Thomas G. Wilson

Intracellular localization and tissue specificity of the Methoprene- tolerant (Met) gene product in Drosophila melanogaster

The Methoprene-tolerant (Met) gene product in Drosophila melanogaster facilitates the action of juvenile hormone (JH) and JH analog insecticides. Previous work resulted in the cloning and identification of the gene as a member of the bHLH-PAS family of transcriptional regulators. A Met(+) cDNA was expressed in Escherichia coli, and polyclonal antibody was prepared against the purified protein. A single band on a Western blot at the expected size of 79kD was detected in extracts from Met(+) larvae but not from Met(27) null mutant larvae, demonstrating the antibody specificity. Antibody detected MET in all stages of D. melanogaster development and showed tissue specificity of its expression. MET is present in all cells of early embryos but dissipates during gastrulation. In larvae it is present in larval fat body, certain imaginal cells, and immature salivary glands. In pupae it persists in fat body cells and imaginal cells, including abdominal histoblast cells. In adult females MET is present in ovarian follicle cells and spermathecae; in adult males it is present in male accessory gland and ejaculatory duct cells. In all of these tissues MET is found exclusively in the nucleus. Some of these tissues are known JH target tissues but others are not, suggesting either the presence of novel JH target tissues or another function for MET.

Stress-reactivity and juvenile hormone degradation in Drosophila melanogaster strains having stress-related mutations.

Juvenile hormone (JH) degradation was studied under normal and stress conditions in young and matured females of Drosophila melanogaster strains having mutations in different genes involved in responses to stress It was shown that (1) the impairment in heat shock response elicits an alteration in stress-reactivity of the JH system; (2) the impairment JH reception causes a decrease of JH-hydrolysing activity and of stress-reactivity in young females, while in mature ones stress reactivity is completely absent; (3) the absence of octopamine results in higher JH-hydrolysis level under normal conditions and altered JH stress-reactivity; (4) the higher dopamine content elicits a dramatic decrease of JH degradation under normal conditions and of JH stress-reactivity. Thus, the impairments in any component of the Drosophila stress reaction result in changes in the reponse of JH degradation system to stress. The role of JH in the development of the insect stress reaction is discussed.

Lufenuron, a chitin-synthesis inhibitor, interrupts development of Drosophila melanogaster

The chitin-synthesis inhibitor lufenuron was administered to Drosophila melanogaster to better understand the effects of chitin-synthesis interruption during the development and reproduction of this insect. When larvae were fed a diet containing a low concentration (< 1 ppm) of lufenuron, mortality was observed during either larval or pupal development, depending on the dose. Survivor adults were unable to fly, probably due to abnormal cuticular development in the wing hinge regions of the thorax. Larvae fed a higher concentration (10 ppm) completed development within that instar, but died during ecdysis to the next instar, presumably due to inadequate cuticle synthesis. Third instar larvae pupariated, but the puparium was abnormal, and pupation did not occur. Adults fed 10 ppm showed normal viability but slightly depressed oogenesis; additionally, although their eggs were fertilized, they failed to hatch. Examination of the embryos showed that they completed development but were unable to perforate the surrounding vitelline membrane, probably due to a weakened chitinous mouth hook assembly that was insufficiently rigid to effect hatching. These results identify stages during D. melanogaster development when chitin synthesis and deposition are critical. This information will be useful for identifying chitin-synthesis mutants of this insect.

GENETIC EVIDENCE THAT MUTANTS OF THE METHOPRENE-TOLERANT GENE OF DROSOPHILA MELANOGASTER ARE NULL MUTANTS

The Methoprene-tolerant (Met) mutation of Drosophila melanogaster results in resistance to juvenile hormone (JH) or JH analogs and appears to alter JH reception during late larval development. Several alleles of Met have been recovered from methoprene selection screens after mutagenesis with ethyl methanesulfonate, X-rays, or transposable genetic elements. The phenotype of files carrying any of these alleles is similar-resistance to the toxic and morphogenetic effects of methoprene-but otherwise is essentially wild-type. Understanding the function of the Met gene requires that we know whether these alleles are hypomorphic, producing some functional gene product, or amorphic, producing no functional gene product. This determination was made by comparing the methoprene-resistance phenotype produced by representative Met alleles with that produced by a chromosome carrying a deficiency that deletes the Met gene. The level of resistance to either the toxic or the morphogenetic effect of methoprene was similar among files heterozygous for either the deficiency chromosome or for any of the alleles. The results provide genetic evidence that the Met alleles recovered to date are amorphic and suggest that the Met gene may not be mutable to a more severe Met allele that affects the viability, development, or reproduction of the flies.

Stress-reactivity of a Drosophila melanogaster strain with impaired juvenile hormone action

Met(27) is a null allele of the Methoprene-tolerant gene of D. melanogaster that shows resistance to the toxic effects of both juvenile hormone (JH) and a JH analog, methoprene. The mechanism of resistance appears to be altered JH reception. We measured fertility, JH-hydrolyzing activity, and dopamine (DA) levels in Met(27) and Met(+) flies under normal (25 degrees C) and heat-stress (38 degrees C) conditions. We show that under normal conditions Met(27) females have JH-hydrolyzing activity and fertility lower than Met(+), but DA content did not differ between the two strains. At 38 degrees C Met(27) flies show no impairment in JH-hydrolyzing activity in response to stress, but they do show lower DA levels and impaired reproduction. The results with Met(27) are consistent with the previous hypothesis that the alteration in fertility that follows heat stress in D. melanogaster could result from alteration in the JH endocrine system.