Juvenile Hormone (JH) Esterase of the Mosquito Culex quinquefasciatus Is Not a Target of the JH Analog Insecticide Methoprene

Juvenile hormones (JHs) are essential sesquiterpenes that control insect development and reproduction. JH analog (JHA) insecticides such as methoprene are compounds that mimic the structure and/or biological activity of JH. In this study we obtained a full-length cDNA, cqjhe, from the southern house mosquito Culex quinquefasciatus that encodes CqJHE, an esterase that selectively metabolizes JH. Unlike other recombinant esterases that have been identified from dipteran insects, CqJHE hydrolyzed JH with specificity constant (kcat/KM ratio) and Vmax values that are common among JH esterases (JHEs). CqJHE showed picomolar sensitivity to OTFP, a JHE-selective inhibitor, but more than 1000-fold lower sensitivity to DFP, a general esterase inhibitor. To our surprise, CqJHE did not metabolize the isopropyl ester of methoprene even when 25 pmol of methoprene was incubated with an amount of CqJHE that was sufficient to hydrolyze 7,200 pmol of JH to JH acid under the same assay conditions. In competition assays in which both JH and methoprene were available to CqJHE, methoprene did not show any inhibitory effects on the JH hydrolysis rate even when methoprene was present in the assay at a 10-fold higher concentration relative to JH. Our findings indicated that JHE is not a molecular target of methoprene. Our findings also do not support the hypothesis that methoprene functions in part by inhibiting the action of JHE

Construction of occluded recombinant baculoviruses containing the full-length cry1Ab and cry1Ac genes from Bacillus thuringiensis

The administration of baculoviruses to insects for bioassay purposes is carried out, in most cases, by contamination of food surfaces with a known amount of occlusion bodies (OBs). Since per os infection is the natural route of infection, occluded recombinant viruses containing crystal protein genes (cry1Ab and cry1Ac) from Bacillus thuringiensis were constructed for comparison with the baculovirus prototype Autographa californica nucleopolyhedrovirus (AcNPV). The transfer vector pAcUW2B was used for construction of occluded recombinant viruses. The transfer vector containing the crystal protein genes was cotransfected with linearized DNA from a non-occluded recombinant virus. The isolation of recombinant viruses was greatly facilitated by the reduction of background "wild type" virus and the increased proportion of recombinant viruses. Since the recombinant viruses containing full-length and truncated forms of the crystal protein genes did not seem to improve the pathogenicity of the recombinant viruses when compared with the wild type AcNPV, and in order to compare expression levels of the full-length crystal proteins produced by non-occluded and occluded recombinant viruses the full-length cry1Ab and cry1Ac genes were chosen for construction of occluded recombinant viruses. The recombinant viruses containing full-length and truncated forms of the crystal protein genes did not seem to improve its pathogenicity but the size of the larvae infected with the recombinant viruses was significantly smaller than that of larvae infected with the wild type virus

Field trial of a genetically improved baculovirus insecticide

IMPROVEMENT of biological pesticides through genetic modification has enormous potential and the insect baculoviruses are particularly amenable to this approach1,2. A key aim of genetic engineering is to increase their speed of kill, primarily by the incorporation of genes which encode arthropod or bacterially derived insect-selective toxins3–11, insect hormones12,13 or enzymes14,15. We report here the first, to our knowledge, field trial of a genetically improved nuclear polyhedrosis virus of the alfalfa looper, Autogmpha californica (AcNPV) that expresses an insectselective toxin gene (AaHIT) derived from the venom of the scorpion Androclonus australisl6–18. Previous laboratory assays with the cabbage looper, Trichoplusia ni, demonstrated a 25% reduction in time to death compared to the wild-type virus, but unaltered pathogenicity6 and host range19. In the field, the modified baculovirus killed faster, resulting in reduced crop damage and it appeared to reduce the secondary cycle of infection compared to the wild-type virus