Computational protocol: Queen reproductive tract secretions enhance sperm motility in ants

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Protocol publication

[…] Colonies of A. echinatior were excavated in Gamboa, Panama from 2001 to 2014 and reared in Copenhagen at 25°C and relative humidity 60–70%. Winged reproductives were collected shortly before each trial and checked for sexual maturity during dissection [] with watchmaker forceps in Hayes saline (see the electronic supplementary material for details).Accessory testes of 16 males were punctured to collect subsamples of outflowing sperm with a pipette tip previously loaded with 3 µl Hayes saline containing 375 µM of SYTO 13 (Molecular Probes) fluorescent dye (see the electronic supplementary material for details). Mixtures were gently pipetted into a counting chamber (SC-20-01-04-B, Leja), after which spermatozoa were observed with a spinning-disc confocal microscope (Revolution XD, Andor) at 20× magnification. The fluorescent dye was excited with a 488 nm laser and motility recorded at 30 frames per second with an Andor iXon DU-897-BV EMCCD camera. For each male, we obtained two 5 s recordings, between which we changed the field of vision within the same counting chamber, expecting that sperm motility parameters should remain similar unless measurements were affected by technical noise. We analysed recordings with the computer-assisted sperm analyser (CASA) plugin [] for ImageJ ( and assessed measurement repeatability with the R package rptR, method REML [,].Using the same methods, we obtained 5 s recordings of spermatozoa from 10 individual males (two replicate experiments) while swimming in: (i) reproductive tract fluid from a virgin queen collected from the same colony as the focal male, (ii) virgin queen fluid from an unrelated colony and (iii) Hayes saline as a control. To obtain female fluid, we dissected the bursa copulatrix and spermatheca of virgin queens (a), gently punctured these organs in 3 µl Hayes in a 0.2 ml PCR tube, centrifuged at 17 000g for 3 min, and transferred 1.5 µl supernatant (or Hayes only) into 2 µl Hayes containing SYTO 13 (375 µM final concentration) before using 3 µl aliquots as test fluids. Figure 1.Because CASA yields intercorrelated measures of sperm velocity, we performed a principal component analysis (JMP v. 12) incorporating curvilinear velocity (VCL), velocity on the average path (VAP) and straight-line velocity (VSL) and used the first principal component (PC1) as a proxy of overall sperm velocity. PC1, the proportion of motile sperm and sperm linearity (LIN) were subsequently used as dependent variables in linear mixed-effects models fitted by restricted maximum likelihood (see the electronic supplementary material for details). […]

Pipeline specifications

Software tools CASA, ImageJ
Applications Laser scanning microscopy, Microscopic phenotype analysis
Organisms Homo sapiens, Acromyrmex echinatior