Toll signalling promotes blastema cell proliferation during cricket leg regeneration via insect macrophages.

Hemimetabolous insects, such as the two-spotted cricket Gryllus bimaculatus, can recover lost tissues in contrast to the limited regenerative abilities in human tissues. Following cricket leg amputation, the wound surface is covered by the wound epidermis, and plasmatocytes, which are insect macrophages, accumulate in the wound region. Here, we studied the function of Toll-related molecules identified by comparative RNA-seq during leg regeneration. Among 11 Toll genes in the Gryllus genome, expression of Gb'Toll2-1, Gb'Toll2-2, and Gb'Toll2-5 was upregulated during regeneration. RNA interference (RNAi) of Gb'Toll, Gb'Toll2-1, Gb'Toll2-2, Gb'Toll2-3, or Gb'Toll2-4 produced regeneration defects in more than 50% of crickets. RNAi of Gb'Toll2-2 decreased the ratios of S and M phase cells, expression of JAK/STAT signalling genes, and accumulation of plasmatocytes in the blastema. Depletion of plasmatocytes in crickets using clodronate also produced regeneration defects, along with reduced proliferating cells in the regenerating legs. Plasmatocyte depletion also downregulated the expression of Toll and JAK/STAT signalling genes in the regenerating legs. These results suggest that Spz-Toll-related signalling in plasmatocytes promotes leg regeneration through blastema cell proliferation by regulating the Upd-JAK/STAT signalling pathway.