Insecticidal activity of a basement membrane-degrading protease against Heliothis virescens (Fabricius) and Acyrthosiphon pisum (Harris)

Department of Entomology, Iowa State University, 418 Science II, Ames, IA 50011-3222, USA.
Journal of Insect Physiology (Impact Factor: 2.47). 06/2008; 54(5):777-89. DOI: 10.1016/j.jinsphys.2008.02.008
Source: PubMed


ScathL is a cathepsin L-like cysteine protease derived from the flesh fly Sarcophaga peregrina that functions in basement membrane (BM) remodeling during insect development. A recombinant baculovirus expressing ScathL (AcMLF9.ScathL) kills larvae of the tobacco budworm, Heliothis virescens, significantly faster than the wild-type virus. Here, we show that the occurrence of larval melanization prior to death was closely associated with the onset of high cysteine protease activity of ScathL in the hemolymph of fifth instars infected with AcMLF9.ScathL, but not with AcMLF9.ScathL.C146A, a recombinant baculovirus expressing a catalytic site mutant of ScathL. Fragmented fat body, ruptured gut and malpighian tubules, and melanized tracheae were observed in AcMLF9.ScathL-infected larvae. Phenoloxidase activity in hemolymph was unchanged, but the pool of prophenoloxidase was significantly reduced in virus-infected larvae and further reduced in AcMLF9.ScathL-infected larvae. The median lethal dose (LD(50)) for purified ScathL injected into fifth-instar H. virescens was 11.0 microg/larva. ScathL was also lethal to adult pea aphids, Acyrthosiphon pisum with a similar loss of integrity of the gut and fat body. Injection with purified ScathL.C146A or bovine trypsin at 20 microg/larva did not produce any effect in either insect. These results illustrate the potent insecticidal effects of ScathL cysteine protease activity and the potential for use of ScathL in development of insect resistant transgenic plants when combined with an appropriate delivery system.

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Available from: Bryony Bonning, Oct 04, 2015
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    • "In response to bacteria, for example, our previous study shows that PO-catalyzed oxidation of dopamine generates reactive compounds that immobilize, aggregate, and kill Escherichia coli and Bacillus subtilis (Zhao et al., 2007). Against viruses, Trudeau et al. (2001) reported that Helicoverpa zea, a semipermissive host of Autographa californica multi-capsid nucleopolyherdrovirus (AcMNPV), melanizes infection foci, whereas this response was not observed in the fully permissive host Heliothis virescens due to AcMNPV reducing proPO levels in the hemolymph (Li et al., 2008). Shelby and Popham (2006) elaborated on these results by showing that PO inhibitors abolish the virucidal activity of H. virescens plasma against a baculovirus, while Clarke and Clem (2002) report that hemocytes also affect spreading of AcMNPV in Trichoplusia ni and Spodoptera frugiperda. "
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    ABSTRACT: Phenoloxidase (PO) and its activation system are implicated in several defense responses of insects. Upon wounding or infection, inactive prophenoloxidase (proPO) is converted to active PO through a cascade of serine proteases and their homologs. PO generates reactive compounds such as 5,6-dihydroxyindole (DHI), which have a broad-spectrum antibacterial and antifungal activity. Here we report that DHI and its spontaneous oxidation products are also active against viruses and parasitic wasps. Preincubation of a baculovirus stock with 1.25 mM DHI for 3 h near fully disabled recombinant protein production. The LC₅₀ for lambda bacteriophage and eggs of the wasp Microplitis demolitor were 5.6 ± 2.2 and 111.0 ± 1.6 μM, respectively. The toxicity of DHI and related compounds also extended to cells derived from insects that serve as hosts for several of the aforementioned pathogens. Pretreatment of Sf9 cells with 1.0 mM DHI for 4 h resulted in 97% mortality, and LC₅₀ values of 20.3 ± 1.2 μM in buffer and 131.8 ± 1.1 μM in a culture medium. Symptoms of DHI toxicity in Sf9 cells included DNA polymerization, protein crosslinking, and lysis. Taken together, these data showed that proPO activation and DHI production is strongly toxic against various pathogens but can also damage host tissues and cells if not properly controlled.
    Insect biochemistry and molecular biology 05/2011; 41(9):645-52. DOI:10.1016/j.ibmb.2011.04.006 · 3.45 Impact Factor
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    • "Harrison et al. [38] showed a 49% reduction in survival time of neonate H. virescens when infected with a AcMNPV recombinant containing the ScathL gene under the control of the p6.9 promoter (AcMLF9.ScathL) when compared to the wild type AcMNPV. Furthermore, Li et al. [66] have shown that purified ScathL was able to kill insects in the absence of baculovirus infection by injecting the protease into the hemocoel. The difference in larval survival time from the work by Harrison et al. [38] and this work, might be due to the diferent promoters used for the expression of the ScathL gene and the different viral susceptibilty of the insects tested, since S. frugiperda has been shown to be 1000 × less susceptible to AcMNPV by oral inoculaton when compared to the more susceptible T. ni larvae [67]. "
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    ABSTRACT: Baculovirus comprise the largest group of insect viruses most studied worldwide, mainly because they efficiently kill agricultural insect pests. In this study, two recombinant baculoviruses containing the ScathL gene from Sarcophaga peregrina (vSynScathL), and the Keratinase gene from the fungus Aspergillus fumigatus (vSynKerat), were constructed, and their insecticidal properties analysed against Spodoptera frugiperda larvae. Bioassays of third-instar and neonate S. frugiperda larvae with vSynScathL and vSynKerat showed a decrease in the time needed to kill the infected insects when compared to the wild type virus. We have also shown that both recombinants were able to increase phenoloxidase activity in the hemolymph of S. frugiperda larvae. The expression of proteases in infected larvae resulted in destruction of internal tissues late in infection, which could be the reason for the increased viral speed of kill. Baculoviruses and their recombinant forms constitute viable alternatives to chemical insecticides. Recombinant baculoviruses containing protease genes can be added to the list of engineered baculoviruses with great potential to be used in integrated pest management programs.
    Virology Journal 06/2010; 7(1):143. DOI:10.1186/1743-422X-7-143 · 2.18 Impact Factor
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    • "Mortality was associated with systemic melanisation, which was a result of the cysteine proteinase activity of ScathL. Recombinant ScathL was also found to be insecticidal against H. virescens and pea aphids (Acyrthosiphon pisum) (Li et al., 2008). "
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    ABSTRACT: A cDNA encoding a cathepsin L-like cysteine proteinase (DcCathL) was prepared from gut tissue of larvae of wheat bulb fly (Delia coarctata: Diptera). The predicted protein is a homologue of the product of Drosophila melanogaster gene Cp-1 (CG6692), and is similar to a sub-family of cysteine proteinases found in other insects which have roles in tissue remodelling during development, and moulting. Recombinant DcCathL was produced using the yeast Pichia pastoris as expression host, and showed hydrolytic activity in vitro towards the synthetic substrate Z-Phe-Arg-AMC with a pH optimum of 4.5. DcCathL was insecticidal to lepidopteran larvae when injected into haemolymph, causing mortality that was accompanied by systemic melanisation, suggesting that DcCathL was affecting the immune-related proteolytic activation cascade leading to production of active phenoloxidase. This process is normally negatively regulated by serpins in the haemolymph. Recombinant serpins from cabbage moth (Mamestra brassicae) did not inhibit DcCathL, and were susceptible to degradation by the enzyme in vitro in buffer and extracted haemolymph. When M. brassicae larvae were co-injected with a lethal dose of DcCathL and exogenous recombinant serpins, no mortality or systemic melanisation was observed, suggesting that the insecticidal effects of DcCathL in vivo result from degradation of endogenous serpins.
    Insect biochemistry and molecular biology 06/2009; 39(8):535-46. DOI:10.1016/j.ibmb.2009.05.003 · 3.45 Impact Factor
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