Bacillus thuringiensis (Bt) produces Cry toxins that are widely used as biological pesticides and for genetically modified crops. Recent studies show that ATP-binding cassette transporter subfamilies C2 (ABCC2) and C3 (ABCC3), and the cadherin-like receptor function as susceptibility determinants for Cry1 toxins. However, the underlying mechanism of susceptibility determination for Cry toxins in insects is ambiguous. To investigate whether all the BmABCC2, BmABCC3 and cadherin-like receptor (BtR175) are susceptibility determinants for Cry1Aa, we performed Cry1Aa toxin diet overlay assays using Bombyx mori strains in which one or two of the putative receptors were disrupted by transcription activator-like effector nuclease-mediated gene editing. Whereas larvae with knockout of BmABCC2 or BmABCC3 individually showed no resistance to Cry1Aa, larvae with knockout of both BmABCC2 and BmABCC3 exhibited a high level of resistance to Cry1Aa. Analysis of a progeny population of BtR175 knockout homozygotes, which were crossed with heterozygotes for BtR175 knockout allele, revealed that knockout of this receptor resulted in moderate resistance to Cry1Aa. Using a cell swelling assay with Sf9 cells ectopically expressing the receptors, we estimated the susceptibility-conferring activities of the expression of BmABCC2, BmABCC3, or the toxin binding region of BtR175 (BtR175-TBR) alone, as well as co-expression of BtR175-TBR and BmABCC2, or BtR175-TBR and BmABCC3. The susceptibility-conferring ability of Sf9 cells only expressing BmABCC2 for Cry1Aa was higher than that of BmABCC3 expression alone. Using the same system, synergistic susceptibility conferring activity resulting from co-expression of BtR175-TBR and BmABCC3 was indicated to be almost comparable to that resulting from co-expression of BtR175-TBR and BmABCC2. In addition, a putative BtR175 knockout progeny population exhibited resistance, although BtR175-TBR showed low receptor activity when expressed in Sf9 cells. These results suggest that co-expression of BtR175-TBR and BmABCC2, as well as co-expression of BtR175-TBR and BmABCC3, act synergistically to confer susceptibility to Cry1Aa in B. mori larvae.

 Long-term cryopreservation of the Bombyx mori silkworm is currently performed using ovaries and mature sperm. These methods have been in practice in the cryopreservation of many silkworm strains. Development of multiple methods for the safe preservation of bioresources is desirable. In this study, we investigated the potential for silkworm testis cryopreservation. We show here that the viability of cryopreservation using testis varied among the strains. For viable long-term testis cryopreservation, increasing the success rate for low fertility strains is essential. We investigated a vitrification method for testis using aluminum cryo-plates. There was the possibility, however, more experiments are required to improve this vitrification method for silkworm testis cryopreservation.