Control wells contained (1) bacteria, peptone and antibiotic [streptomycin

(100 μg/mL) and ampicillin (80 μg/mL)]; (2) bacteria and peptone; and/or (3) peptone alone. Bacteria were grown in 20 mL tryptone soy buffer (TSB) with shaking for 17 h at 30 °C and then 100 μL of the E. coli k12 solution was transferred to 10 mL of TSB and incubated for a further 4 h. The bacteria were then Dabrafenib washed in PBS and diluted in TSB to a final concentration of 1 × 105 cells/mL. Fifty microliters of midgut sample were then incubated with 10 μL of bacterial suspension in triplicate in the wells of a sterile flat-bottom, 96-well microtiter plate (Nunc, Fisher Scientific UK, Leicestershire, UK). The optical densities were measured at 550 nm (OD550) at 37 °C and read at hour intervals from time zero for 12 h. All data points were subsequently blanked against time zero to account

for the opacity of the midgut samples and then the E. coli k12 readings were subtracted from all sample readings and multiplied by 100. Samples for the nitrite and nitrate determinations were collected in the same manner as for the antibacterial assays. The anterior midgut samples dissected nine days after feeding were homogenized in a tube with 200 μL of Milli-Q water and centrifuged at 8000 g for 1 min at 4 °C. Aliquots of 10 μL from supernatant Erlotinib ic50 were diluted in 90 μL of Milli-Q water. Nitrate and nitrite contents of samples were determined following the manufacturer’s instructions using the Griess Reagent System Assay Kit (Promega, WI, Methocarbamol USA), and absorbance of the product was measured at 550 nm (Moncada, 1992). Nitrite and nitrate contents were quantified as μmoles using a range of sodium nitrate standards and the specific activity was calculated as mg/mL of protein concentration in the anterior midgut samples. Protein content of samples was quantified with a protein assay kit (BCA∗ Protein Assay Reagent,

Pierce, USA) using bovine serum albumin (BSA) standards. The results were analyzed with GraphPad Prism 5 using 1 Way ANOVA or unpaired T test, or Mann Whitney test (nonparametric test) depending on the data distribution and number of treatments. Data were reported as mean ± standard error (SE) or as individual values with medians for parasite and microbiota populations. Differences among groups were considered not statistically significant when p > 0.05. Probability levels are specified in the text and figure legends. The physalin B treatment by oral, topical and contact application did not alter the physiology of the insects even when the insects were challenged by T. cruzi Dm28c clone. The mortality of all treated insects (around 9.6%) was similar to control (8.2%) during the 30 days and no alterations in the ecdysis process were observed. Experiments to investigate the direct effects of physalin B on T.