Mettler ae163

The aphid clone used was A. pisum LL01, a long-established alfalfa-collected clone containing only the primary endosymbiont B. aphidicola. Aphids were maintained on young broad bean plants (Vicia faba L. cv. Aguadulce) at 21 °C, with a photoperiod of 16 h light–8 h dark to obtain strictly parthenogenetic aphid matrilines, that were reared and synchronized as previously described [20 (link)].
For toxicity analyses, three groups of ten 1st instar nymphs (aged between 0 and 24 h) were collected and placed in ad hoc feeding chambers containing an AP3 artificial diet [49 (link)] supplemented with different doses (5 µM to 80 µM) of solubilized BCR4 peptide. Toxicity was evaluated by scoring survival daily over the whole nymphal life of the pea aphid (7 days) [50 (link)]. Growth was also measured by weighing adult aphids on a Mettler AE163 analytical microbalance (Mettler Toledo, Columbus, OH, USA) at the closest 10 µg, following the protocol described previously [51 (link),52 (link)].
Aphid mortality data for all BCR4 concentrations were analyzed separately in a parametric survival analysis with a log-normal fit. Aphid weights were analyzed by ANOVA followed by Tukey–Kramer HSD test for comparing multiple means. All analyses were performed with JMP software version 11 (SAS Institute Cary USA, MacOS version).

After the initial intramuscular bolus injection of anesthetic, the animals were weighed prior to the experiment. This initial weight was used for comparison to repeated weighing immediately after the cessation of the experiment.
The animals were killed 2 h after rewarming to 36°C (bladder), and 1–3 g tissue samples from brain, heart, lung, liver, kidney, gut, and muscle (long adducting muscle) were harvested and immediately put in liquid nitrogen. Later, the sample wet weight was determined (Mettler AE 163, Mettler-Toledo AG, Greifensee, Switzerland). After drying in incubator at 36°C for 24 h, the dry weight was determined, and dry/wet ratio was calculated.