Induction of dauer pupae by fenoxycarb in the silkworm, Bombyx mori.

Topical application of fenoxycarb (1 &mgr;g per animal) at 129 or 132 h of the fifth instar larvae of the silkworm, Bombyx mori, did not induce morphological abnormalities in the pupal stage, but these animals became dauer (permanent) pupae. This condition of B. mori and the endocrine events leading to permanent pupae are discussed in this work. Application of fenoxycarb at 132 h of the fifth instar elicited a high ecdysteroid titre in the pharate pupal stage and a steadily high ecdysteroid titre in the pupal stage. The fenoxycarb-induced permanent pupae had non-degenerating prothoracic glands that secreted low amounts of ecdysteroid and did not respond to recombinant prothoracicotropic hormone (rPTTH) late in the pupal stage. The Bombyx PTTH titre in the haemolymph, determined by a time-resolved fluoroimmunoassay, was lower than that of controls at the time of pupal ecdysis, but higher than controls later in the pupal stage in fenoxycarb-treated animals. After application of fenoxycarb, its haemolymph level, measured by ELISA, reached a peak at pupal ecdysis, then remained low. These results suggest that the fenoxycarb-mediated induction of permanent pupae is only partially a brain-centred phenomenon. It also involves alterations in the hormonal interplay that govern both the initiation of pupal-adult differentiation and changes in the steroidogenic pathway of the prothoracic glands of B. mori.

A duplexed microsphere-based fluorescent immunoassay.

Microsphere-based immunoassays are described for the simultaneous measurement of the clinically important drugs digoxin and theophylline. Competitive immunoassays were performed using haptenized microspheres and antibodies labeled with horseradish peroxidase. Enzyme-catalyzed reporter deposition (CARD) resulted in immunofluorescence signal amplification. Two encoding dyes were used to differentiate analytical signals from microspheres containing assays for the two analytes. An epifluorescence microscope and a CCD camera interfaced with a computer were utilized to measure fluorescence signals of individual microspheres. The microspheres from a duplexed assay were mounted on microscope slides as well as inserted into wells etched into the distal ends of optical imaging fibers. Fluorescence images from both formats were captured. In the experiments using microscope slides, the immunoassays were successfully duplexed and only marginal interferences at high analyte concentrations were observed. Preliminary results suggest that simultaneous determination of the two analytes using a fiber-based sensor-array format is feasible, but requires further development before precise quantitative analyses are possible.

A combinatorial approach to discover new chelators for optical metal ion sensing

This paper reports the synthesis and characterization of metal-binding indicators with diverse optical responses on exposure to various heavy metal ions. A combinatorial approach, based on azo coupling of diazonium salts with either phenolic compounds or aromatic amines, generated a library of azo dyes. Each reaction mixture, containing the product(s) of azo coupling, was incubated with a series of solutions, each containing a different heavy metal ion. The absorbance and, in some cases, fluorescence spectra of the resulting complexes were recorded. The metal chelates showed extensive diversity in their UV-visible absorbance spectra upon binding to selected metal ions. Of the azo dyes prepared, the terdentate dyes were particularly useful, providing distinct spectral responses to three or more metal ions in a panel of seven.

Rapid assays for environmental and biological monitoring.

Rapid, inexpensive, sensitive, and selective enzyme-linked immunosorbent assays (ELISAs) now are utilized in environmental science. In this laboratory, many ELISAs have been developed for pesticides and other toxic substances and also for their metabolites. Compounds for which ELISAs have recently been devised include insecticides (organophosphates, carbaryl, pyrethroids, and fenoxycarb), herbicides (s-triazines, arylureas, triclopyr, and bromacil), fungicides (myclobutanil), TCDD, and metabolites of naphthalene and toluene. New rapid assays have been developed for mercury.

Projuvenoids: synthesis and biological evaluation of sulfenylated, sulfinylated, and sulfonylated carbamates.

Applying the proinsecticide principle developed earlier for neurotoxic carbamate insecticides, a series of new N-sulfenylated, N-sulfinylated, and N-sulfonylated derivatives of fenoxycarb were synthesized and evaluated for juvenile hormone mimicking activity. Laboratory evaluations of the compounds using Pieris brassicae and Sitophilus oryzae, as well as field experiments using Bemisia tabaci, showed that several symmetrical biscarbamates with either a sulfenyl or sulfinyl bridge possessed higher activity than the parent carbamate. From the unsymmetrical compounds containing biologically inert derivatizing moieties, one of the sulfenylated biscarbamates also showed improved activity against P. brassicae. The changes in the biological activity of the sulfur-containing derivatives compared to that of the parent compound are attributed to the modified physicochemical characteristics, i.e., increased lipophilicity facilitating penetration, transport, as well as protection of the compound from metabolism.

Development of rapid mercury assays. Synthesis of sulfur- and mercury-containing conjugates.

We have devised rapid analyses for mercury exploiting the high affinity of dithiocarbamate chelators for the mercuric ion. Our first assay is based on a sandwich chelate formed by a ligand supported on the well of an ELISA plate, Hg2+ ion of the investigated sample, and another ligand bound to a reporter enzyme. The second assay utilizes competitive binding of the analyte Hg2+ ions versus an organomercury conjugate to a chelating conjugate. Low ppb sensitivity and high selectivity for Hg2+ ions have been achieved in our pilot studies.

Cinnamyl derivatives and monoterpenoids as nonspecific ovipositional deterrents of the onion fly.

Laboratory dose-response choice tests and discriminate-dosage bioassays revealed wide variation in the effectiveness of cinnamyl, cinnamoyl, monoterpene, and phenethyl alcohol derivatives as ovipositional deterrents toDelia antiqua (Meigen), the onion fly. (E)-Cinnamic acids were not detectably deterrent. When formulated in particles of polyethylene glycol, (E)-cinnamaldehyde had a BR90 (concentration eliciting 90% deterrency) of 1.0% and (E)-4-methoxycinnamaldehyde had a BR90 of 0.38%. Among nine monoterpenoids tested,p-cymene was inactive, citronellal had a BR90 of 3.7%, and terpinene-4-ol had a BR90 of 0.46%. Para-substituted phenethyl alcohols gave increasing deterrence in the order: -NO2, CH3O-, -Cl, -CH3, -H. Wide varieties of structures were deterrent: C-8 to C-13, intermediate in polarity, and possessing either oxygen-containing or nitrile functional groups. The air concentration of (E)-cinnamaldehyde at its BR90 was 1.7 ng/ml. This relatively high concentration, the diversity in deterrent structures, and the lack of differences in deterrency among positional and optical isomers suggest that ovipositional deterrency in onion flies is mediated by receptors broadly tuned for detecting phenylpropenoid, phenolic, monoterpenoid, and perhaps other classes of allelochemicals.