02480nas a2200313   4500008004600000245018400046210006900230300001600299490000700315520135500322653001201677653003401689653002701723653004001750653003001790653002201820653002301842653002301865653001901888653003201907653002001939653007101959653002102030100001102051700001302062700002102075700002002096856005002116       Submitted                   eng d     00aImpact of a basement membrane-degrading protease on dissemination and secondary infection of Autographa californica multiple nucleopolyhedrovirus in Heliothis virescens (Fabricus)0 aImpact of a basement membranedegrading protease on dissemination  a1109 - 11190 v883 aScathL is a cathepsin L-like cysteine protease from the flesh fly, Sarcophaga peregrina, that digests components of the basement membrane (BM) during insect metamorphosis. A recombinant baculovirus that expresses ScathL (AcMLF9.ScathL) kills larvae of the tobacco budworm, Heliothis virescens, significantly faster than the wild-type virus and triggers melanization and tissue fragmentation in infected larvae shortly before death. As BMs are a potential barrier to the spread of baculovirus secondary infection to other tissues in the host, this study tested the hypothesis that the rapid death of insects infected with AcMLF9.ScathL was caused by accelerated secondary infection resulting from the degradation of host BMs by ScathL. Viruses expressing catalytically active or inactive ScathL were used to examine the effects of ScathL activity on budded virus release into the haemocoel during infection, the production of polyhedra in infected larvae and the rate of infection of the gut, trachea, haemocytes, fat body and Malpighian tubules. It was concluded that the enhanced insecticidal efficacy of the recombinant baculovirus that expresses ScathL does not result from altered tissue tropism or accelerated systemic infection. Implications for the role of the BM as a barrier to baculovirus dissemination within the host insect are discussed.10aAnimals10aBasement Membrane/*metabolism10aCathepsins/*metabolism10aCysteine Endopeptidases/*metabolism10aDigestive System/virology10aFat Body/virology10aHemocytes/virology10aHemolymph/virology10aLarva/virology10aMalpighian Tubules/virology10aMoths/*virology10aNucleopolyhedrovirus/enzymology/genetics/pathogenicity/*physiology10aTrachea/virology1 aLi, H.1 aTang, H.1 aHarrison, R., L.1 aBonning, B., C.  uhttps://sarcophagidae.myspecies.info/node/46502790nas a2200373   4500008004600000245020300046210006900249300001400318490000700332520144700339653002401786653001201810653005201822653001601874653003601890653002201926653002501948653003101973653003002004653002302034653002902057653001302086653002002099653004202119653004102161653004902202653002102251100001902272700001602291700002102307700001602328700002202344856005002366       Submitted                   eng d     00aCharacterisation of functional and insecticidal properties of a recombinant cathepsin L-like proteinase from flesh fly (Sarcophaga peregrina), which plays a role in differentiation of imaginal discs0 aCharacterisation of functional and insecticidal properties of a   a589 - 6000 v373 aScathL is a cathepsin L-like cysteine proteinase from Sarcophaga peregrina (flesh fly), which is involved in differentiation of imaginal discs, through proteolysis of components of basement membranes. An expression system based on the methylotrophic yeast Pichia pastoris was used to produce recombinant ScathL. Although the expression construct contained the full proprotein coding sequence for ScathL, the proprotein was only detected in culture supernatant at early stages of expression by Western blotting. The purified recombinant protein contained only a polypeptide similar to mature ScathL, as a result of autocatalytic processing. After activation by reducing agents, the enzyme hydrolysed the cathepsin L substrate Z-Phe-Arg-AMC, with optimal activity at pH 5.5. ScathL showed decreasing activity with increasing ionic strength above 0.3M NaCl, and lost activity irreversibly at pH &gt; or = 7.5. The enzyme showed limited activity towards protein substrates, digesting only to large fragments. ScathL was insecticidal towards larvae of the tomato moth, Lacanobia oleracera, following injection into the haemolymph. It caused melanisation, although no evidence of extensive proteolysis in haemolymph or gut was observed. Production of a inactive mutant form of ScathL showed that enzyme activity was necessary for the complete proprotein processing observed during production as a recombinant protein, and for insecticidal activity.10aAmino Acid Sequence10aAnimals10aCysteine Endopeptidases/genetics/isolation &amp10adevelopment10aDiptera/*enzymology/growth &amp10aEnzyme Activation10aHemolymph/metabolism10aHydrogen-Ion Concentration10aInsecticides/pharmacology10aLarva/drug effects10aLepidoptera/drug effects10aMutation10aPichia/genetics10apurification/*metabolism/pharmacology10apurification/metabolism/pharmacology10aRecombinant Proteins/genetics/isolation &amp10aSalts/metabolism1 aPhilip, J., M.1 aFitches, E.1 aHarrison, R., L.1 aBonning, B.1 aGatehouse, J., A.  uhttps://sarcophagidae.myspecies.info/node/46602767nas a2200301   4500008004600000245012100046210006900167300001400236490000700250520166600257653001201923653002801935653004901963653004302012653005602055653004102111653004802152653002902200653004502229653003802274653002202312100001302334700001102347700001602358700002102374700002002395856005002415       Submitted                   eng d     00aTissue specificity of a baculovirus-expressed, basement membrane-degrading protease in larvae of Heliothis virescens0 aTissue specificity of a baculovirusexpressed basement membranede  a431 - 4430 v393 aScathL is a cathepsin L-like cysteine protease from the flesh fly, Sarcophaga peregrina, which digests components of the basement membrane during insect metamorphosis. A recombinant baculovirus (AcMLF9.ScathL) expressing ScathL kills larvae of the tobacco budworm Heliothis virescens significantly faster than the wild type virus and triggers melanization and tissue fragmentation shortly before death. The tissue fragmentation was assumed to be a direct consequence of basement membrane degradation by ScathL. The goal of this study was to investigate the tissue specificity of ScathL when expressed by AcMLF9.ScathL using light, transmission and scanning electron microscopy. Baculovirus expression of ScathL resulted in damage to the basement membrane overlying the midgut, fat body and muscle fibers in larvae infected with AcMLF9.ScathL, but not in larvae infected with the control virus AcMLF9.ScathL.C146A or wild type virus AcMNPV C6. Injection of recombinant ScathL and high levels of baculovirus-mediated expression of ScathL resulted in complete loss of the gut. Extensive damage to the basement membrane mediated by ScathL likely resulted in loss of viability of the underlying tissue and subsequent death of the insect. These results confirm the conclusion of an earlier study (Philip, J.M.D., Fitches, E., Harrison, R.L., Bonning, B.C., Gatehouse, J.A., 2007. Characterisation of functional and insecticidal properties of a recombinant cathepsin L-like proteinase from flesh fly (Sarcophaga peregrina), which plays a role in differentiation of imaginal discs. Insect Biochem. Mol. Biol. 37, 589-600) of the remarkable specificity of this protease.10aAnimals10aBaculoviridae/*genetics10aBasement Membrane/*metabolism/ultrastructure10adevelopment/*metabolism/ultrastructure10aDigestive System/cytology/metabolism/ultrastructure10aEndopeptidases/*genetics/*metabolism10aFat Body/cytology/metabolism/ultrastructure10aInsecticides/*metabolism10aLarva/cytology/metabolism/ultrastructure10aLepidoptera/cytology/*growth &amp10aOrgan Specificity1 aTang, H.1 aLi, H.1 aLei, S., M.1 aHarrison, R., L.1 aBonning, B., C.  uhttps://sarcophagidae.myspecies.info/node/47102772nas a2200361   4500008004600000245014100046210006900187300001400256490000700270520157500277653001201852653001801864653003601882653002101918653002701939653003401966653004702000653002202047653002802069653002802097653002702125653003402152653001902186653003502205100001102240700001302251700001802264700001502282700002102297700002202318700002002340856005002360       Submitted                   eng d     00aInsecticidal activity of a basement membrane-degrading protease against Heliothis virescens (Fabricius) and Acyrthosiphon pisum (Harris)0 aInsecticidal activity of a basement membranedegrading protease a  a777 - 7890 v543 aScathL 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.10aAnimals10aBaculoviridae10aBasement Membrane/*drug effects10aBiological Assay10aCatechol Oxidase/blood10aCathepsins/genetics/*toxicity10aCysteine Endopeptidases/genetics/*toxicity10aDiptera/chemistry10aEnzyme Precursors/blood10aHemiptera/*drug effects10aInsecticides/*toxicity10aLarva/drug effects/metabolism10aLethal Dose 5010aMoths/*drug effects/metabolism1 aLi, H.1 aTang, H.1 aSivakumar, S.1 aPhilip, J.1 aHarrison, R., L.1 aGatehouse, J., A.1 aBonning, B., C.  uhttps://sarcophagidae.myspecies.info/node/365