longipalpis larvae could exploit these microorganisms as nutrients in nature. L. longipalpis were collected Alpelisib at Gruta da Lapinha, Minas Gerais, Brazil. Adult sand flies received continuously a 70% (w/v) sugar solution in cotton wool. Females were routinely fed on hamsters (Mesocricetus auratus) anesthetized with xylazine

(10 mg/kg) plus ketamine (200 mg/kg). Engorged females were transferred to rearing containers ( Barretto and Coutinho, 1940), with a piece of cotton wool soaked in sugar solution on it. Dead females were removed after oviposition. Larvae received a mixture of grinded rabbit faeces, rabbit food and earth (1:1:1), which is left at room temperature for 15 days for aging before use. From the third instar onwards, larvae were fed with a mixture (1:1) of soya protein (Carrefour, Brazil) and cereal flakes (Neston, Nestlé, Brazil). This food is offered as a pellet in the middle of the container, to avoid the spreading of fungus which grows on it intensively. The colony was maintained at 26 °C ± 1 °C,

70–80% humidity and natural light. Fourth instar larvae with the gut full of food and mycelia growing on the white food were collected from the same rearing cages for all experiments. More details about sand fly capture and rearing in laboratory conditions are described in Volf and Volfova (2011). All substrates and chemical substances used were acquired from Sigma (USA) and were of analytical grade. All larvae samples were immobilized by placing them on ice, after which they were dissected in cold 150 mM NaCl. Protein concentration was determined according to Smith et al. (1985), using bovine CP-868596 cost serum ovalbumin as a standard. Enzyme activities were evaluated by the release of 4-methylumbelliferone (4-MU) according to Baker and Woo (1992). The enzymes evaluated were (enzyme, substrate concentration): α-glycosidase, 4-methylumbelliferyl-α-d-glucopiranoside

20 μM (Sigma cat. no. M9766); β-mannosidase, 4-methylumbelliferyl-β-d-mannopiranoside 20 μM (M0905); N-acetyl-β-glucosaminidase, 4-methylumbelliferyl-β-N-acetyl-d-glucosaminide Ribonucleotide reductase 20 μM (M2133); neuraminidase, 2′-(4-Methylumbelliferyl)-α-d-N-acetylneuraminic acid 20 μM (M8639); β-glycosidase, 4-methylumbelliferyl-β-d-glucopiranoside 20 μM (M3633) and α-mannosidase, 4-methylumbelliferyl-α-d-mannopiranoside 20 μM (M3657). Lysozyme or chitinase activity was measured by the release of 4-MU from 4-methylumbelliferyl-β-d-N′,N″,N″′-triacetyl-chitotrioside 30 μM (M5639). The activity of β-1,3-glucanase was determined by measuring the release of reducing groups ( Fox and Robyt, 1991) from 0.04% (w/v) laminarin (from Laminaria digitata, Cat. no. L9634). All enzymes were assayed at 30 °C under conditions such that activity was proportional to protein concentration and to time. Controls without enzyme or without substrate were included. One unit of enzyme (U) is defined as the amount that hydrolyses 1 μmol of substrate (or bonds)/min.