Only three studies have been published to date (Mahmood and Borovsky, 1992, Fazito do Vale et al., 2007 and Moraes et al., 2012). In one of these studies, we described, for the first time, the anatomy of the digestive tube of L. longipalpis larvae and determined the pH along the midgut ( Fazito do Vale et al., 2007). In addition, we investigated how proteins are digested from the beginning of this process in the alkaline anterior midgut (pH ⩾ 9.0) to its end in the acidic posterior midgut (pH ⩾ 6.5) selleck screening library ( Fazito do
Vale et al., 2007). The aim of the present study was to study carbohydrate digestion by L. longipalpis larvae. The main glycolytic activities were identified and partially characterized. Special attention was given to the compartmentalization of the main carbohydrases found to provide an overview of the different stages of digestion. Akt inhibitor Taking into account the hydrolytic activities encountered in the
larval intestine and the material ingested by the larvae, we offer a discussion about the origin and the type of carbohydrates usually ingested by the larvae in nature. All experiments were performed using fourth instar larvae from a colony of L. longipalpis (Teresina/Piauí state, Brazil) maintained according to the methodology described by Modi and Tesh (1983). The standard larval diet was that proposed by Young et al. (1981). The food offered to the larvae (from the second to the fourth instars) was supplemented with a mixture of powdered cereals prepared with grains of wheat, barley and oats (Neston from Nestle®). In this case, care was necessary to avoid excessive growth of fungi. Homogenates of the total midgut were prepared by dissecting the larvae in 0.9% (w/v) NaCl. The dissected midguts were washed in 300 mM NaCl containing 0.03 mM CaCl2 and transferred to the same solution in a micro centrifuge
Ponatinib in vivo tube to be homogenized with an abrasive micro homogenizer made of glass. At least 15 midguts were pooled for each sample preparation. All material was stored in an ice bath during the procedures. The supernatant obtained after centrifugation for 10 min at 14,000×g at 4 °C was used in the experiments. The assays were performed by mixing 100 μL of 1.5% (w/v) starch (Sigma No. S9765), glycogen (Sigma No. G8751) or dextran (Sigma No. D1662) (each dissolved in water) in a micro centrifuge tube with 150 μL of 0.1 M buffer. The reaction was started by adding 50 μL of the sample. Each 50 μL aliquot of sample contained the equivalent of 1 midgut. This incubation mixture, comprising a final volume of 300 μL, was incubated at 30 °C for 1 h. The reducing carbohydrates released from the substrate by the action of the amylase were quantified using the dinitrosalicylic acid method (Miller, 1959). After the incubation, 500 μL of the dinitrosalicylic reagent (DNS reagent) was added to the tubes, which were then heated in boiling water for 10 min.