Investigation of Natural Genetic Variation in the Circadian System of Drosophila melanogaster. II. Biometrical Analyses of Locomotor Activity Rhythms Recorded in Constant Darkness

The locomotor activity rhythms of 24 isochromosomal strains of Drosophila melanogaster were recorded in constant conditions, using an experimental design suitable for the serial screening with so many strains using limited equipment. The data obtained were subjected to biometrical genetic analysis. The results show that four characteristics of the rhythms (period, phase, definition, and waveform) display genetically based variation in expression and that each appears to be affected by a range of genes.     

An Investigation of Natural Genetic Variation in the Circadian System of Drosophila melanogaster: Rhythm Characteristics and Methods of Quantification

Variation in four characteristics of the circadian locomotor activity rhythm was investigated in 24 true-breeding strains of Drosophila melanogaster with a view to establishing methods of phenotypic measurement sufficiently robust to allow subsequent biometric analysis. Between them, these strains formed a representative sample of the genetic variability of a natural population. Period, phase, definition (the degree to which a rhythmic signal was obscured by noise), and rhythm waveform were all found to vary continuously among the strains, although within each strain the rhythm phenotype was remarkably consistent. Each characteristic was found to be sufficiently robust to permit objective measurement using several different methods of quantification, which were then compared.     

An Investigation of Natural Genetic Variation in the Circadian System of Drosophila melanogaster: Rhythm Characteristics and Methods of Quantification

Variation in four characteristics of the circadian locomotor activity rhythm was investigated in 24 true-breeding strains of Drosophila melanogaster with a view to establishing methods of phenotypic measurement sufficiently robust to allow subsequent biometric analysis. Between them, these strains formed a representative sample of the genetic variability of a natural population. Period, phase, definition (the degree to which a rhythmic signal was obscured by noise), and rhythm waveform were all found to vary continuously among the strains, although within each strain the rhythm phenotype was remarkably consistent. Each characteristic was found to be sufficiently robust to permit objective measurement using several different methods of quantification, which were then compared.     

Genetic Variants Affecting Phenoloxidase Activity in Drosophila melanogaster

In Drosophila melanogaster, two new variants affecting the activity of phenoloxidase were found in natural populations at Gomel in Belorussia and at Krasnodar in Russia. Prophenoloxidases, A ₁ and A ₃ , in these variants had the same mobilities on native electrophoresis as the wild type. However, enzymatic activities in their activated states were much lower than in the wild type, whereas the existence of prophenoloxidase proteins was demonstrated. Egg-to-adult and relative viabilities in the variants did not decrease at temperatures between 18 and 29°C. Genetic analyses indicated that the genes showing the phenotype of variants are new alleles of Mox and Dox-3 on the second chromosome.     

Circadian Rhythms of Locomotor Activity in the Nile Tilapia Oreochromis niloticus

Behavioral rhythms of the Nile tilapia were investigated to better characterize its circadian system. To do so, the locomotor activity patterns of both male and female tilapia reared under a 12:12 h light-dark (LD) cycle were studied, as well as in males the existence of endogenous rhythmicity under free-running conditions (DD and 45 min LD pulses). When exposed to an LD cycle, the daily pattern of activity differed between individuals: some fish were diurnal, some nocturnal, and a few displayed an arrhythmic pattern. This variability would be typical of the plastic circadian system of fish. Moreover, reproductive events clearly affected the behavioral rhythms of female tilapia, a mouth-brooder teleost species. Under DD, 50% (6 of 12) of male fish showed circadian rhythms with an average period (τ) of 24.1±0.2 h, whereas under the 45 min LD pulses, 58% (7 of 12) of the fish exhibited free-running activity rhythms with an average τ of 23.9±0.5 h. However, interestingly in this case, activity was always confined to the dark phase. Furthermore, when the LD cycle was reversed, a third of the fish showed gradual resynchronization to the new phase, taking 7–10 days to be completely re-entrained. Taken together, these results suggest the existence of an endogenous circadian oscillator that controls the expression of locomotor activity rhythms in the Nile tilapia, although its anatomical localization remains unknown.     

Entrainment Properties of the Locomotor Activity Rhythm of Drosophila melanogaster Under Different Photoperiodic Regimens

In this paper we report the results of an experiment to assess how closely repeated brief light pulses (LPs) mimic the effects of 12:12 h light/dark (LD) cycles (PPc). The locomotor activity rhythm of individual fruit flies from a laboratory population of Drosophila melanogaster was monitored under four different photoperiodic regimens, created using 12 h of light and 12 h of darkness or brief light pulses (LPs). The phase relationship (Ψ) and the stability (precision) of the locomotor activity rhythm during entrainment were estimated in order to compare the state of the circadian clocks under the four different photoperiodic regimens. The flies (n = 72) were subjected to four different LD cycles: (i) 12 h of light and 12 h of darkness (complete photoperiod, PPc); (ii) a single brief LP of 15 min duration presented close to the onset of activity (SLP-1); (iii) a single brief LP of 15 min duration administered close to the offset of activity (SLP-2); and (iv) two brief LPs administered 12 h apart (skeleton photoperiod, PPs). The locomotor activity rhythm of the flies was first monitored under constant darkness (DD) for about 10 days and then under the four different photoperiodic regimens for about 10 days, and finally in DD for the remainder of the experiment. The Ψ of the locomotor activity rhythm and its precision under PPc and PPs did not differ significantly, but they were significantly different from the SLP-1 and SLP-2 conditions. The results provide interesting insights into photoentrainment mechanisms of circadian clocks in D. melanogaster, and suggest that skeleton photoperiods, but not single brief LPs, mimic the actions of complete photoperiods.     

Cyclic Presence and Absence of Conspecifics Alters Circadian Clock Phase But Does Not Entrain the Locomotor Activity Rhythm of the Fruit Fly Drosophila melanogaster

Circadian clocks use a wide range of environmental cues, including cycles of light, temperature, food, and social interactions, to fine-tune rhythms in behavior and physiology. Although social cues have been shown to influence circadian clocks of a variety of organisms including the fruit fly Drosophila melanogaster, their mechanism of action is still unclear. Here, the authors report the results of their study aimed at investigating if daily cycles of presence and absence (PA) of conspecific male visitors are able to entrain the circadian locomotor activity rhythm of male hosts living under constant darkness (DD). The results suggest that PA cycles may not be able to entrain circadian locomotor activity rhythms of Drosophila. The outcome does not change when male hosts are presented with female visitors, suggesting that PA cycles of either sex may not be effective in bringing about stable entrainment of circadian clocks in D. melanogaster. However, in hosts whose clock phase has already been set by light/dark (LD) cycles, daily PA cycles of visitors can cause measurable change in the phase of subsequent free-running rhythms, provided that their circadian clocks are labile. Thus, the findings of this study suggest that D. melanogaster males may not be using cyclic social cues as their primary zeitgeber (time cue) for entrainment of circadian clocks, although social cues are capable of altering the phase of their circadian rhythms. (Author correspondence: vsharma@jncasr.ac.in, vksharmas@gmail.com)     

A Mutation in Drosophila simulans that Lengthens the Circadian Period of Locomotor Activity

The length of the Thr-Gly repeat within the period gene of Drosophilids, coevolves with its immediate flanking region to maintain the temperature compensation of the fly circadian clock. In Drosophila simulans, balancing selection appears to maintain a polymorphism in this region, with three repeat lengths carrying 23, 24 or 25 Thr-Gly pairs, each in complete linkage disequilibrium with a distinctive flanking region amino acid moiety. We wondered whether separating a specific length repeat from its associated flanking haplotype might have functional implications for the circadian clock. We fortuitously discovered a population of flies collected in Kenya, in which a chimeric Thr-Gly haplotype was segregating that carried the (Thr-Gly)24 repeat, but the flanking region of a (Thr-Gly)23 allele. One of the five isofemale lines that carried this 'mutant' Thr-Gly sequence showed a dramatically long and temperature-sensitive free-running circadian period. This phenotype was mapped to the X chromosome, close to the D. simulans per gene, but there was also a significant effect of a modifying autosomal locus or loci. It seems remarkable that such a mutant phenotype should be discovered in a screen of chimeric Thr-Gly regions.     

The Genetic Basis of Resistance to Diazinon in Natural Populations of Drosophila melanogaster

Isofemale strains of Drosophila melanogaster were established from single inseminated females collected from populations along the east coast of Australia. Strains were tested for resistance to the organophosphorus insecticide diazinon at larval and/or adult stages of the life cycle. Considerable phenotypic variation was observed within and between population samples but there was no association between collection site of a sample and resistance status. Adult and larval resistance levels were uncorrelated. Resistance levels in adults were low (2-fold) and polygenically based. Larval resistance levels, due to single genes (or gene complexes) on chromosomes II and III, were significant (15-fold). Evidence indicates that the gene on chromosome II is Cyp6g1.     

Circadian Rhythms in Locomotor Activity of the Hagfish, Eptatretus burgeri VI. The Effects of Cutting the Spinal Cord

The present study is designed to clarify the mechanism by which the circadian pacemaker controls the locomotor activity of the hagfish and also to estimate the role of brain and spinal cord in the swimming behavior of the animal. We examined the effect of cutting the spinal cord at the 6 different positions on the circadian rhythm and the locomotor behavior of the animal. The most frontal cut was located between the brain and spinal cord, and the other 5 cuts were given to every 1/6 the length of the spinal cord. The relation between the locomotor activity and the cut position of spinal cord was summarized as follows. (1) When the ratio of frontal part before the cut was 0/6-1/6, the animal locomoted under initiative of caudal part, in random direction at the bottom and showed neither nocturnal rhythm in LD nor circadian rhythm in DD. (2) When the ratio of the frontal part before the cut was 4/6-5/6, the animal swam up to the surface under initiative of frontal part, and showed both nocturnal rhythm in LD and circadian rhythm in DD. (3) When the frontal ratio of spinal cord was 2/6 or 3/6, the animal showed both kinds of swimming behavior of (1) and (2). These results suggest that the descending system from the brain enable the hagfish to swim up to the surface and to express the rhythmicity of locomotor activity under control of the circadian pacemaker when at least frontal 2/6 of the spinal cord is connected to the brain by neuronal networks not by humoral factors.     

Genetic sexing in Drosophila melanogaster using the alcohol dehydrogenase locus and a Y-linked translocation

Summary By incorporating ethanol (4% v/v) into the larval rearing medium of a specially constructed Drosophila melanogaster strain it was possible to produce only male adults; the female larvae died.In this strain, the male determining chromosome was linked with a positive Alcohol dehydrogenase (ADH) allele by a translocation. The females were homozygous for the null allele and hence sensitive to ethanol.This genetic sexing method is discussed in relation to its use in the genetic control of insects.     

Quantification of Locomotion and the Effect of Food Deprivation on Locomotor Activity in Drosophila

A new method to quantify locomotor behavior in Drosophila is presented, and compared with previous methods. It is based upon a radar wave, reflected by moving flies. A problem associated with the new apparatus is that its output is dependent on fly size. However, for the case the weight of the experimental flies has been determined, a correction is proposed. The method has been used by studying the effect of starvation upon locomotion in Drosophila melanogaster. It was found that starved flies are much more active than well fed flies. The importance of this effect under several conditions is discussed.     

A Comprehensive Study of Genic Variation in Natural Populations of Drosophila melanogaster. III. Variations in Genetic Structure and Their Causes between Drosophila melanogaster and Its Sibling Species Drosophila simulans

The natural populations of Drosophila melanogaster and Drosophila simulans were compared for their genetic structure. A total of 114 gene-protein loci were studied in four mainland (from Europe and Africa) and an island (Seychelle) populations of D. simulans and the results were compared with those obtained on the same set of homologous loci in fifteen worldwide populations of D. melanogaster. The main results are as follows: (1) D. melanogaster shows a significantly higher proportion of loci polymorphic than D. simulans (52% vs. 39%, P<0.05), (2) both species have similar mean heterozygosity and mean number of alleles per locus, (3) the two species share some highly polymorphic loci but they do not share loci that show high geographic differentiation, and (4) D. simulans shows significantly less geographic differentiation than D. melanogaster. The differences in genetic differentiation between the two species are limited to loci located on the X and second chromosomes only; loci on the third chromosome show similar level of geographic differentiation in both species. These two species have previously been shown to differ in their pattern of variation for chromosomal polymorphisms, quantitative and physiological characters, two-dimensional electrophoretic (2DE) proteins, middle repetitive DNA and mitochondrial DNA. Variation in niche-widths and/or genetic strategies of adaptation appear to be the main causes of differences in the genetic structure of these two species.     

Genetic Control of Development of Malpighian Tubules in Drosophila melanogaster

Malpighian tubules of insects are a functional analog of mammalian kidneys and serve as a classical model for studying the structure and functions of transport epithelium. The review contains the data on structural organization, functioning, and formation of the Malpighian tubules during embryogenesis in Drosophila melanogaster. Various systems of genes are described that control the program of development of the renal (Malpighian) tubules in D. melanogaster. A special attention is paid to the ways of signal transduction and factors involved in cell differentiation, proliferation, and morphological transformation during development of the Malpighian tubules. Evolutionarily conservative genetic systems are considered that are involved in the control of development of both the renal epithelium ofDrosophila and mammalian kidneys. A relationship was noted between the disturbed balance of genetic material and congenital defects of the human excretory system.     

Investigation of cis-acting sequences regulating expression of the gene encoding yolk protein 3 in Drosophila melanogaster.

Summary The regulatory sequences leading to the ovarian and fat body expression of yolk proteins 1 and 2 (YP1 and 2) of Drosophila melanogaster have been characterised in some detail. These genes (yp1 and yp2) share many enhancer elements, and some important regulatory sequences lie within the coding regions. We have begun to investigate the cis-regulation of the gene encoding yolk protein 3 (yp3). We describe a system for P element transformation using the complete and unaltered yp3 gene rather than reporter genes and describe sequences conferring correct expression in the ovary and carcass.     

Multi-Oscillatory Control of Eclosion and Oviposition Rhythms in Drosophila melanogaster: Evidence from Limits of Entrainment Studies

The eclosion and oviposition rhythms of flies from a population of Drosophila melanogaster maintained under constant conditions of the laboratory were assayed under constant light (LL), constant darkness (DD), and light/dark (LD) cycles of 10:10 h (T20), 12:12 h (T24), and 14:14 h (T28). The mean (±95% confidence interval; CI) free-running period (τ) of the oviposition rhythm was 26.34 ± 1.04 h and 24.50 ± 1.77 h in DD and LL, respectively. The eclosion rhythm showed a τ of 23.33 ± 0.63 h (mean ± 95% CI) in DD, and eclosion was not rhythmic in LL. The τ of the oviposition rhythm in DD was significantly greater than that of the eclosion rhythm. The eclosion rhythm of all 10 replicate vials entrained to the three periodic light regimes, T20, T24, and T28, whereas the oviposition rhythm of only about 24 and 41% of the individuals entrained to T20 and T24 regimes, respectively, while about 74% of the individuals assayed in T28 regimes showed entrainment. Our results thus clearly indicate that the τ and the limits of entrainment of eclosion rhythm are different from those of the oviposition rhythm, and hence this reinforces the view that separate oscillators may regulate these two rhythms in D. melanogaster.