Effect of Various Sodium Dodecyl Sulfate Sample Buffers on the Exospore and Spore Polypeptide Profiles of Nosema locustae (Microspora: Nosematidae)

Abstract

Several studies using sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis (PAGE) have produced conflicting results regarding exospore polypeptide profiles. Streett and Briggs (1982, Journal of Invertebrate Pathology 40:159165) did not detect exospore polypeptides in samples of intact spores treated with 1% SDS and 0.01 M dithiothreitol. In contrast, Irby (1983, Master's Thesis, North Carolina State University, 99 p.) observed unique exospore profiles for several species of microsporidia treated with 2% SDS and 5% beta-mercaptoethanol. Irby concluded from his investigation that Streett and Briggs (1982, loc. cit.) did not use a sufficient amount of reducing agent (dithiothreitol). Consequently, for this investigation, Nosema locustae Canning spore homogenates and intact spore suspensions were treated with sample buffers containing various concentrations of SDS and reducing agent. Nosema locustae was propagated in Melanoplus differentialis (Thomas) and isolated by differential centrifugation (Henry and Oma, 1981, Pest control by Nosema locustae, a pathogen of grasshoppers and crickets. In Microbial control of pests and plant diseases, H. D. Burges (ed.). Academic Press, New York, New York, pp. 573586). A spore suspension (1.2 x 109 spores) was homogenized and separated by electrophoresis as reported earlier (Streett and Briggs, 1982, loc. cit.). Both intact and homogenized spore pellets (each with 1.2 x 109 spores) were resuspended in 1.2 ml of 0. 1 M Tris buffer pH 7.2, and divided into 6 aliquots for centrifugation at 500 g for 5 min in microcentrifuge tubes. The supernatant was removed and each aliquot of intact and homogenized spores was treated with 60 ,l of sample buffer. The 6 sample buffers used to treat N. locustae intact and homogenized spore preparations were: 1% SDS with 0.01 M dithiothreitol; 2% SDS with 0.01 M dithiothreitol; 2% SDS with 0.02 M dithiothreitol; 4% SDS with 0.02 M dithiothreitol; 2% SDS with 5% beta-mercaptoethanol; 4% SDS with 10% beta-mercaptoethanol. N. locustae spore profiles were compared and evaluated visually. An exospore polypeptide profile was detected when intact spores were treated with each of the sample buffers except 1% SDS with 0.01 M dithiothreitol (Fig. 1A). When examined with phase contrast microscopy intact spores treated with 1% SDS and 0.01 M dithiothreitol were refractile and appeared normal. However, intact spores treated with the other sample buffers were not refractile, which indicates either spore germination or a lack of an exospore layer. The exospore polypeptide profile for treated intact spore preparations ranged from approximately 11,000 daltons to 86,000 daltons. This generally corroborates the results reported by Irby (1983, loc. cit.) for N. locustae spores treated with 2% SDS and 5% beta-mercaptoethanol. The spore polypeptide profiles of homogenized N. locustae treated with the various sample buffers are shown in Figure IB. N. locustae homogenized spores treated with SDS sample buffers using dithiothreitol as a reducing agent are nearly identical with the exception of 1% SDS with 0.01 M dithiothreitol. The 1% SDS and 0.01 M dithiothreitol sample buffer appears to be inadequate for dissociating either exospore or spore polypeptides. Spore polypeptide profiles for homogenized spores treated with SDS sample buffers containing dithiothreitol ranged from approximately 10,000 daltons to 200,000 daltons. No significant differences are detected in the profiles of N. locustae homogenized spores treated with 2% SDS and either 5% or 10% beta-mer-