DrosophilaCG10527 mutants are resistant to juvenile hormone and its analog methoprene

Juvenile hormone (JH) is critical for development, metamorphosis, and reproduction in insects. While the physiological importance of JH has been appreciated for decades, its biosynthetic pathway and molecular action remain poorly understood. DrosophilaCG10527 encodes a protein with high homology to crustacean farnesoic acid methyltransferase (FAMeT) that converts farnesoic acid to methyl farnesoate (MF), a precursor of JH, but its in vivo functions remain unclear. Here we report that CG10527 is expressed widely in secondary cells in the male accessory glands, in ovarian follicle cells, and in glial cells in the nervous system. Furthermore, CG10527 is expressed abundantly in the corpora allata where JH is synthesized. To understand the physiological functions of CG10527, we generated specific CG10527 deletions. Phenotypic analysis showed that CG10527 null mutants are fully viable and fertile in both sexes, indicating that CG10527 is not essential for survival and fertility. Surprisingly, CG10527 mutants showed no defects in the biosynthesis of MF and JH. However, CG10527 mutants were 3-5 times more resistant than wild-type flies to topically applied MF and JH as well as the JH analog methoprene at both sub-lethal and lethal doses. Taken together, our data indicate that DrosophilaCG10527 plays little, if any, role in JH biosynthesis but may participate in the JH signaling pathway.     

Tissue-specific effects of the juvenile hormone analogue ZR 515 during metamorphosis in Drosophila cell cultures

The juvenile hormone analogue ZR 515 has specific effects on ecdysone-induced metamorphic differentation of Drosophila cells cultured in vitro. The number of vesicles containing imaginal cuticular structures is reduced to 10% of control levels. Similarly, the differentiation of adult fat body is partly inhibited by ZR 515. The differentiation of adult tubular and fibrillar muscles, however, is not affected. ZR 515 does not inhibit cuticle secretion by tracheal cells and larval epidermal cells.     

The effects of juvenile hormone analogues on the embryogenesis of Drosophila melanogaster

Two juvenile hormone analogues (JHA), 6,7-Epoxy-3-methyl-7-ethyl-1-(3,4-(methyl-enedioxy)phenoxy)-2-cis/trans-octene (Hoffman-LaRoche RO 20-3600) and 6,7-Epoxy-3,7-dimethyl-1-(p-ethyphenoxy)-2-cis/trans-octene (Stauffer R-20458), produced embryonic and some first larval instar lethality when topically applied to freshly laid Drosophila eggs at concentrations above 0·1μg RO-20-3600/μl acetone or 1·0 μg R-20458/μl acetone. With progressively later JHA treatments during embryonic development, lethality decreased while the proportion of larvae dying at the time of hatching increased. No delayed postembryonic lethality was observed after the first larval instar. JHA applications to freshly laid eggs produced abnormal head and tracheal formation in some embryos treated with RO-20-3600 and caused developmental arrest during the beginning of posterior gut formation in most eggs treated with R-20458. Treatment with either JHA after the beginning of blastoderm formation resulted in normal looking first instar larvae which failed to hatch.     

Zinc accumulation in heterozygous mutants of fumble, the pantothenate kinase homologue of Drosophila

Coenzyme A (CoA) functions in the intracellular trafficking of acetyl groups. In humans, mutations in the pantothenate kinase-2 gene, which encodes a key enzyme in CoA biosynthesis, are associated with neurodegeneration and premature death. Diagnosis is based on iron accumulation in the globus pallidus observed by magnetic resonance imaging. We investigated the elemental composition of the fumble mutant, a model of the human disease. Surprisingly, flies carrying a fumble loss-of-function allele had a three-fold increase in total zinc levels per dry weight when compared to control strains, but no change in total iron, copper or manganese levels. Accordingly, zinc supplementation had an adverse impact on the development of fumble mutant larvae, but zinc chelation failed to protect.     

Sensitivity and adaptation in the Drosophila phototransduction and photoreceptor degeneration mutants trp and rdgB

We studied rdgB, a retinal degeneration mutant, and trp, a phototransduction mutant, separately and in combination in Drosophila. First we showed that trp did not block degeneration in white-eyed rdgB mutants. Thus, rdgB was useful in determining the defects which trp caused in the compound eye receptors R7 and R8; this is because rdgB selectively eliminates R1-6 photoreceptors which would, if present, dominate the compound eye responses. R7 and R8 both express the trptransient receptor potential phenotype in trp mutants. The trp mutation does not change receptor spectral sensitivities, nor does it alter the dark stability of R1-6's and R7's metarhodopsins as judged by dark adaptation studies. The dark adaptation is not significantly affected by trp. However, trp slows the dark adaptation of R8 considerably and seems to make the blue-induced inactivation of R1-6 less stable.     

Activity of juvenile hormone and juvenile hormone analogues on the growth of Trypanosoma cruzi

The effects of juvenile hormone-III (JH-III) and the juvenile hormone analogues (JHA) methoprene and fenoxycarb on the growth and macromolecular biosynthesis in Trypanosoma cruzi were studied in vitro. It was observed that JH-III and JHA blocked growth and ³H-thymidine incorporation without killing the cells within certain concentrations (≤1 × 10⁻⁴M), but they caused cellular death at concentrations over 1 × 10⁻³M. The inhibitory effect on growth was partially reversible. On the other hand, the inhibitory action of JH-III, methoprene and fenoxycarb was an unspecific effect according to the results obtained with Leishmania mexicana mexicana (promastigotes) and human peripheral blood lymphocytes. The JHA have a good possibility of being used in the control of trypanosomiasis.     

Use of Drosophila mutants to investigate the effect of disuse on the maintenance of muscle

The effect of continued muscular inactivity and prolonged paralysis on the structure and function of muscles was investigated in Drosophila melanogaster. A number of flightless mutants was examined to see whether their flight muscles degenerated as a result of disuse. No sign of progressive deterioration was observed in any of these mutants. Further, by producing mosaic flies in which part of the body expressed the temperature-sensitive paralytic mutation shibire^ST139, reversible local paralysis was obtained, and maintained for prolonged periods. Flies in which parts of the leg or flight musculature had been paralysed for several days were examined; no effect of such inactivity on the structure and function of the muscles was observed in any of the flies. These results indicate that in Drosophila continued muscular inactivity does not result in extensive degeneration of the musculature.     

Anacystis nidulans mutants resistant to aromatic amino acid analogues.

Three classes of mutants of Anacystis nidulans were selected on the basis of resistance to fluorophenylalanine and 2-amino-3-phenylbutanoic acid. The most frequent type exhibited DAHP synthetase (7-phospho-2-keto-3-deoxy-D-arabino-heptonate-D-erythrose-4-phosphate-lyase [pyruvate phosphorylating], EC 4.1.2.15) activity identical to that of the parental strain. The second type was characterized by extremely low levels of the activity. The third type had a DAHP synthetase showing decreased sensitivity to inhibition by L-tyrosine. The enzyme was purified 140-fold from wild-type and feedback-insensitive strains, and the kinetics of the reaction was examined. The activity of the wild-type enzyme was inhibited 75% in the presence of 2.0 X 10-3 M tyrosine, and the altered enzyme was inhibited 10%. The following apparent constants were obtained from kinetic studies with partially purified wild-type enzyme: S0.5 for D-erythrose-4-phophate equal to 7.1 X 10-4 M; S0.5 for phosphoenolpyruvate equal to 1.4 X 10-4 M. Inhibition by tyrosine was mixed with respect to binding of both D-erythrose-4-phosphate and phosphoenolpyruvate. In addition, tyrosine promoted cooperative interactions in the binding of phosphoenolpyruvate. For the altered enzyme the following apparent constants were obtained: S0.5 for D-erythrose-4-phosphate equal to 7.1 X 10-4 M; S0.5 for phosphoenolpyruvate equal to 2.9 X 10-4 M. Inhibition by tyrosine was mixed with respect to D-erythrose-4-phosphate and competitive with respect to phosphoenolpyruvate. Tyrosine did not promote cooperative effects in the binding of phosphoenolpyruvate to the altered enzyme.     

Inhibition of reproduction and embryogenesis in the firebrat, Thermobia domestica, by juvenile hormone analogues

Application of compounds with juvenile hormone (JH) activity to reproducing females may result in shortening of the ovipositor and occasionally in some other changes in appearance. Severe defects occur in the ovaries: the differentiation of oögonia and prefollicular cells seems to be hindered and the mature oöcytes are resorbed. The affected ovarioles diminish and in extreme cases tear into the germarium, which remains attached to the terminal filament, and the rest of the ovariole, which shrinks into a small rudiment adjacent to the oviduct. The development of eggs deposited by treated females is often lethally affected. Disorders in embryogenesis also occur in the eggs contaminated with active substances at any time between deposition and 2 days before hatching. The substances interrupt the normal course of embryogenesis but the embryos survive for some time and may develop into strange creatures; in one case, two embryos were found within a single egg shell. One out of 27 tested substances was active in amounts lower than 1 ng/egg.     

Thermal preference in Drosophila

Environmental temperature strongly affects physiology of ectotherms. Small ectotherms, like Drosophila, cannot endogenously regulate body temperature so must rely on behavior to maintain body temperature within a physiologically permissive range. Here we review what is known about Drosophila thermal preference. Work on thermal behavior in this group is particularly exciting because it provides the opportunity to connect genes to neuromolecular mechanisms to behavior to fitness in the wild.     

Myoblast fusion in Drosophila

The body wall musculature of a Drosophila larva is composed of an intricate pattern of 30 segmentally repeated muscle fibers in each abdominal hemisegment. Each muscle fiber has unique spatial and behavioral characteristics that include its location, orientation, epidermal attachment, size and pattern of innervation. Many, if not all, of these properties are dictated by founder cells, which determine the muscle pattern and seed the fusion process. Myofibers are then derived from fusion between a specific founder cell and several fusion competent myoblasts (FCMs) fusing with as few as 3-5 FCMs in the small muscles on the most ventral side of the embryo and as many as 30 FCMs in the larger muscles on the dorsal side of the embryo. The focus of the present review is the formation of the larval muscles in the developing embryo, summarizing the major issues and players in this process. We have attempted to emphasize experimentally-validated details of the mechanism of myoblast fusion and distinguish these from the theoretically possible details that have not yet been confirmed experimentally. We also direct the interested reader to other recent reviews that discuss myoblast fusion in Drosophila, each with their own perspective on the process [1], [2], [3] and [4]. With apologies, we use gene nomenclature as specified by Flybase (http://flybase.org) but provide Table 1 with alternative names and references.     

Assays on Rhodnius for juvenile hormone activity

Contrary to expectation the effect of a given quantity of juvenile hormone (JH) topically applied to the abdomen of Rhodnius is greatly reduced if the cuticle is thin; either newly stretched by a large meal or wasted by prolonged starvation. The active substance apparently enters the body too rapidly and is eliminated in metabolism.The activity of the juvenile hormone and the most active JH-mimics is increased ten to fifteenfold if they are topically applied in dilute solution in peanut oil or triolein.Assays of the juvenile hormones of Cecropia at a concentration of 1 : 1000 in peanut oil (suitably diluted with octane) show that synthetic (racemic) C₁₈-JH of Röller (JH-1) is considerably less active than the natural enantiomorph; and the synthetic C₁₇-JH of Meyer (JH-2) has only about two-thirds of the activity of JH-1. Using this method of application, the natural juvenile hormone applied to the fifth instar larva of Rhodnius will cause a 50 per cent suppression of adult characters (grade 10 in the assay) at a dose of about 15 ng.     

Studies on the diapause of Trogoderma granarium: Effects of juvenile hormone analogues on growth and metamorphosis

Trogoderma granarium larvae reared at 30°C on wheat flour containing appropriate concentrations of farnesyl methyl ether (FME) resembled normal diapause larvae in their growth characteristics and reactions to diapauseterminating conditions. When these larvae were transferred to 37°C either without food or with normal food, normal adults resulted. But when the diapause-like state was broken at 37°C in the presence of the original FME-containing food the insects either died before pupal-adult ecdysis or gave abnormal adults. At 35°C FME did not inhibit the larval-pupal ecdysis, but only a few adults emerged and these were abnormal.Farnesol did not inhibit metamorphosis at 30°C and caused less abnormality than did FME.Based on these results, the hypothesis that the density-dependent diapause of this insect is caused by the presence, in faecal pellets, and thereby in contaminated food, of juvenile hormone (JH) analogues is rejected, but the possibility that diapause results from a high physiological level of JH brought about by causes other than the presence of JH analogues in the environment is examined.     

Anomalies in the selection of mutants of Schizosaccharomyces pombe resistant to 8-azaguanine

Summary Anomalies in selection render 8-azaguanine unsuitable as a selective agent for screening forward mutations in continuous cultures of Sch. pombe. This system may, however, provide the basis for an enrichment method for the simultaneous isolation of mutants at two loci.     

New yeast/E. coli/Drosophila triple shuttle vectors for efficient generation of Drosophila P element transformation constructs

We have generated a set of novel triple shuttle vectors that facilitate the construction of Drosophila-P-element transformations vectors. These YED-vectors allow the insertion of any kind of sequence at any chosen position due to the presence of a yeast casette which ensures replication and allows for homologous recombination in Saccharomyces cerevisiae. As a proof of principle we generated several reporter constructs and tested them in transgenic flies for expression and correct subcellular localization. YED-vectors can be used for many purposes including promoter analysis or the expression of tagged or truncated proteins. Thus, time-consuming conventional restriction site based multi-step cloning procedures can be circumvented by using the new YED-vectors. The new set of triple shuttle vectors will be highly beneficial for the rapid construction of complex Drosophila transformation plasmids.     

Role of svp in Drosophila Pericardial Cell Growth

The Drosophila dorsal vessel is a segmentally repeated linear organ, in which seven-up (svp) is expressed in two pairs of cardioblasts and two pairs of pericardial cells in each segment. Under the control of hedgehog (hh) signaling from the dorsal ectoderm, svp participates in diversifying cardioblast identities within each segment. In this experiment, the homozygous embryos of svp mutants exhibited an increase in cell size of Eve positive pericardial cells (EPCs) and a disarranged expression pattern, while the cardioblasts pattern of svp-lacZ expression was normal. In the meantime, the DA1 muscle founders were absent in some segments in svp mutant embryos, and the dorsal somatic muscle patterning was also severely damaged in the late stage mutant embryos, suggesting that svp is required for the differentiation of Eve-positive pericardial cells and DA1 muscle founders and may have a role in EPC cell growth.     

Asymmetrical Reinforcement and Wolbachia Infection in Drosophila

Reinforcement refers to the evolution of increased mating discrimination against heterospecific individuals in zones of geographic overlap and can be considered a final stage in the speciation process. One the factors that may affect reinforcement is the degree to which hybrid matings result in the permanent loss of genes from a species' gene pool. Matings between females of Drosophila subquinaria and males of D. recens result in high levels of offspring mortality, due to interspecific cytoplasmic incompatibility caused by Wolbachia infection of D. recens. Such hybrid inviability is not manifested in matings between D. recens females and D. subquinaria males. Here we ask whether the asymmetrical hybrid inviability is associated with a corresponding asymmetry in the level of reinforcement. The geographic ranges of D. recens and D. subquinaria were found to overlap across a broad belt of boreal forest in central Canada. Females of D. subquinaria from the zone of sympatry exhibit much stronger levels of discrimination against males of D. recens than do females from allopatric populations. In contrast, such reproductive character displacement is not evident in D. recens, consistent with the expected effects of unidirectional cytoplasmic incompatibility. Furthermore, there is substantial behavioral isolation within D. subquinaria, because females from populations sympatric with D. recens discriminate against allopatric conspecific males, whereas females from populations allopatric with D. recens show no discrimination against any conspecific males. Patterns of general genetic differentiation among populations are not consistent with patterns of behavioral discrimination, which suggests that the behavioral isolation within D. subquinaria results from selection against mating with Wolbachia-infected D. recens. Interspecific cytoplasmic incompatibility may contribute not only to post-mating isolation, an effect already widely recognized, but also to reinforcement, particularly in the uninfected species. The resulting reproductive character displacement not only increases behavioral isolation from the Wolbachia-infected species, but may also lead to behavioral isolation between populations of the uninfected species. Given the widespread occurrence of Wolbachia among insects, it thus appears that there are multiple ways by which these endosymbionts may directly and indirectly contribute to reproductive isolation and speciation.