3 4 dihydroxy dl phenylalanine

Methyl-α-l-fucopyranoside, methyl-β-l-fucopyranoside, d-glucose and d-galactose were purchased from Carbosynth, Compton, United Kingdom; blood group A/B/O trisaccharides, and Fucα1-GlcNAc and Fucα1-GlcNAc were purchased from Carbohydrate Synthesis, Oxford, United Kingdom; l-fucose was purchased from Applichem, Darmstadt, Germany. N-acetyl-d-glucosamine, d-mannose, d-mannose-agarose, biotin and streptavidin, bovine serum albumin, 3,4-dihydroxy-dl-phenylalanine, zymosan A from Saccharomyces cerevisiae and luminol were purchased from Sigma-Aldrich, St Louis, USA. Biotinylated saccharides (biotinylated α/β-d-mannoside, α/β-d-galactoside and α-l-fucoside) were purchased from Synthaur LLC, Moscow, Russia. Fluorescein isothiocyanate (FITC) and DyLight 488 were purchased from ThermoScientific, Rockford, USA. Basic chemicals were purchased from Sigma-Aldrich, St Louis, USA; Duchefa, Haarlem, Netherlands; ForMedium, Hunstanton, United Kingdom and Applichem, Darmstadt, Germany.

Assays were performed with larvae from a previously established local colony [46 (link)] and fed ad libitum with a conventional diet for this species [47 (link)]. The inclusion criteria for inoculation were a length between 1.2 and 1.5 cm, active behavior, and no apparent melanization. Fungal challenges included 1 × 10⁵ yeast-like cells in 10 μL of PBS, and these were injected in the last left pro-leg with a Hamilton syringe and a 26-gauge needle, as reported [46 (link)]. Animals were inspected for 14 days under hydration ad libitum with chopped apple and a 37 °C temperature. Larvae were considered dead when irritability to external stimuli was lost and the body surface was extensively melanized [46 (link)]. Groups of 30 larvae were used to analyze the virulence of each of the Sporothrix strains used in this work. One additional group inoculated only with PBS was included as a control. Colony-forming units, hemocyte concentration, phenoloxidase activity, melanin production, and cytotoxicity were measured, as previously reported [19 (link),47 (link)], using anticoagulated hemolymph. Phenoloxidase activity was assayed using 20 mM 3,4-dihydroxyDL-phenylalanine (Sigma-Aldrich), whilst cytotoxicity was quantified with the Pierce LDH Cytotoxicity Assay (Thermo Fisher Scientific).