RNA extraction and cDNA production
Duplicates of thirty milligrams of tissue from each life stage of Anopheles albimanus were prepared to isolate RNA using a SV Total RNA Isolation System kit (Promega, Z3100). The amount of individuals used per life stage were:   120 larvae 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) and treated with RQ1 RNase-Free DNase (Promega, M6101). Treated RNA was used to generate the first strand of cDNA following protocol instructions by GoScript Reverse Transcription System kit (Promega, A5000).  Random and Oligo(dT)15 Primers were used in each cDNA synthesis according to the manufacturer instructions. 
Gene expression analysis 
The mRNA expression levels of  spermatogenesis orthologues genes AALB006050 (zpg) and AALB005972 (boll) were measured by qPCR (SYBR Green Master Mix 2X, Roche) using primers listed in Table 1 at 0.4uM and comparing expression levels with actin as an internal reference in a LightCycler 96 (Roche). Melt curve analysis were performed and confirmed only a single product was amplified with each primer pair. Primer efficiencies were calculated using Thermofisher qPCR Efficiency calculator online. Reactions were performed in triplicates using 2.5uL cDNA diluted 10 times fold in a total volume of 10uL per reaction. Analysis of gene expression was performed by using the 2-ddCt method.  
Statistical analysis
An Unpaired t test were performed with Graph Pad Prism (GraphPad Prism version 7 for Windows, GraphPad Software, La Jolla California USA, www.graphpad.com). All significant values have a p value less than 0.05.