Species-specific evolution of lepidopteran TspC5 tetraspanins associated with dominant resistance to Bacillus thuringiensis toxin Cry1Ac

● TspC5 tetraspanins show species-specific evolution among lepidopteran insects, suggesting potential functional divergence. ● L31S mutants of Helicoverpa and Heliothis TspC5s mediate Cry1Ac resistance, highlighting a conserved role in conferring Bt resistance. ● piggyBac -mediated transformation combined with genome resequencing allows for the rapid establishment of homozygous transgenic insect strains. Transgenic crops producing insecticidal proteins from the bacterium Bacillus thuringiensis (Bt) have proven to be highly effective in managing some key pests. However, the evolution of resistance by the target pests threatens the sustainability of Bt crops. The L31S mutation in a tetraspanin encoded by HarmTspC5 (previously known as HaTSPAN1 ) has been shown to confer dominant resistance to the Bt protein Cry1Ac in Helicoverpa armigera , a globally damaging lepidopteran pest. However, the broader implications of the L31S mutation in the tetraspanins of other lepidopteran species remain unclear. The evolutionary analyses in this study indicate that TspC5s have evolved in a species-specific manner among the lepidopteran insects. To investigate the role of TspC5s in conferring dominant resistance to Cry1Ac, we used the piggyBac -based transformation system to generate four transgenic H . armigera strains that express exogenous TspC5 variants from three phylogenetically close species ( Helicoverpa zea , Helicoverpa assulta and Heliothis virescens ) and one phylogenetically distant species ( Plutella xylostella ). In comparison with the background SCD strain of H . armigera , the transgenic strains expressing HzeaTspC5-L31S, HassTspC5-L31S, or HvirTspC5-L31S exhibited significant resistance to Cry1Ac (10.0-, 21.4-, and 81.1-fold, respectively), whereas the strain expressing PxylTspC5-L27S remained susceptible. Furthermore, the Cry1Ac resistant phenotypes followed an autosomal dominant inheritance pattern and were closely linked to the introduced mutant TspC5s . These findings reveal the conserved role of TspC5s from Helicoverpa and Heliothis species in mediating the dominant resistance to Cry1Ac, and they provide crucial insights for assessing resistance risks related to mutant tetraspanins and devising adaptive resistance management strategies for these major lepidopteran pests.