Carlo M. Ignoffo

Insect cuticle-degrading enzymes from the entomogenous fungusBeauveria bassiana

Beauveria bassiana is a promising agent for biological control of agricultural insect pests. Its broad host range includes both the Greater Wax Moth (Galleria mellonella) and the cabbage looper (Trichoplusia ni). Five strains ofB. bassiana from the Agricultural Research Service Culture Collection were obtained and grown on defined media and a medium containing purified cuticle from eitherG. mellonella orT. ni. The production of cuticle-degrading enzymes (such as proteases, chitinases, and esterase) was examined. Natural strain variability in enzyme levels was significant, and there was no evidence of coordinated expression. Further, enzyme expression differed considerably as a function of cuticle source. The results suggest that it may be possible to tailor biological control agents against specific targets. Natural strain variability could be used to rationally develop improved mycoinsecticides.

Entomopathogens as insecticides.

Entomopathogens, diseases of insects, are suggested as a possible new generation of safe, selective insecticides. Over a thousand pathogens have been isolated from insects. Many of these, associated with major insect pests, are potential candidates for development into microbial insecticides. Phases in the development of a microbial insecticide are discussed as well as factors (production, safety, efficacy, registration, non-technical) which may influence this development.

Cuticular and non-cuticular substrate influence on expression of cuticle-degrading enzymes from conidia of an entomopathogenic fungus,Nomuraea rileyi

Larval cuticle fromTrichoplusia ni, Helicoverpa (=Heliothis)zea, andHeliothis virescens and a cellulose substrate were used to quantify release of proteolytic, chitinolytic, and lipolytic enzymes by germinating conidia of the entomopathogenic fungus,Nomuraea rileyi. There was no significant difference in conidial viability incubated withT. ni, H. zea or cellulose substrates. Conidial viability onH. virescens cuticle, however, was significantly lower (ca. 19–25%) than the other three substrates. The presence of cuticle substrates, especially cuticle ofT. ni, stimulated germination. The nature of the substrate influenced both the time and quantity of the enzymes expressed. Specific proteases (aminopeptidase, chymoelastase, trypsin) generally were expressed earlier and/or in greater quantities on cuticular than on the cellulose substrate. Although both chitinolytic enzymes (endochitinase, N-acetylglucosaminidase) were detected on all three cuticular substrates, their activity was substantially lower than that of the proteolytic enzymes. Lipase activity was only minimally present. Early concurrent release of both proteases and chitinases suggested that both may be important in the penetration of the larval integument by germinating conidia ofN. rileyi. Expression of proteases and chitinases, especially aminopeptidase and endochitinase was probably a specific response to cuticle, because little or no activity was expressed on the non-host, cellulose substrate.

Effects of cuticle source and concentration on expression of hydrolytic enzymes by an entomopathogenic fungus, Nomuraea rileyi

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.

Insect cuticle and yeast extract effects on germination, growth, and production of hydrolytic enzymes byNomuraea rileyi

Larval cuticle ofHelicoverpa (Heliothis)zea and yeast extract added to a minimal medium (MM) induced germination of conidia ofNomuraea rileyi whereas sterile distilled water or MM alone did not. Yeast extract increased mycelial yield, but when cuticle was added, mycelial yield significantly decreased. Proteases and chitinases ofN. rileyi were only expressed when cuticle was added to the MM.

Use of a colorimetric system to detect enzymes expressed by germinating conidia of entomopathogenic fungi.

An apiZYM™ system, with 19 substrates, was used to detect enzymes expressed by germinating conidia of Nomuraea rileyi (5 isolates), Nomuraea atypicola, Nomuraea anemonoides, Beauveria bassiana and Metarhizium anisopliae. Similar enzyme profiles were obtained for two of the N. rileyi isolates (Mississippi, Ecuador) regardless of whether culture medium (Sabouraud-maltose-yeast) or cuticle (from larvae of Trichoplusia ni, Heliothis zea or Heliothis virescens) were used as substrates. Centroid-clustering analysis revealed three distinct enzyme profiles.

INSECT CELLS AND THEIR POTENTIAL AS STABILIZATION BARRIERS FOR DNA OF MULTIPLE AND SINGLE NUCLEOPOLYHEDROVIRUSES AGAINST ULTRAVIOLET-B–SIMULATED SUNLIGHT INACTIVATION

SummaryA cell line from Trichoplusia ni (TN-CLI) infected with the Autographa californica multiple nucleopolyhedrovirus (AcMNPV-HPP) and a cell line from Helicoverpa zea (BCIRL-HZ-AM1) infected with the Helicoverpa zea single nucleopolyhedrovirus (HzSNPV/BrCL2) were subjected to ultraviolet-B (UV-B) irradiation at a predetermined level of exposure that would inactivate greater than 95% of the virus suspended in the liquid. The working hypothesis was that the homologous insect cells would utilize their inherent deoxyribonucleic acid (DNA) repair mechanism(s) to prevent, repair, or at least mitigate the damaging effects of UV-B light on viral DNA synthesis. We attempted to determine this by using infected cells that were subjected to UV-B irradiation at different postinoculation periods under two experimental conditions of exposure: (1) shielded, and (2) nonshielded. Of the two cell lines infected with their respective homologous viruses, the virus from TN-CL1 cells was the least sensitive to UV-B light because the extracellular virus (ECV) and occlusion body (OB) levels of virus-infected TN-CL1 cells were higher than those of the virus-infected BCIRL-HZ-AM1 cells. Production of ECV and OB from both cell lines was lower in the exposed, nonshielded treatment than in the exposed, shielded treatment. However, AcMNPV-HPP was produced in enough quantity to indicate that TN-CL1 might impart a level of protection to the virus against UV light.

A semi-defined medium for culturing Nomuraea rileyi

A semi-defined medium was successfully used to culture several natural and mutant isolates of the entomopathogenic fungus, Nomuraea rileyi. The medium contains only two complex ingredients at very low levels, yet permits germination and mycelial growth of N. rileyi. The medium also facilitates recovery and detection of hydrolytic enzymes. Use of the formulation could simplify nutritional, biochemical and physiological studies with N. rileyi and also might be used to culture other entomopathogenic fungi.

Purification and characterization of trypsin from an entomopathogen, Nomuraea rileyi NRRL 13755

Nomuraea rileyi isolate NRRL-13755 produced a large amount of trypsin enzyme when cultured on basal salt medium containing 1% (w/v) gelatin. The trypsin was purified nearly 60-fold, with a recovery of about 13% of the initial activity from the culture supernatant. This protease exhibited a remarkably high specific activity of nearly 370,000 IU/mg protein. The native molecular weight was estimated by gel permeation chromatography to be 30 kDa, and the subunit molecular weight was determined to be about 30 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The pH and temperature optima were determined to be 8.5 and 35°C, respectively. With a relative trypsin activity of 100%, this purified preparation showed about 10% chymoelastase and nearly 50% chymotrypsin activity. Metal-chelating agents such as EDTA and EGTA at 2mm inhibited the enzyme activity by 40%, whereas N-carbobenzoxy-glycyl-l-phenylalaninamide (CBZ-gly-phe-NH2) (2mm) and DTT (2mm) had no effect on activity. Trypsin inhibitor from turkey egg-white at 100 μg/ml strongly inhibited the enzyme activity.