Mechanisms Underlying Tolerant Difference Between Helicoverpa Armigera And Spodoptera Exigua To Cry1ca And Cry1Ac

The genetically modified cotton varieties expressing the gene that encode Cry1Ac toxin have been planted for many years in China. These varieties could effectively control the target pests, such as Helicoverpa arimigera and Pectinophora gossypiella, and consequently increased cotton yield, quality and the incomes of cotton farmers, reduced the environmental pollution and prevented pesticide poisoning. However, the damage of some non-target pests, such as Spodoptera exigua, has become more and more serious. One of the main reasons is that most of Cry toxins of transgenic cotton are modified by Cry1Ac gene, whose products have little effecacy on S. exigua larvae. It has been widely documented that Cry1Ab, Cry1Ca and CrylBa had significantly higher toxicities to S. exigua larvae than Cry1Ac. Among them Cry1Ca exhibited the highest toxicity and should be an ideal toxin to control S. exigua larvae.Tolerant levels of pests to various Cry toxins may be affected by several facts. For example, solubilization of protoxins in midgut lumen, activation of protoxins by midgut proteases, further detoxification of toxins by proteases, binding to primary receptors after traversing the peritrophic membrane, insertion into the cell membrane, formation of pores and interection with microorganism in midgut may influence toxic effects and tolerant levels. In the present paper, we compared the interections of Cry toxins with their receptors, midgut proteases and microorganism in Helicoverpa armigera and S. exigua larvae. We tried to reveal the mechanisms underlying tolerant differences in toxins. The main results are summarized as follows.1. Interactions of cadherins from H. armigera and S. exigua with Cry1Ca and Cry1AcUsing transcriptome data and RACE, a full-length cDNA encoding cadherin was obtained from S. exigua and named as SeCad2. The sequence analysis proved that SeCad2shared59%of similarity to the cadherin from H. armigera (HaBtR). Three conservative toxin binding sites were found in HaBtR and SeCad2. The3rd binding site of HaBtR and SeCad2had amino acid sequence of1423GVLSLNFQ1430and1436GVISLNFQ1443 respectively, leucine-1425(L) in HaBtR was replanced by isoleucine-1438(I) in SeCad2. We expressed HaBtR and SeCad2fragments containing the three toxin binding sites in vitro. Western blot, dot-blot and the synergistic effects analysis proved that SeCad2prefered binding with Cry1Ca, whereas HaBtR prefered binding with Cry1Ac. It was consistent with the difference in sensitivity among H. armigera and S. exigua larvae to Cry1Ca and CrylAc. So we concluded that there were great relationships between the binding preference of cadherins and the toxicities of Cry toxins.2. Molecular cloning and characterization of alkaline phosphatase in S. exiguaA full-length cDNA encoding alkaline phosphatase in S. exigua was cloned and characterized. This gene, named as ALP I, highly expressed in the midgut of the larvae. We compared the binding capacity between SeALP1and Cry1Ca or Cry1Ac. As expected, SeALP1prefered binding with Cry1Ca, but not Cry1Ac.3. Influence of midgut proteases on toxic effects of Cry toxinsEnzyme preparations from midguts of H. armigera and S. exigua could detoxificated CrylCa and CrylAc. However, no significant difference in enzyme activity was found between the preparations. Several protease inhibitors, when used at an appropriate amount, could enhance the Cry toxin toxicity.4. Penicillin ingestion influences toxic effects of several Cry toxins on S. exiguaToxicities of CrylAbs CrylBa and CrylCa from Bacillus thuringiensis to the2nd-instar larvae of Spodoptera exigua were evaluated. The LC50values were0.4425,0.6757and0.1502μg/cm2respectively. Cry1Ba was least toxic, followed by Cry1Ab, and CrylCa was most toxic. Penicillin affected larval tolerance to the3Cry toxins. At the concentration of500μg/cm2, penicillin was mixed directly with the toxins could significantly increase larval tolerance. At the concentration of60μg/cm2, however, penicillin must be first added alone to the diet and fed the larvae several generations, in order to obtain higher tolerance than control larvae.In summary, our results indicated that the interactions between Cry toxins and their recepters resulted in the tolerant difference between H. armigera and S. exigua larvae. The effects of other factors need further researches to confirm.