A toxin complex protein from Photorhabdus akhurstii conferred oral insecticidal activity against Galleria mellonella by targeting the midgut epithelium

The nematode-bacterium pair Heterorhabditis indica-Photorhabdus akhurstii is a malleable model system to investigate mutualistic relations. A number of toxins produced by P. akhurstii allow the bacterium to kill the insect host. However, a few of these heterologously expressed toxins are orally active against different insects which possibly caused neglected attention to Photorhabdus toxins compared to Bt (Bacillus thuringiensis). In the current study, a functional subunit of orally active toxin complex (Tc) protein, TcaB (63 kDa), isolated from two strains of P. akhurstii namely IARI-SGHR2 and IARI-SGMS1, was tested for biological activity against Galleria mellonella. A force feeding-based administration of the toxin translated into LD50 values of 45.63-58.90 ng/g which was even lower compared to injection LD50 values (51.48-64.30 ng/g) at 48 h after inoculation. An oral uptake of 500 ng toxin caused extensive gut damage in G. mellonella during 6-24 h incubation period coupled with a gradual disruption of gut integrity leading to escape of TcaB into the hemocoel. This finding was supported by the cytotoxic and immune-stimulatory effect of TcaB in the insect hemocoel at 6-24 h after force feeding. The circulatory hemocyte numbers and cell viability was markedly reduced to 0.66-0.68 x 10(6) ml(-1) and 49-52 %, respectively, in TcaB force fed insect at 24 h, compared to control (2.55 x 10(6) ml(-1); 100 %). The hemolymph phenoloxidase (PO) activity was elevated by 10.2-fold in force fed larvae than control at 24 h. An in silico docking study revealed that TcaB putatively interacts with a number of G. mellonella receptor proteins in order to become a gut-active toxin. Present research reinforces the potential of gut-active Photorhabdus toxins for their inclusion in sustainable insect management tactics and strengthens the existing Bt-dominated management repository.