RESUMEN
Background: A multitude of Cry toxins (secreted by Bacillus thuringiensis or Bt) has been deployed globally either via transgenic mean or bio-pesticidal formulations in order to manage insect pests. However, Bt resistance development in insects is emerging as a major concern. To avoid this problem, multiple gene pyramiding or protein-engineered chimeric toxin-based strategy has been analyzed. Methods: In the present study, one such chimeric toxin Cry1AcF (contain the swapped domains of Cry1Ac and Cry1F) was used to investigate its in vivo pathogenesis process in lepidopteran pests Spodoptera frugiperda and S. litura. A number of biochemical and molecular analysis were performed. Results: Oral ingestion of Cry1AcF caused greater toxicity in S. frugiperda than S. litura with larvae displaying increased hemolymph melanization. Histopathology of the midgut transverse sections exhibited Cry1AcF-induced extensive gut damage in both the test insects followed by cytotoxicity in terms of reduced hemocyte numbers and viability. Elevated hemolymph phenoloxidase activity indicated the immune-stimulatory nature of Cry1AcF. In order to analyze the role of gut receptor proteins in Cry1AcF intoxication in test insects, we performed RNAi-mediated silencing using bacterially-expressed dsRNAs of individual receptor-encoding genes including CAD, ABCC2, ALP1 and APN. Target-specific induced downregulation of receptor mRNAs differentially altered the insect susceptibility to Cry1AcF toxin in our study. The susceptibility of ALP1 and APN dsRNA pre-treated S. frugiperda was considerably decreased when treated with Cry1AcF in LD50 and LD90 doses, whereas susceptibility of CAD and ABCC2 dsRNA pre-treated S. litura was significantly reduced when ingested with Cry1AcF in different doses. CAD/ABCC2-silenced S. frugiperda and ALP1/APN-silenced S. litura were vulnerable to Cry1AcF alike of control larvae. In conclusion, our results indicate ALP1/APN and CAD/ABCC2 as the functional receptor for Cry1AcF toxicity in S. frugiperda and S. litura, respectively.
Asunto(s)
Inmunotoxinas , Animales , Spodoptera/genética , Larva/genética , Inmunotoxinas/genética , Interferencia de ARN , Proteínas Bacterianas/genética , Proteína 2 Asociada a Resistencia a Múltiples MedicamentosRESUMEN
Crystal (Cry) toxins produced from the soil bacterium, Bacillus thuringiensis (Bt), have gained worldwide attention for long due to their insecticidal potential. A number of receptor proteins located on the epithelial cells of the larval midgut were shown to be crucial for Cry intoxication in different insect pests belonging to order Lepidoptera, Diptera and Coleoptera. A beehive pest, Galleria mellonella, serves as an excellent insect model for biochemical research. However, information on the Cry receptor-like genes in G. mellonella is limited. In the present study, the full-length sequences of four putative Cry receptor genes (ABC transporter, alkaline phosphatase, aminopeptidase N and cadherin) were cloned from G. mellonella. All these receptor genes were substantially upregulated in the midgut tissue of fourth-instar G. mellonella larvae upon early exposure (6 h) to a sub-lethal dose of Cry1AcF toxin. Oral and independent delivery of bacterially-expressed dsRNAs corresponding to four receptor genes in G. mellonella suppressed the transcription of target receptors which in turn significantly reduced the larval sensitivity to Cry1AcF toxin. As the laboratory populations of G. mellonella develop Bt resistance in a relatively short time, molecular characterization of Cry receptor genes in G. mellonella performed in the present study may provide some useful information for future research related to the genetic basis of Bt resistance in the model insect.
Asunto(s)
Bacillus thuringiensis , Mariposas Nocturnas , Animales , Bacillus thuringiensis/química , Bacillus thuringiensis/genética , Proteínas Bacterianas/química , Endotoxinas/metabolismo , Endotoxinas/farmacología , Proteínas Hemolisinas/genética , Proteínas Hemolisinas/metabolismo , Proteínas Hemolisinas/farmacología , Proteínas de Insectos/genética , Proteínas de Insectos/metabolismo , Insectos/metabolismo , Larva/genética , Larva/metabolismo , Mariposas Nocturnas/genética , Mariposas Nocturnas/metabolismo , Receptores de Superficie CelularRESUMEN
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 × 106 ml-1 and 49-52 %, respectively, in TcaB force fed insect at 24 h, compared to control (2.55 × 106 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.