, 2007). The immunoscreening method has selleck screening library also some possible source of errors: (a) undetected proteins because of lack reacting antibodies caused by extremely low amounts of antigens or because they were not enough immunogenic; (b) contaminants detected because they are highly immunogenic; (c) non-microvillar proteins detected because they share epitopes or were accidentally associated with microvillar proteins; (d) failure of inserted-cDNA-phage expression. In spite of the limitations discussed above, both methods allowed the characterization of a substantial number of midgut
microvillar proteins of different taxa (Candas et al., 2003, McNall and Adang, 2003, Krishnamoorthy et al., 2007, Ferreira et al., 2007, Bayyareddy et al., 2009, Popova-Butler and Dean, 2009 and Pauchet et al., 2009). This study describes the immunoscreening of a S. frugiperda expression midgut cDNA library with antibodies against isolated microapocrine vesicle proteins. Sequences obtained together with data obtained by pyrosequencing S. frugiperda midgut mRNA were used to identify the proteins secreted selleck and those putatively involved in the secretory machinery. S. frugiperda (Lepidoptera: Noctuidae) were laboratory
reared according to Parra (1986). The larvae were individually contained in glass vials with a diet based on kidneys beans (Phaseolus vulgaris), wheat germ, yeast, and agar, and were maintained under a natural photoregime Urocanase at 25 °C. Adults were fed a 10% honey solution. Fifth (last)-instar larvae of both sexes were used in the determinations. Larvae were immobilized by placing them on ice, after which they were rinsed in water and blotted with filter paper. Their guts were dissected in cold 125 mM NaCl, and the peritrophic membrane with contents and the midgut tissue were pulled apart. The midgut tissue was suspended above a centrifuge tube and rinsed with a 125 mM NaCl solution. This rinsing saline has been previously shown to correspond to ectoperitrophic contents (Ferreira et al., 1994). The rinsing saline was then centrifuged at 600g for 10 min at 4 °C. The resulting
supernatant was centrifuged at 25,000g for 30 min at 4 °C. The pellet was suspended in Milli-Q water and labeled microapocrine vesicles. Midgut tissue and peritrophic membrane with contents were homogenized in Milli-Q water with the aid of a Potter–Elvehjem homogenizer. After that, the peritrophic membrane with contents were centrifuged at 10,000g for 10 min at 4 °C. The supernatant was used in all cases, except when otherwise indicated. Microvilli were isolated from midgut tissue with a procedure derived from that of Schmitz et al. (1973), as detailed in Ferreira et al. (2007). The preparations could be stored for at least 3 months at −20 °C without noticeable change in the activity of the enzymes assayed. Aminopeptidase and trypsin were assayed in 50 mM Tris–HCl buffer (pH 7.