Safeseal micro tube

Endocrinological response to the TAP was measured using salivary cortisol and testosterone. Saliva samples were taken twice prior to the TAP and three times following aggression induction (Fig. 1). Saliva for cortisol analysis was collected using Salivette sampling devices (Sarstedt, Numbrecht, Germany). Saliva for testosterone analysis was sampled in SafeSeal micro tubes (Sarstedt, Numbrecht, Germany), because collection with the Salivette cotton swabs may introduce artifacts in the analyses of testosterone^{38 (link)}. To limit the influence of diurnal variation on hormonal levels, all experimental procedures and samplings were performed in the afternoon between 3:00 and 6:00 PM^{39 (link),40 (link)}. Participants were instructed to refrain from drinking and eating for at least half an hour and from physical exercise and smoking two hours before the experiment. Saliva samples were stored uncentrifuged at −20 °C until assay. Cortisol and testosterone levels were assayed at the Department of Psychology of the Dresden University of Technology by using a luminsescence immunoassay, with a lower limit of detection of 1.8 pg/ml for testosterone and 0.276 nmol/L for cortisol. The mean and intra- and inter-assay coefficients of variation were 8% for both hormones.

Fourteen days post infection, the mosquitoes were immobilized with CO₂ and the legs and wings of each mosquito were removed. Next, mosquito saliva was collected by inserting the mosquito proboscis into a 200 µL pipet tip containing 5 µL of a 50% FBS and 25% sugar solution in sterilized tap water. After 45 min, the mosquito bodies were stored at −80 °C in individual SafeSeal micro tubes (Sarstedt) containing 0.5 mm zirconium oxide beads (Next Advance). Individual mosquito saliva samples were mixed with 55 µL DMEM HEPES complete and stored at −80 °C.

Four batches of 5 flies each were mixed with 700 µl MTBE/methanol (10/3, v/v) in 2 ml safe-seal micro tubes (Cat#: 72.695.500, Sarstedt, Nürmbrecht, DE) and disrupted with a metal bead (5 mm diameter; Cat#: 504942, Askubal Korntal-Münchingen, DE) in a Retsch MM 400 mixer mill (3 min, 30 Hz, 4°C) and lipids were extracted by shaking for 20 min at 1400 rpm and 4°C in a ThermoMixer (Eppendorf, Hamburg, DE). After the addition of 200 µl H₂O, samples were again incubated 20 min at 1400 rpm and 4°C. Phase separation was induced by centrifugation for 10 min at 16,000 x g and 4°C. The upper organic phase was collected and dried under a stream of nitrogen. Lipids were washed by dissolving in 500 µl chloroform/methanol (2/1, v/v) and again dried under a stream of nitrogen and stored at −20°C or prepared immediately for LC-MS analysis. For MS analysis, lipid extracts were dissolved in 0.5 ml chloroform/methanol (2/1, v/v), 50 µl of the sample were diluted with 100 µl isopropanol and 10 µl were injected for analysis using UPLC-QTOF-MS as described (Knittelfelder et al., 2014 (link)). Data analysis was performed using the Lipid Data Analyzer (LDA) software (Hartler et al., 2011 (link)). The abundance of each TG species was normalized to the intensity of the internal standard TG 51:0 (Larodan, Solna, SWE) and to animal number.