Contents - Faperta

298 Biotechnological Approaches for Pest Management and Ecological Sustainability
Viral Vectors
Densoviruses have been used for genetic transformation of insects, particularly mosquitoes.
They have a narrow host range, are relatively stable in the environment, and no
microinjection is necessary (Barreau, Jousset, and Cornet, 1994). Alphavirus, Sindbis (SIN ),
serves as an mRNA template for translation (Strauss, Rice, and Strauss, 1984). This has
been used for stable cytoplasmic expression of foreign DNA. Foreign DNA can be inserted
downstream of this promoter (dsSIN ). Alpine viruses are infectious to humans, and this
may be inappropriate for genetic transformation of insects. Alphavirus, Sindbis, can be
used to deliver high levels of gene expression in vivo in a number of nonhost arthropod
species without causing cytopathic effects in infected cells or impairing development
(Lewis et al., 1999). Recombinant Sindbis virus has a great potential for analyzing the
change in the function of developmental genes during diversifi cation. Densoviruses from
Aedes aegypti (L.) (AcDNV ) and Junonia coenia (Hubner) (JCDNV ) have also been used to
create transducing vectors to express the reporter gene in insect cells (Giraud, Devauchelle,
and Bergoin, 1992; Afanasiev et al., 1994). Using recombinant Sindbis virus, the function of
the homeotic gene Ultrabithorax in the development of butterfl y, Precis coenia (Hubner),
wings, and beetle, Tribolium castaneum (Herbst.), embryos has been investigated. Ectopic
Ultrabithorax expression in butterfl y forewing imaginal discs was suffi cient to cause transformation
of forewings in adults, including scale morphology and pigmentation. Expression
of Ultrabithorax in beetle embryos outside of its endogenous expression domain affects
normal development of the body wall cuticle and appendages. Homeotic genes probably
play an important role in diversifi cation of arthropod appendages. Ultrabithorax is suffi -
cient to confer hindwing identity in butterfl ies and alters normal development of anterior
structures in beetles.
Baculoviruses
Corsaro, DiRenzo, and Fraser (1989) optimized transfection conditions for cloning a
UND-K derivative of the IPLB-HZ 1075 cell line of the corn earworm, Helicoverpa zea
(Boddie), using a calcium phosphate coprecipitation technique and the DNA genome of
the H. zea S-type nuclear polyhedrosis virus (NPV). The technique permitted relatively
effi cient in vitro manipulation of the virus genome. Human 5- lipoxygenase (EC 1.13.11.34),
the key enzyme involved in the transformation of arachidonic acid to the potent biologically
active leukotrienes, has been overexpressed in cells of Spodoptera frugiperda (J.E. Smith)
using the Autographa californica Speyer NPV strain E2 expression system (Funk et al., 1989).
Although infected cells were able to express mutant 5-lipoxygenase protein, enzyme activity
was not substantially altered, suggesting the nonessential nature of certain histidines
in binding iron at the putative ferric catalytic site. The major antibacterial proteins attacin,
cecropin, and lysozyme are secreted into the hemolymph of the saturniid, Hyalophora
cecropia (L.) upon challenge with bacteria. Attacin has been isolated and the corresponding
amino acid sequence suggested that basic attacin is synthesized as a 233-residue pre-
proprotein confi rmed by cloning the cDNA fragment encoding the basic attacin in the
A. californica NPV downstream of the polyhedrin promoter and expressing the protein in
S. frugiperda cells. Attacin has also been produced in last-instar larvae of T. ni after injection
of recombinant virus (Gunne, Hellers, and Steiner, 1990). Based on protein processing
pattern, it has been suggested that a protease produced by the S. frugiperda cells cleaves
Arg45-Arg46, producing mature attacin. The instability of the attacin proteins is rationalized
in terms of their random-coil structure, which was deduced from circular dichroism