Crickets were searched in 13 places in Europe and found in 9 of them across a latitudinal gradient. Crickets were searched during day and night in urban and suburban areas, and all the adult individuals seen were collected. The wild plants available as food, the altitude, the temperature and the humidity were collected in the place of capture. The guts of both the crickets from the wild and the laboratory experiments were dissected for microbiome extraction and analyses. We are now processing 212 faecal samples and guts from the wild crickets from WP2 and 295 from WP4 following the standardised methods used in one of the projects of the host centre, the Earth Holobiome Initiative. This is basically based on extracting the DNA from the samples and preparing the library for shotgun analyses. We have also polished the laboratory protocol for vitamin B12 measurements, which has involved a series of steps (i.e. extraction, purification and measurement using ultra-high-performance liquid chromatography) to avoid biasing our results due to the presence of pseudovitamin B12 in these animals. In addition, we carried out an experiment where temperature and food conditions were manipulated in the laboratory. To do this, we reared the crickets born from industrial populations (F0) in 3 different temperatures (27, 30 and 33 C) and two diet conditions (low and high protein). The next generation of crickets (F1) was then reared and mated in each of their environmental conditions (Sample size 20 couples per treatment combination) and for females the number of eggs was counted. Finally, we dissected both male and female crickets for reproductive tissue measurements. We also dissected the guts for later genomic analyses and the rest of the animal was frozen for vitamin B12 analyses.
