) hepatic ribosomes

1. Isolation conditions for rainbow trout (Salmo gairdneri) liver ribosomes were optimized. 2. Optimal initial buffer (Buffer I) concentrations were 250 mM sucrose, 50 mM Tris (pH 7.6, 25 degrees C), 75 mM KCl, and 5 mM MgSO4.7H2O. 3. Optimal concentrations for post-105 supernatant buffer (Buffer III) were 25 mM Tris (pH 7.6, 25 degrees C), 75 mM KCl, and 8 mM MgSO4.7H2O.     

Preparation of high molecular weight plant DNA and its use for artificial chromosome construction

Summary The availability of a substantial amount of high molecular weight DNA is an essential prerequisite for the construction of yeast artificial chromosome (YAC) libraries. Parameters concerning protoplast isolation and DNA extraction have been systematically analyzed. Conditions have been established for the obtainment of high molecular weight DNA from Arabidopsis thaliana and Nicotiana plumbaginifolia protoplasts either embedded in agarose plugs or in liquid suspension. Restriction fragments were obtained by partial and total digestion with different endonucleases, and separated by pulsed-field gel electrophoresis. Ligation of partially EcoRI-digested DNA (range 30–300 kbp) followed by transformation of yeast spheroplasts gave rise to YACs with an average size of 60 kbp. The introduction of a DNA size-selection step before ligation led to production of YACs in the range of 100–200 kbp. Clones of up to 460 kbp were obtained by blunt-end ligation of pre-selected unrestricted DNA.Abbreviations 2,4-D 2,4-dichloro phenoxyacetic acid - 6BAP 6-benzylaninopurine - BFP bovine serum albumin 0.1%, Ficoll 400 0.1%; polyvinylpyrrolidone 0.1% - CHEF clumped homogeneous electric field - DTT dithiothreitol - EDTA ethylenediaminetetraacetic acid - HMW high molecular weight - km kanamycin - LMP agarose low melting point agarose - MS Murashige and Skoog mediun - npt neomycin phosphotransferase - PEG polyethyleneglycol - PFGE pulsed-field gel electrophoresis - RFLP restriction fragrant lenght polymorphism - SDS sodium dodecyl sulphate - SSC sodium chloride 150 mM, sodiun citrate 15 nM, pH 7 - TAE TRIS-Acetate pH 8 40 mM, EDTA 2 mM - TE TRIS-HCl pH 8 10 mM, EDTA 1 mM - YAC yeast artificial chromosome     

The spore wall and polar tube proteins of the microsporidian Nosema grylli: the major spore wall protein is released before spore extrusion

Incubation of Nosema grylli spores in alkaline--saline solution (10 mM KOH, 170 mM KCl) leads to solubilization of the major spore wall protein of 40 kDa (p40). Both the compounds of this solution are crucial for p40 solubilization. After spore incubation in 170 mM KCl no proteins were released in the medium. In contrast, 10 mM KOH causes a release of many spore proteins but only a small amount of p40. A long storage of spores (over a year) in water or 0.02% sodium azide results in a sharp decrease of p40 content. Specific polyclonal antibodies were obtained by immunization of rabbits with isolated p40. The specificity of serum was confirmed by immunoblotting. IFA showed reliable reaction on the envelopes of sporonts and sporoblasts, whereas only part of spores reacted with antibodies. This distinction may be due to changing surface antigens during spore maturation. Solubilization of p40 under alkaline conditions could be associated with spore extrusion, since a subsequent transfer of spores to neutral solution leads to their discharge. Subsequent wash of discharged spores with 1-3% SDS, 9 M urea and treatment by 100% 2-ME result in solubilization of protein of 56 kDa (p56). The maximum concentration of 2-ME is important for isolation of pure p56. Evidence has been provided that p56 is a protein of N. grylli polar tubes. Treatment of discharged spores by 2-ME in the presence of SDS results in solubilization of four additional proteins with molecular weights about 46, 34, 21 and 15 kDa.     

Morphological variants of Moniliophthora roreri on artificial media and the biotroph/necrotroph shift

Moniliophthora roreri (Mr) causes frosty pod rot of Theobroma cacao in a hemibiotrophic association. The Mr biotroph-like phase has not been studied in culture. Mr spores (isolates Co12, Co52, and B3) were germinated on high (V8) and low (BPMM) nutrients with different media hardness (0.5% to 3% agarose). Germination was high on V8 media. Hardness affected germination on BPMM. Most colonies on V8 were slow-growing, failing to sporulate. Colony morphology depended on the isolate. On BPMM, exaggerated mycelia formed of limited length with enlarged cells. On agarose, rapidly expanding sporulating necrotrophic colonies formed rarely. Co12 and B3 spores were germinated on V8 and BPMM with low melting point (LMP) agarose. Slow-growing colonies of B3 on BPMM were unstable on LMP agarose, often forming slow-growing/rapidly expanding hybrids. Slow-growing colonies are hypothesized to represent the biotrophic phase. One nucleus was common in Mr cells, other than spores. Binucleate cells were occasionally observed in aged cells of slow-growing mycelia. Co52 cells often had more than two nuclei per cell after germination. Mr mycelia cells typically carry a single nucleus, being considered haploid. Biotroph- and necrotroph-like mycelia displayed differential gene expression but results were inconsistent with published in vivo results and require further study.     

Spore lytic enzyme released from Clostridium perfringens spores during germination.

The exudate of fully germinated spores of Clostridium perfringens was found to contain a large amount of a spore lytic enzyme which acted directly on alkali-treated spores of the organism to cause germination. Although no detectable amount of the enzyme was found in dormant spores during germination in a KCl medium, the enzyme was produced rapidly and released into the medium. The optimal conditions for enzyme activity were pH 6.0 and 45 degrees C. Maximum activity occurred in the presence of various univalent cations at a concentration of 50 mM. The enzyme was readily inactivated by several sulfhydryl reagents. A strong reducing condition was generated in the ionic germination of the spores, a minimum Eh level of -350 mV being reached 30 min after initiation of germination. Furthermore, adenosine triphosphate-dependent pyruvate:ferredoxin oxidoreductase (EC 1.2.7.1) was identified in both dorman and germinated spores. The relationship between the release of active enzyme and the generation of reducing conditions during germination is discussed.     

Inhibition of Na+, K+ Activated ATPase: By Macrocyclic Polyethers

A NADH coupled assay was used to evaluate the effect of crownethers on the kinetics of Na+, K+ activated ATPase. The coupledassay reaction mixture contained lactic acid dehydrogenase,pyruvate kinase (0.06 mg protein/ml), phosphoenolpyruvate (0.8mM), NADH (1.41 mM), ATPase (0.079 mg protein/ml), ATP (variable)in a 50mM Tris HC1 buffer, pH 7.8, containing 100 mM NaCl, 10mM KCl, 5 mM MgCl₂ and 3 mM EDTA. Two crown ethers and two cryptand polyethers were used, thedicyclohexano-18- crown-6 (18-C-6) inhibited over the rangefrom 18 mM (9%) to 100 mM (66%), dicyclohexano- 21-crown-7 (21-C-7)from 9 mM (13%) to 110 mM (93%). The cryptand 4,7,13,16,21-pentaoxa-l,10-diazabicyclo-(8,8,5)-tricosane(k221) inhibited over the range from 2 mM (9%) to 20 mM (82%),while its analogue, 4,7,13,16,21,24-hexaoxal, 10-diazabicyclo-(8,8,8)-tricosane(k222) inhibited from 0.3 mM (11%) to 18 mM (65%). The kineticplots exhibits uncompetitive inhibition for 18-C-6, and suggestthe same for 21-C-7, k221 and k222, although noncompetitiveinhibition cannot be excluded from the latter. (Received January 6, 1981; Accepted February 10, 1981)     

Mechanisms of sorbate inhibition of Bacillus cereus T and Clostridium botulinum 62A spore germination.

The mechanism by which potassium sorbate inhibits Bacillus cereus T and Clostridium botulinum 62A spore germination was investigated. Spores of B. cereus T were germinated at 35 degrees C in 0.08 M sodium-potassium phosphate buffers (pH 5.7 and 6.7) containing various germinants (L-alanine, L-alpha-NH2-n-butyric acid, and inosine) and potassium sorbate. Spores of C. botulinum 62A were germinated in the same buffers but with 10 mM L-lactic acid, 20 mM sodium bicarbonate, L-alanine or L-cysteine, and potassium sorbate. Spore germination was monitored by optical density measurements at 600 nm and phase-contrast microscopy. Inhibition of B. cereus T spore germination was observed when 3,900 micrograms of potassium sorbate per ml was added at various time intervals during the first 2 min of spore exposure to the pH 5.7 germination medium. C. botulinum 62A spore germination was inhibited when 5,200 micrograms of potassium sorbate per ml was added during the first 30 min of spore exposure to the pH 5.7 medium. Potassium sorbate inhibition of germination was reversible for both B. cereus T and C. botulinum 62A spores. Potassium sorbate inhibition of B. cereus T spore germination induced by L-alanine and L-alpha-NH2-n-butyric acid was shown to be competitive in nature. Potassium sorbate was also a competitive inhibitor of L-alanine- and L-cysteine-induced germination of C. botulinum 62A spores.     

A New Method for Inoculation of Poor Germinator, Nosema sp. NIS M11 (Microsporida: Nosematidae), into an Insect Cell Culture

ABSTRACT. Spores of Nosema sp. NIS M11, primed with 0.1 N KOH solution, were mixed with either physiological salt solutions or IPL-41 medium, an insect cell culture medium, for germination. In the latter medium, only a few spores germinated, while high percentages of spore germination were obtained in physiological salt solutions, particularly in Rinaldini's solution. By using the salt solutions as inoculation media, KOH-primed NIS M11 spores were inoculated into the Spodoptera frugiperda SF21AEII cell line. The initial infection levels were consistently higher than that obtained by using IPL-41 medium. Among the salt solutions, Rinaldini's solution, containing KCl in place of NaCl, gave the highest percentage of initial cell infection. Increased osmolarity of salt solutions did not improve the efficiency of spore germination and infection of N. sp. NIS M11.     

Sodium and buffer cations inhibit dephosphorylation of (Na+ + K+)-ATPase

Effects of various cations on the dephosphorylation of (Na+ + K+)-ATPase, phosphorylated by ATP in 50 mM imidazole buffer (pH 7.0) at 22 degrees C without added Na+, have been studied. The dephosphorylation in imidazole buffer without added K+ is extremely sensitive to K+-activation (Km K+ = 1 microM), less sensitive to Mg2+-activation (Km Mg2+ = 0.1 mM) and Na+-activation (Km Na+ = 63 mM). Imidazole and Na+ effectively inhibit K+-activated dephosphorylation in linear competitive fashion (Ki imidazole 7.5 mM, Ki Na+ 4.6 mM). The Ki for Na+ is independent of the imidazole concentration, indicating different and non-interacting inhibitory sites for Na+ and imidazole. Imidazole inhibits Mg2+-activated dephosphorylation just as effective as K+-activated dephosphorylation, as judged from the Ki values for imidazole in the two processes. Tris buffer and choline chloride, like imidazole, inhibit dephosphorylation in the presence of residual K+ (less than 1 microM), but less effectively in terms of I50 values and extent of inhibition. Tris inhibits to the same extent as choline. This indicates different inhibitory sites for Tris or choline and for imidazole. These findings indicate that high steady-state phosphorylation levels in Na+-free imidazole buffer are due to the induction of a phosphorylating enzyme conformation and to the inhibition of (K+ + Mg2+)-stimulated dephosphorylation.     

Na+-like effect of imidazole on the phosphorylation of (Na+ + K+)-ATPase

A high basal level of phosphorylation (approx. 70% of the optimal Na+-dependent phosphorylation level) is observed in 50 mM imidazole-HCl (pH 7.0), in the absence of added Na+ and K+ and the presence of 10-100 microM Mg2+. In 50 mM Tris-HCl (pH 7.0) the basal level is only 5%, irrespective of the Mg2+ concentration. Nevertheless, imidazole is a less effective activator of phosphorylation than Na+ (Km imidazole-H+ 5.9 mM, Km Na+ 2 mM under comparable conditions). Imidazole-activated phosphorylation is strongly pH dependent, being optimal at pH less than or equal to 7 and minimal at pH greater than or equal to 8, while Na+-activated phosphorylation is optimal at pH 7.4. This suggests that imidazole-H+ is the activating species. Imidazole facilitates Na+-stimulated phosphorylation. The Km for Na+ decreases from 0.63 mM at 5 mM imidazole-HCl to 0.21 mM at 50 mM imidazole-HCl (pH 7; 0.1 mM Mg2+ in all cases). Imidazole-activated phosphorylation is more sensitive to inhibition by K+ (I50 = 12.5 microM) than Na+-activated phosphorylation (I50 = 180 microM). Mg2+ antagonizes activation by imidazole-H+ and also inhibition by K+. The Ki value for Mg2+ (approx. 0.3 mM) is the same for the two antagonistic effects. Tris buffer (pH 7.0) inhibits imidazole-activated phosphorylation with an I50 value of 30 mM in 50 mM imidazole-HCl (pH 7.0) plus 0.1 mM Mg2+. We conclude that imidazole-H+, but not Tris-H+, can replace Na+ as an activator of ATP-dependent phosphorylation, primarily by shifting the E2----E1 transition to the right, leading to a phosphorylating E1 conformation which is different from that in Tris buffer.     

Effects of alpha- and beta-D-glucose on germination of spores of Bacillus megaterium QM B1551.

The effects of D-glucose anomers on the germination of dormant spores of Bacillus megaterium QM B1551 were studied, alpha-D-Glucose (1 mM) slightly initiated the germination of the dormant spores during 10 min incubation at 37 degrees C, while about 60% of the dormant spores became germinated with beta-D-glucose (1 mM) in the same conditions. From the above observations and the finding that only a trace amount of alpha- or beta-D-glucose may bind with the dormant spores, it is speculated that the beta-D-glucose-stereospecific receptor site for the germination exists on the surface of the dormant spores of the bacillus.     

Neisseria gonorrhoeae M.Ngo AI DNA methyltransferase: physical and catalytic properties of the homogeneous enzyme

A DNA methyltransferase, M.NgoAI, was purified to homogeneity from Neisseria gonorrhoeae strain WR220 by successive column chromatography. Its Mr is 25,000, as determined by both gel filtration and denaturing polyacrylamide gel electrophoresis. Maximal enzymatic activity was obtained in 50 mM Tris.HCl (pH 7.4), 10 mM EDTA, with incubation at 37 degrees C. An apparent Km value for S-adenosylmethionine and 5' -GGCC sites was determined to be 1.25 microM and 89.6 nM, respectively.     

Determination of dipicolinic acid in bacterial spores by derivative spectroscopy

Dipicolinic acid was extracted from approximately 0.1 mg spores or 0.5 ml of sporulating culture with 20 mM HCl for 10 min at 100 degrees C. The suspension was diluted with 5 mM Ca2+, 100 mM Tris, pH 7.6, centrifuged, and the first derivative of the uv absorbance spectrum recorded from 275 nm to 285 nm. DPA concentration was determined from the difference between the maximum at 276.6 nm and the minimum at 280 nm. The use of the difference between two first derivative values removed possible interference from sloping baselines. Turbidity, nucleic acids, and bacteriological media did not interfere. Analysis time for four extracts was 4 min using a spectrophotometer reading at 0.1-nm intervals. Dipicolinate at 0.1 mM gave 0.184 absorbance/nm at 25 degrees C. The coefficient of variation was 1.5%, and the detection limit 1 microM.     

Pheromone binding proteins in the European and Asian corn borers: no protein change associated with pheromone differences.

Pheromone binding proteins (PBPs) are thought to play a role in the recognition of sex pheromone in male moth antennae. By binding selectively to different components of pheromone blends, these PBPs could play a role in differentiating between structurally related compounds. In this study we have characterized the pheromone binding proteins of two pheromone strains of the European corn borer (Ostrinia nubilalis) and also the closely related Asian corn borer (O. furnacalis). We have been able to detect only one PBP gene, which encodes a mature protein that is identical in amino acid sequence in individuals from different pheromone strains and different species. This result suggests that the PBP is not detecting differences between the two isomeric compounds of the European corn borer pheromone or the difference in double bond position between the pheromone molecules of the European and Asian corn borers.