Abstract
Insecticidal effects of Bacillus thuringiensis Cry toxins in hemocoel of larvae have not been properly evaluated. In the present study, hemocoelic injection of four representative Cry toxins i.e., Cry1Aa, Cry1Ab, Cry1Ac, and DOR5 to an economically important lepidopteran insect pest Achaea janata, induced larval mortality, reduced larval growth rate and gave rise to smaller pupae, all in a dose-dependent manner. We observed extensive degeneration as well as the disintegration of larval tissues, most notably, fat body, and the possible involvement of lysosomal enzymes in tissue histolysis. The resultant “hypoproteinemia” and most relevantly, the drastic reduction of 80–85 kDa hexamerin proteins levels of hemolymph could be attributed to the pathological state of the fat body induced by Cry toxin injection. Formation of non-viable larval-pupal intermediates and emergence of defective adults also indicate toxicity effects of Cry toxins during metamorphosis. Thus, findings from our study suggest Cry toxins in larval hemocoel are also toxic to A. janata larval survival and subsequent development.
Highlights
Insecticidal proteins called crystal (Cry) toxins, produced as crystal inclusions by gram-positive bacteria Bacillus thuringiensis (Bt), are toxic to a wide range of insects including those belonging to the order Lepidoptera (Bravo et al, 2011)
Feeding bioassay with 2 days old A. janata larvae showed that recombinant Cry toxins i.e., Cry1Aa, Cry1Ab, and Cry1Ac feeding causes larval mortality, DOR5 Cry toxins showed much higher insecticidal activity with fairly low LC50 (4.9 ng/cm2 of leaf surface) (Table 1)
Hemocoelic injection with doses of 200 ng/0.2 g body weight of Cry1Aa (Figure 1A), 150 ng/0.2 g body weight each of Cry1Ab (Figure 1B) and Cry1Ac (Figure 1C), and 100 ng/0.2 g body weight of DOR5 (Figure 1D) toxins resulted in 50–70% larval mortality
Summary
Insecticidal proteins called crystal (Cry) toxins, produced as crystal inclusions by gram-positive bacteria Bacillus thuringiensis (Bt), are toxic to a wide range of insects including those belonging to the order Lepidoptera (Bravo et al, 2011). Development of Bt-based sprayable products and Bt transgenic crops have provided valuable alternatives to chemical insecticides as they are more insect species-specific and least harmful to the natural enemies (James, 2009; Bravo et al, 2011). As a consequence of the uncontrolled widespread use of Bt-based biopesticides in agriculture, a number of insect species including those belonging to Lepidoptera have developed resistance to Bt Cry toxins (Ferré and Van Rie, 2002; Tabashnik et al, 2005, 2013). As an Abbreviations: 5E, early fifth instar; 5L, late fifth instar; ACP, acid phosphatase; ALP, alkaline phosphatase; APN, aminopeptidase N; BCIP, 5-bromo-4-chloro-3′-indolyl phosphate; BSA, bovine serum albumin; Bt, Bacillus thuringiensis; GPI, glycosylphosphatidyl-inositol; IgG, immunoglobulin G; NBT, nitro blue tetrazolium chloride; PP, pre-pupa; SDS-PAGE, sodium dodecyl sulfate polyacrylamide gel electrophoresis
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