Robert T. Gudauskas

Effects of Bacillus thuringiensis Nuclear-Polyhedrosis Virus Mixtures on Trichoplusia ni Larvae

Effects of exposure to Bacillus thuringiensis and a nuclear-polyhedrosis virus (NPV) on larvae of the cabbage looper, Trichoplusia ni , were determined in laboratory experiments. Mortality curves Were established for each pathogen and served as the basis for the tests. Third-instar larvae averaging 15 mg body weight were inoculated by being on a diet containing desired concentrations of the pathogens. The ELD 50 for B. thuringiensis was 300 IU/ml of diet and for NPV, 2.27 × 10 3 polyhedral inclusion bodies/ml of diet. Mortality data from larvae exposed to both pathogens simultaneously at generally low dosages indicated that the effects of the pathogens in combination were additive. Results from experiments in which larvae were stressed by exposure to NPV for 24-96 hr prior to exposure to B. thuringiensis showed that increased mortality was an additive effect of the two pathogens. Pupae from larvae exposed to B. thuringiensis were significantly smaller than those from larvae exposed to NPV alone or to no pathogen.

The effect of heat, buffer salt and H-ion concentration, and ultraviolet light on the infectivity of Heliothis and Trichoplusia nuclear-polyhedrosis viruses

Abstract The thermal inactivation point of Heliothis nuclear-polyhedrosis virus (HPV) was determined to be between 75° and 80°C for 10 min; that of the Trichoplusia nuclear-polyhedrosis virus (TPV) was between 82° and 88°C. Infectivity of HPV was reduced when the virus was buffered at pH 2 or 12 for 30 min to 24 hr but was unaffected at pH 5, 7, or 9. The concentration of phosphate buffer used appeared to have little effect. TPV polyhedra dissolved after 30 min in 0.1 m phosphate at pH 11, but infectivity was not lost until a pH of 12 was reached. HPV was inactivated by ultraviolet light after 5 min at 2 inches from the light source. Infectivity of TPV was greatly reduced by exposures of 4–10 min to ultraviolet at 2 inches; total inactivation did not occur after 60 min, but did after 120 min. Effect of ultraviolet on both viruses was progressively lessened as distance from light source to virus increased. No reduction in infectivity of either virus occurred at an exposure distance of 32 inches.

Biochemical comparison of virion proteins from five nuclear polyhedrosis viruses infecting plusiine larvae (Lepidoptera: Noctuidae)

Abstract The virions of six isolates of five nuclear polyhedrosis viruses (NPV) infecting plusiine larvae (Lepidoptera: Noctuidae) were reproducibly separated by sucrose density gradient centrifugation and fractionation. Purity of the preparations was established by electron microscopy. Virion proteins were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE); each produced 12 distinct polypeptides ranging from 10,300 to 82,900 mw. Qualitative and quantitative differences were found between most of the polypeptide patterns. The singly embedded viron (SEV)-type isolates had two major components with mw in the range of 32,900–35,200; multiply embedded virion (MEV)-type isolates had a major component of ca. 12,500 mw. SEV isolates showed almost no within-group differences, while minor differences were found among the MEV banding patterns in both intensity and presence of certain bands. Capsids and envelopes from MEV had two to four polypeptides with mw between 10,800 and 26,900. The presence of more than one polypeptide and electron microscopy of sample composition suggested that the capsid and envelope are composed of several distinct proteins.

Analysis of deoxyribonucleic acid from five nuclear polyhedrosis viruses infecting plusiine larvae (Lepidoptera: Noctuidae)

Deoxyribonucleic acid (DNA) from isolates of five nuclear polyhedrosis viruses (NPV) from lepidopterous hosts of the noctuid subfamily Plusiinae was analyzed by ion-exchange and paper chromatography. Viruses and production hosts were: Trichoplusia ni singly embedded virion type (SEV) from T. ni, Pseudoplusia includens SEV from P. includens, T. ni multiply embedded virion type (MEV) from T. ni, Autographa californica MEV from A. californica, A. californica MEV from T. ni, and Rachiplusia ou MEV from R. ou. Neither uracil nor 5-methyl cytosine was detected in the DNAs. Adenine:thymine (A:T) and guanine:cytosine (G:C) ratios were nearly constant for all the NPVs. AT:GC ratios for the SEVs were 1.60 and 1.57 and were clearly separable from those of the MEVs which ranged from 1.32 to 1.38. No differences in DNA composition within SEV or MEV groups were apparent.

Serological comparison of polyhedron protein and virions from four nuclear polyhedrosis viruses of plusiine larvae (Lepidoptera: Noctuidae)

Abstract Purified polyhedron proteins and purified, ultrasonicated virions of four nuclear polyhedrosis viruses (NPVs), separable into two morphologic groups of singly and multiply embedded virion types (SEVs and MEVs), were investigated by immunodiffusion and immunoelectrophoresis. The four viruses were Pseudoplusia includens SEV, Trichoplusia ni SEV, T. ni MEV, and Autographa californica MEV. In immunodiffusion, SEV polyhedron proteins formed two precipitin bands with antiserum to SEV polyhedron proteins, while MEV polyhedron proteins formed only one. All four proteins formed one precipitin band with antiserum to MEV polyhedron protein, with a spur between SEV and MEV proteins. In immunoelectrophoresis, mobilities of SEV proteins were significantly different from those of MEVs. Precipitin arc patterns were similar to those in immunodiffusion when electrophoresis was carried out at 4 C; at room temperature, a single arc of precipitation formed with all four proteins. SEV virions formed five possibly identical precipitin bands in immunodiffusion with antiserum to SEV virions. MEV virions formed three possibly identical precipitin bands when reacted with antiserum to MEV virions. Little or no cross-reactions were observed between SEV and MEV virions or between virions and polyhedron proteins. In immunoelectrophoresis, SEV virions formed three precipitin arcs in reactions with SEV antisera and none with MEV antisera; MEV virions formed two arcs with MEV antisera and none with SEV antisera. When antisera were subjected to electrophoresis, five arcs were formed by SEVs and three by MEVs in homologous systems, and none were formed in heterologous systems.

Arginine Metabolism by the Corn Stunt Spiroplasma

When corn stunt spiroplasma (CSS) was grown in spiroplasma medium A (SMA) containing 5% horse serum, 16% sucrose, 1.5% PPLO broth, and 0.002% phenol red indicator, color and pH of the medium changed from red (pH 7.5) to yellow (pH 5.8) by 4 days after inoculation with CSS. In spiroplasma medium B (SMB), identical to SMA except for supplementation with 47 mM arginine-HCl, color and pH changed within 4 days but reverted to red (pH 7.5) 6 days later, suggesting that arginine was metabolized. Growth of CSS in both SMA and SMB was a characteristic sigmoid pattern; maximum titers were reached the fifth day after inoculation and were 2.3 × 108 cells/ml in SMA and 4.5×108 cells/ml in SMB. Helicity and motility of CSS were lost after 8 days in SMA, while in SMB some cells were still helical and motile up to 15 days. Daily analyses of CSS cultures by photometric methods showed that arginine was depleted from SMB and production of ammonia and ornithine was higher in SMB than in SMA; citrulline was detected in SMB but urea was not found in either medium. Results from additional tests using an amino acid analyzer and thin-layer chromatography further substantiated arginine metabolism and indicated that an arginine deiminase pathway was operative in CSS.