Determinants of oöcyte degeneration in Drosophila melanogaster

During normal oögenesis in many insects some of the oöcytes fail to mature; instead they degenerate and are resorbed. In this work oöctte degeneration was investigated in Drosophila melanogaster females and found to be limited to early vitellogenic stages (stages 8–10). Even when retained for up to 18 days by females, mature (stage 14) oöcytes showed unaltered protein patterns after separation by SDS polyacrylamide electrophoresis, indicating that protein breakdown, which is characteristic of degeneration, does not occur in chorionated oöcytes.A number of environmental parameters were shown to influence the percentage of degenerating oöcytes in females. Strong responses as reflected by increased stage-8 and 9 oöcyte degeneration were found in females subjected to suboptimal (but not starvation) medium, virgin females, females mechanically unable to oviposit, and females unable to locate suitable oviposition sites. Little or no response was seen in females subjected to crowding, however, since all of these environmental parameters except adult crowding have been shown to decrease fecundity, and therefore the rate of oöcyte production, it is suggested that oöcyte degeneration is a strategy for decreasing the rate of oöcyte production in Drosophila.     

Genetic analysis of X-linked mutagen-sensitive mutants of Drosophila melanogaster

Mutants at 2 new loci which control mutagen-sensitivity are described. Mutants at both loci are female-sterile and are hypersensitive to killing by MMS; neither increases the frequency of sex-linked recessive lethals. A screen of previously described female-sterile and meotic mutants has revealed that a number of these are also sensitive to mutagens. In addition, several new mutants have been identified on the basis of sensitivity to either HN2 or MMS. An anlysis of complementation data suggests that all of the X-linked genes controlling sensitivity to MMS may now have been identified. Among the new mei-41 alleles are mutants which show verly little meiotic nondisjunction or loss. Cytogenetic mapping of previously known mutants is also described. The mutants mus(1)104^D1 and mei-41^D5 are located in th eregion 14B13±?14D1,2 on the polytene chromosome map, and they map very close to each other genetically. Cytogenetically mus(1)101^D1 is between salivary chromosome bands 12A6,7 and 12D3, mus(1)103^D1 is between bands 12A1,2 and 12A6,7, and mus(1)-109^A1 is in section 8F3-9A2.     

MMS sensitive strains in Drosophila melanogaster

Larval populations of Drosophila melanogaster were treated with the monofunctional alkylating agent methyl methyl methanesulfonate (MMS). Four different stocks were tested. The method methyl used permits the direct registration of MMS-induced lethality of every genotype present in the treated population. Up to 5-fold differences in sensitivity between different genotypes were obeserved. Larvae of the wild-type strain. Antibes, which was reported to have increased UV sensitivity in embryonic stages, were MMS sensitive. In another stock, marked with spa^pol, female larvae were about twice as sensitive as male larvae.     

Drosopterins in phototaxis-selected strains of Drosophila melanogaster

The age-dependent drosopterin concentration was investigated in phototaxis-selected strains (Röko, Röpo, Röne, Stab) of D. melanogaster using extracted head homogenates. The results are compared to the wild stock derived strain Plus. The drosopterin concentration increases with the age of the flies. Females have larger concentrations than males. There are no differences in the drosopterin concentrations among the phototaxis-selected strains. Plus shows a higher drosopterin content than all other strains. This difference might be based on the genetical background, which is different in these strains. The data indicate that the phototactic behaviour is not related to the drosopterin concentration of the eyes.     

Drosophila melanogaster deoxyribonucleoside kinase activates gemcitabine

Drosophila melanogaster multisubstrate deoxyribonucleoside kinase (Dm-dNK) can additionally sensitize human cancer cell lines towards the anti-cancer drug gemcitabine. We show that this property is based on the Dm-dNK ability to efficiently phosphorylate gemcitabine. The 2.2 Å resolution structure of Dm-dNK in complex with gemcitabine shows that the residues Tyr70 and Arg105 play a crucial role in the firm positioning of gemcitabine by extra interactions made by the fluoride atoms. This explains why gemcitabine is a good substrate for Dm-dNK.     

Mutagenicity tests with metrifonate in Drosophila melanogaster

The organophosphorus insecticide metrifonate (O,O-dimethyl(1-hydroxy-2,2,2-trichloroethyl)phosphonate), also known as trichlorfon or Dipterex ®, was tested for its ability to induce sex-linked and autosomal recessive lethal mutations in Drosophila melanogaster. There was no evidence of an increase in the frequency of lethal mutations after feeding adult males with metrifonate, but the high toxicity of the compound meant that only low concentrations could be used in this test system.     

An operating cost of learning in Drosophila melanogaster

Although the fitness benefits of learning are well understood, we know little about its costs; yet both are essential to understand the evolution of animal learning. We tested the hypothesis that learning has an operating cost, such that an animal repeatedly forced to use its learning ability would show a reduction in some fitness component(s), relative to an animal of the same genotype that does not have to learn. Five ‘High-learning’ lines of Drosophila melanogaster, which had been selected for improved learning ability, were exposed to 12 consecutive 48-h cycles of alternating conditioning treatment under mild nutritional stress. Their learning score first increased, reaching a maximum around day 12 (i.e. the sixth conditioning cycle), and then progressively declined. These changes were not due to ageing, as they were not observed in flies from the same lines maintained under standard conditions. From around day 12, the productivity (egg-laying rate) of the flies in the conditioning treatment became progressively reduced, relative to flies from the same lines not exposed to conditioning, but otherwise kept under the same food-limited conditions. This reduction in productivity was not observed when these treatments were applied to five ‘Low-learning’ lines, which had not been exposed to selection, and which show no detectable response to conditioning under our experimental conditions. Furthermore, exposure to repeated cycles of conditioning revealed an apparent trade-off between the learning score and productivity among the ‘High-learning’ lines. These results indicate an operating cost of learning, paid only by genotypes that show learning, rather than general effects of stress caused by the conditioning regime. Potential proximate explanations include (1) the impairment of oviposition decisions caused by the accumulation of memory interference and (2) energy costs of collecting, processing and storing information.     

Evolutionary significance of courtship conditioning in Drosophila melanogaster

Males of the fly Drosophila melanogaster initially court mated, unreceptive females but later develop an avoidance reaction against them and even become temporally unresponsive to virgin females. This conditioned inhibition has been described as an associative process, the conditioned stimulus being a mixture of pheromones on the female's cuticle. To assess the evolutionary significance of courtship conditioning we recorded and analysed the male's behaviour during the conditioning process. The time traces of individual males were marked by an abrupt behavioural change. The time he spent courting suddenly decreased, and the frequency of ‘turn-away’ events at the same time sharply increased. Thus, the gradual decline of courtship observed as a group average does not reflect a slow change in motivation of the individual male but rather the interindividual differences of an active, experience-guided all-or-none decision to stop courting and to avoid the female. Three recently collected D. melanogaster strains were each maintained under two distinct mating conditions. Males were kept together with females for either ca. 2 weeks or 18 h. After 21 generations males of the two regimes differed markedly in their behaviour towards mated females. With long interaction periods between males and females, selection favoured courtship conditioning, while with short periods no such selection was observed. Slowly recovering receptivity of mated females may be needed for the maintenance of courtship conditioning. Courtship conditioning in D. melanogaster appears to be a fitness-relevant behaviour adapted to high-density populations with females mating a second time.     

Ecdysteroids in adult males and females of Drosophila melanogaster

Ecdysteroid titres in whole flies and different tissues of adult male and female Drosophila were determined at various times after eclosion using a radioimmunoassay. The ecdysteroid titre decreased as the flies matured after eclosion. The differences in titre between males and females can be accounted for by their difference in body weight. The ecdysteroids were found to be distributed throughout several tissues. At eclosion not all of the ecdysteroid complement present could be accounted for by that found localised in tissues. After maturation of the flies the ecdysteroids in various tissues can account for the majority of that detected in whole-fly extracts. Ecdysteroids were produced during in vitro culture of various tissues, but the quantities detected were low by comparison with ring glands of wandering 3rd-instar larvae. Neither the ovaries nor the abdominal body walls (fat body) seem to be a major source of hormone, and they are only able to convert minute quantities of ecdysone to the biologically active form, 20-hydroxyecdysone, in vitro. The amounts of 20-hydroxyecdysone present were measured using high performance liquid chromatography and radioimmunoassay. We tentatively suggest that the differential experession of the yolk-protein-genes in the fat bodies of males and females does not result from differences in hormone titres between them.     

Visual learning behaviour in Drosophila melanogaster wildtype AS

Visual learning in Drosophila populations is characterized by a fast rate of acquisition, and conditioning indices near 0.3 are obtained. Heterogeneity of the populations with respect to this behaviour is small. Reversed conditioning is possible. Prolonged extinction procedures fail to reduce the conditioning index to zero. Memory lasts for a minimum of 14 hours.     

Molecular characterization of choline acetyltransferase from Drosophila melanogaster

Purified acetyl-CoA: choline O-acetyltransferase (EC 2.3.1.6) from Drosophila melanogaster has been shown to contain two major polypeptides of 67 and 54 K Daltons. However, all enzyme activity is found in a single molecular weight form of approx 67 K Daltons as determined by sucrose gradient sedimentation and molecular exclusion chromatography. The latter showed both the 67 and 54 K Dalton polypeptides on polyacrylamide gel electrophoresis in sodium lauryl sulfate (10% acrylamide). Analysis of purified choline acetyltransferase on polyacrylamide gel electrophoresis in sodium lauryl sulfate (15% acrylamide) revealed the presence of an additional polypeptide at 13 K Daltons. Tryptic-peptide maps of the 67, 54 and 13 K Dalton components showed all three to be structurally related. In addition to several common tryptic peptides, the 13 K Dalton polypeptide contained three tryptic-peptides that were also found in the 67 K Dalton polypeptide, but were absent from the 54 K Dalton polypeptide. This evidence suggests that native Drosophila choline acetyltransferase may exist in two forms, one a single polypeptide chain with a molecular weight of 67 K Daltons and the other consisting of two noncovalently bound polypeptide chains with molecular weights of 54 and 13 K Daltons. The latter form is the major one isolated and may be generated by limited proteolysis of the single chain 67 K Dalton form.     

The stability of Drosophila melanogaster courtship across matings

Sex effects on behaviour were investigated by repeatedly mating the same male and female Drosophila melanogaster. Male influence is mainly on percentage wing vibration while females largely determine wing vibration duration and bout number. This courtship pattern is stable across matings involving the same individuals.     

Operant conditioning in Drosophila melanogaster wild-type and learning mutants with defects in the cyclic AMP metabolism

Wild-type Drosophila melanogaster and the learning mutants dunce, amnesiac and rutabaga, were tested using a new operant conditioning paradigm for single flies. All strains are able to learn to different extents, but no evidence of memory was found in the mutants amnesiac and rutabaga, while dunce has a reduced but extended memory. The relationship between this characteristic and cAMP levels are discussed. The three mutants have previously been shown, using classical conditioning paradigms to be deficient in olfactory learning and/or memory, and show reduced visual learning. The variability of the response of the mutants in the different paradigms is discussed in relation to the generality of the Aplysia model of the cellular mechanism underlying learning. In the operant conditioning paradigm described here, 93% of the wild-type flies learned to criterion. The performance of individual flies was consistent.     

Characterization of the Drosophila melanogaster Dis3 ribonuclease

The Dis3 ribonuclease is a member of the hydrolytic RNR protein family. Although much progress has been made in understanding the structure, function, and enzymatic activities of prokaryotic RNR family members RNase II and RNase R, there are no activity studies of the metazoan ortholog, Dis3. Here, we characterize the activity of the Drosophila melanogaster Dis3 (dDis3) protein. We find that dDis3 is active in the presence of various monovalent and divalent cations, and requires divalent cations for activity. dDis3 hydrolyzes compositionally distinct RNA substrates, yet releases different products depending upon the substrate. Additionally, dDis3 remains active when lacking N-terminal domains, suggesting that an independent active site resides in the C-terminus of the protein. Finally, a study of dDis3 interactions with dRrp6 and core exosome subunits in extracts revealed sensitivity to higher divalent cation concentrations and detergent, suggesting the presence of both ionic and hydrophobic interactions in dDis3-exosome complexes. Our study thus broadens our mechanistic understanding of the general ribonuclease activity of Dis3 and RNR family members.     

Developmental profiles of PERIOD and DOUBLETIME in Drosophila melanogaster ovary

The clock protein PERIOD (PER) displays circadian cycles of accumulation, phosphorylation, nuclear translocation and degradation in Drosophila melanogaster clock cells. One exception to this pattern is in follicular cells enclosing previtellogenic ovarian egg chambers. In these cells, PER remains high and cytoplasmic at all times of day. Genetic evidence suggest that PER and its clock partner TIMELESS (TIM) interact in these cells, yet, they do not translocate to the nucleus. Here, we investigated the levels and subcellular localization of PER in older vitellogenic follicles. Cytoplasmic PER levels decreased in the follicular cells at the onset of vitellogenesis (stage 9). Interestingly, PER was observed in the nuclei of some follicular cells at this stage. PER signal disappeared in more advanced (stage 10) vitellogenic follicles. Since the phosphorylation state of PER is critical for the progression of circadian cycle, we investigated the status of PER phosphorylation in the ovary and the expression patterns of DOUBLETIME (DBT), a kinase known to affect PER in the clock cells. DBT was absent in previtellogenic follicular cells, but present in the cytoplasm of some stage 9 follicular cells. DBT was not distributed uniformly but was present in patches of adjacent cells, in a pattern resembling PER distribution at the same stage. Our data suggest that the absence of dbt expression in the follicular cells of previtellogenic egg chambers may be related to stable and cytoplasmic expression of PER in these cells. Onset of dbt expression in vitellogenic follicles coincides with nuclear localization of PER protein.     

Biochemical aspects of oögenesis in Drosophila melanogaster

The growth of ovaries of a wild type strain of Drosophila melanogaster has been studied from eclosion to 7 days later. Wet weight, protein, DNA, RNA, and carbohydrate have been determined at successive intervals. All experiments were performed on flies grown axenically on a killed yeast medium. The results indicate that wet weight increases rapidly from day 1 to day 4 and reaches a maximum at day 6. This increase is associated with the appearance of vitellogenic and post-vitellogenic stages of oögenesis. The increase in protein and RNA generally follows the wet weight curve and remains more or less constant after reaching a maximum at day 6. The DNA content reaches a maximum at day 3 and falls rapidly thereafter. The carbohydrate content increases slowly, reaching a maximum at day 7. The data are compared with available evidence from other strains and genotypes of D. melanogaster.     

Mutations induced by a hormonal imbalance in Drosophila melanogaster

Pterin treatment of chrysalids in diapause modified the juvenile hormone-ecdysone ratio. The treatment of Drosophila donor mutant with a mixture of reduced folic acid, pterins and extract of Pierides chrysalids in diapause induced the formation of short sequences, by the intermediary of variations in hormonal balance. The effect of this variation was seen in the germinal lines especially at the gonial stage, where recessive, visible or lethal mutations were induced in the form of clusters. Genetically active fractions were found in the 4S–8S and 18S–28S sedimentation zones after saccharose gradient ultracentrifugation. The short DNA sequences coded for tRNA and rRNA. Consequently, it is these DNA sequences from the Drosophila donor mutant that altered the genetic information of the host.2 tpes of recessive visible mutation appeared: those affecting the differentiation of the imaginal discs and those affecting the pigment biosynthetic chain. Recessive lethals were induced by treatment.3 hypotheses are proposed: the first suggests the formation of a short DNA sequence complexing at a specific locus in the acceptor. The second involves transporable factors belonging to the acceptor itself, behaving as a particular transporable factor. The third supposes the induction of alterations at loci of rRNA and tRNA synthesis at the origin of perturbations in protein synthesis. The present data do not allow us to choose between 3 hypotheses.In conclusion, it seems that a ‘hormonal imbalance’ can have grave consequences not only for the individual itself but also for its descendants.     

The chemical basis for sex recognition in Drosophila melanogaster

Chemical cues were recognized to play a predominant role in initiating male courtship behaviour in Drosophila melanogaster as measured by the number and duration of wing-vibration responses elicited in test males. The effect was associated with compounds specific to the female cuticle, for which we describe a simple extraction procedure. Female active extracts were compared with behaviourally non-active extracts from males, using gas-liquid and thin-layer chromatography. Using these preparative methods, long-chain hydrocarbons were isolated and activity was found only among unsaturated molecules. One, heptacosadiene, inducing the highest level of courtship, appears to be the main aphrodisiac pheromone of the female D. melanogaster. This compound is specific to females of the species and is the most abundant of their cuticular hydrocarbons.