Gryllus locorojo or Gryllus assimilis? – PCR-based differentiation of two commonly confused crickets for pet and livestock feeding

A real-time polymerase chain reaction (PCR)-based protocol (Gloco-PCR) was validated to specifically detect Gryllus locorojo, a Gryllus species on the European market often mistaken for Gryllus assimilis. Whereas the latter species is allowed in the EU for feeding farmed animals, G. locorojo is only permitted for pets according to the current legislation. The method was developed on the basis of the cytochrome oxidase I gene, (COI), which was sequenced with thoroughly characterised G. locorojo and G. assimilis samples. The method is highly sensitive, detecting 0.8 pg G. locorojo-DNA or 0.1% G. locorojo incurred in feed, respectively. Authentic G. assimilis specimens were used to ensure that the G. locorojo method (Gloco-PCR) discriminates this closely related sister taxon, with a comfortable Ct-difference of 10-15. For cross analysis of true G. assimilis, similar primers with another probe were employed (Gassim-PCR) and the annealing temperature was increased from 60 °C to 62 °C. Under these conditions, authentic G. assimilis crickets were detectable with Ct-values around 20, while G. locorojo samples showed a low detection at cycles around Ct 35. An investigation of ten ‘G. assimilis’ samples collected from Germany and four other European countries revealed that all of them were of the G. locorojo type. This proves the usefulness of our approach and supports the assumption that many G. assimilis crickets marketed in the EU indeed belong to the species G. locorojo. Consequently, European legislation, currently based on a white list of allowed insect species, is critically questioned.