Sperm quantification

In females with sperm present in their spermatheca, the sperm bundle was gently picked up with a dissecting pin and transferred to 500 µl of lysis mix combined with 10 µl of proteinase K and incubated overnight on the heat block at 55 °C. The next morning, DNA was extracted from it using a ChargeSwitch gDNA micro tissue kit (Invitrogen, Carlsbad, CA, USA) following the manufacturer’s instructions.

Sperm quantification was performed as described previously [24]. The number of sperm cells contained in the gDNA extracts from the spermathecae was estimated using a Taqman assay (Applied Biosystems, Foster City, CA, USA) targeting a known Y-chromosome-specific sequence [38]. The following primer and probe were used: forward (5′-TTA CCA CGC TGG CAA ATG C-3′); reverse (5′-CGT GCA ACA GCT CGT GAT G-3′); probe (5′-ACG CCG CAT CCA TCT-3′). Quantitative real-time PCRs were run using TaqMan Universal Master Mix (Applied Biosystems) on a Step-One-Plus Real Time PCR System (Applied Biosystems Foster City, CA, USA). PCR steps were: 50 °C for 2 min, 95 °C for 10 min, 95 °C for 0.15 s, and 60 °C for 1 min, for 30 cycles. Cycle threshold values (CT) were translated into number of Y-chromosome copies using the standard curve method and a pool of male mosquito DNA as standard. The concentration of the pool of male DNA was first adjusted to 50,000 haploid genome copies/ml using a theoretical approximate molecular weight of 0.3 pg per genome, i.e. 1.96e−21 g/bp and 278,253,050 bp [39]. Two independent five-step serial dilutions curves were made per PCR plate. The curve with the best fit was used to translate CT-values into estimated numbers of Y-chromosomes/ml, which were multiplied by two to account for female sperm (assuming a 1:1 male to female sperm ratio) and multiplied by the total volume of the DNA extraction from inseminated spermathecae (75 ml).