Methods
Mosquitoes
We used larvae, pupae and adults  of Anopheles albimanus laboratory strain Sanarate maintained at x°C and x% relative humidity in the insectary of Centro de Estudios en Salud of Universidad del Valle de Guatemala, Guatemala
RNA extraction and cDNA production
RNA was extracted using a the SVTotal RNA isolation system (Promega) from 30mg of tissue of each stage of Anopheles albimanus, this is equal to 120 L1, 40 larvae L2, 30 larvae L3, 7 larvae L4, 5 pupae, 15 female adults and 25 male adults.  RNA quantity and purity was measured with Nanodrop One (Thermofisher). One microgram of Dnase I (Promega) treated RNA was used to generate the first strand of cDNA using the kit GoScript Reverse Transcription System with Random primers and Oligo(dT)15 Primer according to the manufacturer instructions. 
  Gene expression analysis
The mRNA expression levels for each stage was measured by qPCR with Power Up Sybr Green(Thermo Scientific,   ) in a LightCycler 96 (Roche). The following primers and concentrations were used: boule, (XuM),5’ 3’; zpg, (XuM),5’ 3’ and actin, (XuM),5’ 3’. The conditions of the RT-qPCR were as follow: XXX. To monitor primer specificity, melting curves were performed. Reaction were performed in triplicates using XuL cDNA diluted 1:10 in a total volume of 15uL per reaction. We used the Δ ΔCt method to evaluate the gene expression using the actin gene (cita) as a reference gene.
Statistical analysis