Phlebotomus perniciosus

Phlebotomus perniciosus View in CoL colony maintenance

Phlebotomus perniciosus larvae and adult females were obtained from a colony originating from Murcia, Spain, maintained for two decades at the Department of Parasitology, Charles University, Prague, Czech Republic, under controlled conditions as described previously [ 47].

Aza and ecdysone treatment in P. perniciosus larvae Phlebotomus perniciosus larvae were fed a diet containing composted and powdered rabbit feces, as described previously [ 47]. Aza (Sigma-Aldrich, Darmstadt, Germany) and α-ecdysone (Sigma-Aldrich) (the precursor of 20E) were first dissolved in 250 μl of 99% ethanol P.A. and then diluted in 750 μl of 0.9% NaCl (1:4 ethanol/saline), resulting in final concentrations of 1 and 2.5 μg/ml, respectively. Different volumes of diluted Aza were initially tested to determine the appropriate method for incorporating the liquid Aza into the powdered sieved larval food suitable for first-instar larvae (L1).

To standardize the volume of Aza diluent that could be mixed into the L1 food, we aimed for a pasty texture that would allow the larvae to feed and develop normally. Te chosen mixture consisted of 35 mg of larval food plus 125 μl of 0.9% NaCl. Consequently, Aza and ecdysone final concentrations were 1 μg and 2 μg per milligram of L1 food, respectively. Tese concentrations were based on previous studies with Lutzomyia longipalpis [ 42]. Tis mixture was sufficient to feed approximately 200 L 1 larvae in a small plaster pot for 2 days.

Te experimental groups were as follows: (a) control group: L1 food plus 0.9% NaCl; (b) Aza group: L1 food plus 1 μg/mg of Aza; (c) Aza + Ecd group: L1 food containing 1 μg/mg of Aza plus 2 μg/mg of ecdysone; (d) Ecd group: L1 food containing 2 μg/mg of ecdysone. Each group consisted of three pots, each containing approximately 200 larvae. Each pot received L1 food equivalent to 35 mg of the dry powder mixture containing the specified chemicals, provided twice until the first molting to L 2 larvae was observed in the control group (around 15 days after larval hatching week from eggs).

Larval development was monitored daily, considering the size of larvae and the number of caudal setae (two in L1 and four in L2). Adult emergence was observed following standard colony maintenance. Te larvae were collected in pools of 15 individuals from each group at 1 and 7 days after first feeding (DAF) for subsequent gene expression analysis. Te larval collection was random, without distinction of larval stage, ensuring that the results reflected the overall impact of the treatments on the larval populations. Te experiments were conducted in triplicate.