Prakash Y Shankhu1, Chetna Mathur1, Abhishek Mandal2, Doddachowdappa Sagar3, Vishal S Somvanshi1, Tushar K Dutta1. 1. Division of Nematology, ICAR - Indian Agricultural Research Institute, New Delhi, India. 2. Division of Agricultural Chemicals, ICAR - Indian Agricultural Research Institute, New Delhi, India. 3. Division of Entomology, ICAR - Indian Agricultural Research Institute, New Delhi, India.
Abstract
BACKGROUND: Txp40, a 37 kDa protein, previously characterized from the Gram-negative bacterium Photorhabdus akhurstii (symbiotically associates with insect-parasitic nematode, Heterorhabditis indica), conferred insecticidal activity against Galleria mellonella. Here, the biological activity of Txp40 was evaluated against economically important insects, including Helicoverpa armigera, Spodoptera litura and S. exigua. RESULTS: When both intra-hemocoel injected and orally fed to test insects, comparatively greater oral LD50 (187.7-522 ng g-1 ) than injection LD50 (32.33-150.6 ng g-1 ) was obtained with Txp40 derived from P. akhurstii strain IARI-SGMG3. Injection of purified Txp40 caused a dose-dependent reduction in the total circulatory hemocytes and hemocyte viability of fourth-instar larvae of the test insects at 12 h post incubation; unlike healthy cells toxin-treated ones displayed aggregated distribution. Injection of Txp40 significantly elevated the phenoloxidase activity of insect hemolymph, which potentially led to unrestrained melanization reaction and ultimately larval death. Histological analyses showed the primary site of action of Txp40 in the insect midgut. Extensive damage to midgut epithelium 24 h after injection of the Txp40 explains the access of the toxin from hemocoel to midgut via leaky septate junctions. In silico analyses suggested that Txp40 can potentially interact with H. armigera midgut receptor proteins cadherin, ATP-binding cassettes, aminopeptidase N1 and alkaline phosphatase to exert toxicity. CONCLUSION: We propose Txp40 as an attractive alternative to Cry toxins of Bacillus thuringiensis, the transgenic expression of which is reported to cause resistance development in insects.
BACKGROUND: Txp40, a 37 kDa protein, previously characterized from the Gram-negative bacterium Photorhabdus akhurstii (symbiotically associates with insect-parasitic nematode, Heterorhabditis indica), conferred insecticidal activity against Galleria mellonella. Here, the biological activity of Txp40 was evaluated against economically important insects, including Helicoverpa armigera, Spodoptera litura and S. exigua. RESULTS: When both intra-hemocoel injected and orally fed to test insects, comparatively greater oral LD50 (187.7-522 ng g-1 ) than injection LD50 (32.33-150.6 ng g-1 ) was obtained with Txp40 derived from P. akhurstii strain IARI-SGMG3. Injection of purified Txp40 caused a dose-dependent reduction in the total circulatory hemocytes and hemocyte viability of fourth-instar larvae of the test insects at 12 h post incubation; unlike healthy cells toxin-treated ones displayed aggregated distribution. Injection of Txp40 significantly elevated the phenoloxidase activity of insect hemolymph, which potentially led to unrestrained melanization reaction and ultimately larval death. Histological analyses showed the primary site of action of Txp40 in the insect midgut. Extensive damage to midgut epithelium 24 h after injection of the Txp40 explains the access of the toxin from hemocoel to midgut via leaky septate junctions. In silico analyses suggested that Txp40 can potentially interact with H. armigera midgut receptor proteins cadherin, ATP-binding cassettes, aminopeptidase N1 and alkaline phosphatase to exert toxicity. CONCLUSION: We propose Txp40 as an attractive alternative to Cry toxins of Bacillus thuringiensis, the transgenic expression of which is reported to cause resistance development in insects.