A mating plug protein reduces early female remating in Drosophila melanogaster

Mating plugs are formed within the female reproductive tract during mating from male ejaculate constituents or even from male genitalia themselves. Across species, mating plugs have roles in sperm storage and the prevention of female remating. In the fruitfly Drosophila melanogaster, accessory gland proteins such as the sex peptide are known to reduce female remating, however this effect can take some time to establish, hence other ejaculate components must also be involved. We hypothesised a role for the PEBII mating plug protein in the prevention of early female remating. Using RNA interference we produced PEBII knockdown males. We found that these males were significantly less able to prevent female remating in the 4 h following mating. The mating plugs produced by PEBII knockdown males also showed lower levels of autofluorescence in the first 10 min after the start of mating, suggesting they differed in composition to those of control males. Reduced levels of PEBII had no effect, however, on fecundity, progeny production or egg-adult viability in the first 24 after mating, suggesting there were no short-term effects of PEB II on sperm transfer, storage or use. Our results show that PEBII has a subtle but significant role in the prevention of early female remating.     

The function of pulse song and sine song in the courtship of Drosophila melanogaster

Drosophila melanogaster females were subjected to pulse song before being allowed to mix with males. Sine song increases female receptivity, pulse song does not. Pulse song does however increase receptivity if the females are subjected to it while being courted by males which are deaf and which cannot produce any auditory stimulation themselves. It is suggested that sine song is summated and has a priming effect on female receptivity whereas pulse song functions as a species recognition signal in a trigger-like fashion.     

Visual signals in the courtship of Drosophila melanogaster

Drosophila melanogaster carrying either of the mutations sev or dipp⁶ show defective phototactic behaviour owing to deficiencies in the processing of visual information perceived by the central retinula cells (R7, R8). Mutant females show increased time to mating because the deficient visual input via this subsystem has an inhibitory effect on female receptivity. Similarly, deficient input through the peripheral retinula cells (R1–R6) also makes females sexually unreceptive. Thus females require appropriate visual stimulation through both subsystems to become maximally sexually receptive. One major source of this stimulation is the red eye of the male.     

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.     

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.     

The role of auditory stimuli in the courtship of Drosophila melanogaster

Simulated courtship song of male Drosophila melanogaster was played to males or females of this species. Upon receiving the song males increase their locomotor activity and start courting each other, whereas females reduce their locomotor activity. In wingless males the locomotor activity difference between the silent control and the experimental sound situation is much larger than in winged males, due to the inactivity of wingless males in the control situation. Males which had been kept singly up to the time of the experiment exhibit higher locomotor and sexual activity than group housed males. A second component of the male courtship song ‘sine song’ is described, together with experiments which investigate the sensory basis of the effect male courtship song has on males.     

Principles of Genome Evolution in the Drosophila melanogaster Species Group

That closely related species often differ by chromosomal inversions was discovered by Sturtevant and Plunkett in 1926. Our knowledge of how these inversions originate is still very limited, although a prevailing view is that they are facilitated by ectopic recombination events between inverted repetitive sequences. The availability of genome sequences of related species now allows us to study in detail the mechanisms that generate interspecific inversions. We have analyzed the breakpoint regions of the 29 inversions that differentiate the chromosomes of Drosophila melanogaster and two closely related species, D. simulans and D. yakuba, and reconstructed the molecular events that underlie their origin. Experimental and computational analysis revealed that the breakpoint regions of 59% of the inversions (17/29) are associated with inverted duplications of genes or other nonrepetitive sequences. In only two cases do we find evidence for inverted repetitive sequences in inversion breakpoints. We propose that the presence of inverted duplications associated with inversion breakpoint regions is the result of staggered breaks, either isochromatid or chromatid, and that this, rather than ectopic exchange between inverted repetitive sequences, is the prevalent mechanism for the generation of inversions in the melanogaster species group. Outgroup analysis also revealed evidence for widespread breakpoint recycling. Lastly, we have found that expression domains in D. melanogaster may be disrupted in D. yakuba, bringing into question their potential adaptive significance.     

The hormonal control of salivary gland secretion in Drosophila melanogaster: Studies in vitro

The larval salivary gland of Drosophila melanogaster synthesises a complex secretion, known as ‘glue’. which is secreted at puparium formation and then cements the puparium to its substrate. This secretion is made during the third larval instar and is stored in the gland cells as large granules. A few hours before puparium formation it is secreted into the gland's lumen by exocytosis. This process is induced by ecdysone and can be studied in vitro. Secretion is initiated about 3.5 hr after exposure of glands to ecdysone and is complete by 8 hr. The effects of varying the ecdysone concentration, of inhibitors of RNA or protein synthesis, and of withdrawing the hormone at various times after initial exposure on the process of secretion have been studied. We conclude that some event(s) occurring during the first 3 hr exposure to ecdysone is necessary to initiate secretion of the glue into the gland lumen. The possible relationship between this event(s) and the ecdysone induced changes in gene activity (puffs) which occur in the salivary glands at the same time is discussed.     

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.     

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.     

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.     

The relationship between locomotor activity and sexual behaviour in ebony strains of Drosophila melanogaster

Male flies homozygous for the ebony¹¹ mutant allele suffer a severe visual impairment and consequently show poor mating success. However, ebony males show improved mating in darkness because they initiate courtship more quickly than in the light. They also exhibit a competitive superiority over wild-type males under darkened conditoons that correlates with the observed increase in the mutant's locomotor activity. Locomotor activity levels were subsequently shown to correlate with several indices of mating success under both dark and illuminated conditions. The evolutionary implications of this relationship between the two characters are discussed.     

Cyclical expression of Na/K-ATPase in the visual system of Drosophila melanogaster

In the first (lamina) and second (medulla) optic neuropils of Drosophila melanogaster, sodium pump subunit expression changes during the day and night, controlled by a circadian clock. We examined α-subunit expression from the intensity of immunolabeling. For the β-subunit, encoded by Nervana 2 (Nrv2), we used Nrv2-GAL4 to drive expression of GFP, and measured the resultant fluorescence in whole heads and specific optic lobe cells. All optic neuropils express the α-subunit, highest at the beginning of night in both lamina and medulla in day/night condition and the oscillation was maintained in constant darkness. This rhythm was lacking in the clock arrhythmic per⁰ mutant. GFP driven by Nrv2 was mostly detected in glial cells, mainly in the medulla. There, GFP expression occurs in medulla neuropil glia (MNGl), which express the clock gene per, and which closely contact the terminals of clock neurons immunoreactive to pigment dispersing factor. GFP fluorescence exhibited circadian oscillation in whole heads from Nrv2-GAL4 + UAS-S65T-GFP flies, although significant GFP oscillations were lacking in MNGl, as they were for both subunit mRNAs in whole-head homogenates. In the dissected brain tissues, however, the mRNA of the α-subunit showed a robust daily rhythm in concentration changes while changes in the β-subunit mRNA were weaker and not statistically significant. Thus in the brain, the genes for the sodium pump subunits, at least the one encoding the α-subunit, seem to be clock-controlled and the abundance of their corresponding proteins mirrors daily changes in mRNA, showing cyclical accumulation in cells.     

The homoeotic effect of “tumorous head” in Drosophila melanogaster

The morphology of the cuticular abnormalities appearing in the heads of flies from a tumorous head stock of Drosophila melanogaster was investigated. The antenna is sometimes partly replaced by leg structures, and parts of the head are frequently replaced by parts of abdominal tergites. The “rostralhaut” is sometimes replaced by genital elements, such as anal plates and clasper teeth. This is the first reported example of a homoeotic alteration to abdomen or genitalia.     

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.     

The effects of CO2 and chronic cold exposure on fecundity of female Drosophila melanogaster

Carbon dioxide and chilling are sometimes used to immobilise insects for laboratory research. Both of these methods are known to have short-term effects on behaviour and physiology in Drosophila, but their long-term impacts are unknown. We exposed female D. melanogaster adults to high CO₂ concentrations (4 h at 18,000 ppm) and chronic cold (72 h at 4 °C). The carbon dioxide exposure increased chill coma recovery time, but did not result in changes in offspring number, sex ratio, or size. By contrast, the cold exposure resulted in fewer, smaller offspring, and resulted in a male-biased sex ratio compared to controls. There was no significant interaction between CO₂ and cold. We conclude that although caution must be used in choosing an immobilisation method, CO₂ appears to have less long-term impact than cold.     

Strain-differences and inducibility of microsomal oxidative enzymes in Drosophila melanogaster flies

Some basic characteristics of the enzyme system involved in the oxidative metabolism of xenobiotic compounds were investigated in Drosophila melanogaster flies. Attention was focussed on (1) the normal levels of these enzymes and their activities in whole flies, in different parts of the fly's body and in different sexes, (2) the changes in levels and activities of the enzymes elicited by pretreatment of the flies with known enzyme inducers and (3) differences between strains.Four commonly used wild-type (WT) strains, three insecticide resistant strains (IR) and one white-eyed mutant strain were employed. Except in those experiments on sex differences and in spatial distribution in the fly's body of the enzymatic activities, microsomes were isolated from whole-body homogenates of mixtures of female and male flies. Microsomal cytochrome P-450, benzo[a]pyrene (BP) hydroxylation, p-nitroanisole (pNA) demethylation and aminopyrine (AP) demethylation were measured in control flies and in flies pretreated with Aroclor 1254 (AC), phenobarbital (PB) or butylated hydroxytoluene (BHT).In flies of the WT strain Berlin-K, there were no significant differences in BP hydroxylation activity and its inducibility between the two sexes. In males, inducibility of BP hydroxylation activity was similar in the head, thorax and abdomen, but significantly lower in testis. Considerable differences in some enzyme activities were found between the strains. pNA demethylation and AP demethylation were substantially higher in all IR strains, while no correlation could be found between their increased insecticide resistance and BP hydroxylating capacity or cytochrome P-450 content of the microsomes.Response to enzyme inducing compounds was found to be strain-dependent. PB proved to be a more efficient inducer of BP hydroxylation than AC, which does induce pNA demethylation. BHT has inducing properties that are intermediate between PB and AC. IR strain Hikone-R turned out to be an exception, possessing very low BP hydroxylating capacity and a low degree of inducibility of mixed-function oxidase activities. Differential temperature dependence was found for BP hydroxylation as compared with pNA demethylation. While BP hydroxylation was doubled when raising the temperature from 25°C to 35°C, pNA demethylation was reduced by 50%.     

The behavioural basis of overdominance in competitive mating success at the ebony locus of Drosophila melanogaster

Male files homozygous for the gene ebony¹¹ are partially blind, and at a disadvantage in competitive mating. The courtship of the mutant males is deficient in wing vibration stimulation, which is characterized by a low proportion of sine song and a high intra-pulse frequency. Males heterozygous for ebony have normal vision, but show an increase in courtship song, and are superior in competitive mating to wild type males. The auditory characteristics of courtship song produced by heterozygous males are indistinguishable from those of wild type, and their superiority in competitive mating success is due to overdominance involving this specific element of male courtship behaviour.