Apoptosis was determined in cryosections obtained as described above from healthy vitellogenic and atretic follicles, using the ApopTag® Plus Apoptosis Detection Kit (Chemicon) PLX3397 cell line following manufacturer’s instructions, but extending the TdT incubation step to 16 h at 4 °C. Additional controls were also performed excluding the TdT enzyme from the labeling buffer and following the assay as above. Longitudinal sections were
revealed with DAB and photographed under light microscope. Yolk granule fractions from healthy vitellogenic and atretic follicles were obtained as described elsewhere (Ramos et al., 2007). The granules were incubated in the dark for 10 min in Ringer plus 10 mM EGTA containing 5 μg/ml acridine orange. After incubation the yolk granules were deposited on glass slides and observed in a Zeiss Axioplan epifluorescence microscope equipped with a fluorescein filter set and a TK-1270 JVC color video camera. Healthy vitellogenic and atretic follicles were dissected and homogenized on ice in phosphate buffer (0.1 M sodium phosphate, 0.2 M NaCl,
5 mM EDTA) pH 7.0 or acetate buffer (0.1 M sodium acetate, 0.2 M NaCl, 5 mM EDTA) pH 5.0. Ten follicles were used from each sample. Homogenates were submitted to three cycles of freeze and thaw and centrifuged at 20,000 × g see more for 30 min at 4 °C. Supernatants were collected and used as protease preparations. Protease assays were performed by incubating 0.1 follicle equivalents in 50 volumes of acetate buffer pH 4.0 plus 2.5 mM DTT and 10 μM Abz-AEALERMF-EDDnp (Aspartic), or acetate buffer pH 5.0 plus 2.5 mM DTT and 5 μM Z-Phe-Arg-NHMeC ID-8 (Serine and Cysteine). Substrate hydrolysis was monitored in an F-MAX 4500 fluorometer (Molecular Devices, Sunnyvale, CA, USA) at 320 nm excitation and 420 nm emission wavelengths for Abz-AEALERMF-EDDnp or 380 nm excitation and 440 nm emission wavelengths for Z-Phe-Arg-NHMeC.
Steady-state velocities were obtained by linear regression of the substrate hydrolysis curve ( Lima et al., 2001). Healthy vitellogenic and atretic follicles were centrifuged at 20,000 × g for 30 min at 4 °C. Supernatants were collected and used as samples for electrophoresis. Protein concentration was determined by the method of Lowry ( Lowry et al., 1951) using bovine serum albumin as standard. Polyacrylamide gels (10%) were run at 25 mA, applying 10 μg of protein per lane. Gels were silver stained using the protocol described by Dunn and Crisp (1994). Direct injection of conidia into the hemocoel of R. prolixus females at the onset of vitellogenesis did not affect host survival (Log-rank test, p = 0.5553, N = 31–33 subjects). Median survival was 23, 24.5 and 20 days for control (uninjected group); Grace’s injected group and fungal injected group, respectively, confirming the low pathogenicity of A. niger to these insects. Our previous results ( Medeiros et al., 2009) showed that R.