Rearing Neanura muscorum Collembola

Leaf litter was collected in December 2013 from the Monash University, Clayton campus (Victoria, Australia; 37.9119 °S, 145.1317 °E). Invertebrates were extracted from litter using Berlese-Tullgren funnels39 . Neanura muscorum individuals (n = 31) were identified using morphological characteristics and placed into closed 70 ml containers with a plaster of Paris and charcoal (9:1) substratum. This substratum was regularly saturated with reverse osmosis treated water to maintain humidity40 . Containers were kept in temperature-controlled rooms set at 15 °C (mean/sd: 14.64 ± 0.61 °C; verified with iButton Hygrochron® temperature/humidity loggers, Maxim Integrated, San Jose, USA) on a 12:12 hour light:dark photoperiod.
Parental lines (F0) were provided with a combined diet of slime mould (Physarum polycephalum; cultured on 1.5% agarose media at 25 °C on a diet of oats) and algae-covered plane tree (Platanus sp.) bark ad libitum. The latter is a standard diet used in rearing other Collembola species, which enables individuals to select amongst a range of algae, cyanobacteria, and fungi to reach their optimal nutrient target12 13 27 . Collembola cultures were checked daily and eggs were removed to new containers and incubated in the temperature-controlled room until hatching. Four batches of first-generation (F1) eggs were used for experiments (Fig. 1). Subsequent clutches were combined and raised as a F1 breeding population under identical conditions to the parental line. Five batches of eggs laid by the F1 breeding population were then used to create F2 populations for a repetition of the experiments undertaken with the F1 generation (Fig. 1). Neanura muscorum F2 individuals that were not used for experimentation were pooled and kept as laboratory stocks. These stocks continue to be maintained on a combined slime mould and algae-covered plane tree bark diet.