Transcriptome validation and RT-PCR

Validation of RNA-seq was performed using RT-PCR to assess the expression profiles of the eight selected transcripts (Additional file 1: Table S5), following the bioinformatics analysis of the spermathecal transcriptome. Here, the virgin and the inseminated spermathecae were obtained, as before, from Ae. aegypti (Rockefeller strain) available in the Department of Immunology and Microbiology (DIM) at the Johns Hopkins University (Baltimore, MD). Following hatching in distilled water, larvae were fed with Cat Chow (Purina®) and maintained under the conditions indicated above. Another group of females was again separated into two groups (virgin and inseminated) and dissected 7 days after emergence. One hundred virgin and inseminated females (300 spermathecae each) were dissected in RNAse-free 0.1 M PBS (pH 7.6), and the total RNA was extracted using TRIzol® (Invitrogen, Carlsbad, CA). To tease out gene expression in sperm present in the spermathecae, expression levels of the same selected transcripts were also assessed in the luminal content of the spermathecae from inseminated females. For this, reservoirs of 100 inseminated females were disrupted by hand (using forceps), transferred to 1.5 mL microtubes, containing 100 μL of PBS solution, and centrifuged at low speed (below 3000 rpm/956 g for 10 s) to avoid cell damage. The supernatant with the reservoir contents was subjected to total RNA extraction, cDNA synthesis, and expression quantification together with the virgin and the inseminated spermathecae.

To confirm spermatheca-specific profile, the expression profile of each of the eight selected transcripts was also assessed in carcasses (i.e., the body without guts and ovaries), midguts, and ovaries from virgin (non-blood fed) and inseminated (blood fed) females. Carcasses, midguts, and ovaries were collected from 5-day old virgin or inseminated females. Developed ovaries were collected 2 days after the blood meal following feeding on an anesthetized mouse, and as described in the RNAi experiments section below. Total RNA was extracted separately for each sample belonging to the two pools (virgin vs. inseminated), and each pool contained tissues from ten females. Each RNA sample was treated with DNAse I (Invitrogen), precipitated in ethanol/ammonium acetate solution, and finally suspended in RNAse-free water. After RNA quantification (using NanoDrop Lite Spectrophotometer, Thermo Fisher Scientific), 1st strand cDNA was obtained for each sample using Superscript III (Invitrogen) with random hexamers (Thermo Fisher Scientific) and 500 ng of RNA per sample. cDNAs were treated with RNA H (New England Biolabs) for 10 min at 37 °C, and stored at − 70 °C until use.

Relative gene expression profiles were assessed in real time, using SYBR Green PCR Master Mix (Applied Biosystems, Thermo Fisher Scientific) in 20 μL reactions containing 300 nM of each primer, and 100 ng each cDNA. PCR were run using MicroAmp® Fast Optical 96-Well Reaction Plate with Barcode (0.1 mL) (Applied Biosystems, Life Technologies) in the StepOne™ Real-Time PCR System (Applied Biosystems, Life Technologies). The amplification conditions were 94 °C for 2 min, 94 °C for 15 s, 60 °C for 1 min, 95 °C for 15 s, 60 °C for 1 min, and 95 °C for 15 s. Each reaction was performed in triplicate using two biological replicates.

The relative expression profiles were determined using Real Time Quantitative PCR and the 2^-ΔΔCt Method [65]. The ribosomal protein S7 gene (AAEL009496) was used as an endogenous reference, and the results were normalized using the virgin spermathecae control group.