Drosophila genetics

Chemical communication mediates social interactions in
insects [1]. For the fruit fly, D. melanogaster, the chemical
display is a key fitness trait because it leads to mating. An exchange
of cues that resembles a dialogue between males
and females is enacted by pheromones, chemical signals
that pass between individual flies to alter physiology and
behavior [2, 3]. Chemical signals also affect the timing of locomotor
activity [4] and sleep [5]. We investigated genetic
and environmental determinants of chemical communication.
To evaluate the role of the social environment, we extracted
a chemical blend from individual males selected
from groups composed of one genotype and compared
these extracts to those from groups of mixed genotypes.
To evaluate the role of the physical environment, these
comparisons were performed under a light-dark cycle or in
constant darkness. Here, we show that chemical signaling
is affected by the social environment, light-dark cycle, and
genotype as well as the complex interplay of these variables.
Gene-by-environment interactions produce highly significant
effects on chemical signaling. We also examined individual
responses within the groups. Strikingly, the response
of one wild-type fly to another is modulated by the genotypic
composition of his neighbors. Chemical signaling in D. melanogaster may be a ‘‘fickle’’ trait that depends on the individual’s social background.

Selection acting on codon usage can cause patterns of synonymous evolution to deviate considerably from those expected under neutrality. To investigate the quantitative relationship between parameters of mutation, selection, and demography, and patterns of synonymous site divergence, we have developed a novel combination of population genetic models and likelihood methods of phylogenetic sequence analysis. Comparing 50 orthologous gene pairs from Drosophila melanogaster and D. virilis and 27 from D. melanogaster and D. simulans, we show considerable variation between amino acids and genes in the strength of selection acting on codon usage and find evidence for both long-term and short-term changes in the strength of selection between species. Remarkably, D. melanogaster shows no evidence of current selection on codon usage, while its sister species D. simulans experiences only half the selection pressure for codon usage of their common ancestor. We also find evidence for considerable base asymmetries in the rate of mutation, such that the average synonymous mutation rate is 20-30% higher than in noncoding regions. A Bayesian approach is adopted to investigate how accounting for selection on codon usage influences estimates of the parameters of mutation.

Adaptation of insect phenotypes for survival after exposure to xenobiotics can result from selection at multiple loci with additive genetic effects. To the authors' knowledge, no selective sweep analysis has been performed to identify such loci in highly dichlorodiphenyltrichloroethane (DDT) resistant insects. Here we compared a highly DDT resistant phenotype in the Drosophila melanogaster (Drosophila) 91-R strain to the DDT susceptible 91-C strain, both of common origin. Whole genome re-sequencing data from pools of individuals was generated separately for 91-R and 91-C, and mapped to the reference Drosophila genome assembly (v. 5.72). Thirteen major and three minor effect chromosome intervals with reduced nucleotide diversity (π) were identified only in the 91-R population. Estimates of Tajima's D (D) showed corresponding evidence of directional selection in these same genome regions of 91-R, however, no similar reductions in π or D estimates were detected in 91-C. An over...

Despite ongoing high energetic demands, brains do not always use glucose and oxygen in a ratio that produces maximal ATP through oxidative phosphorylation. In some cases glucose consumption exceeds oxygen use despite adequate oxygen availability, a phenomenon known as aerobic glycolysis. Although metabolic plasticity seems essential for normal cognition, studying its functional significance has been challenging because few experimental systems link brain metabolic patterns to distinct behavioral states. Our recent transcriptomic analysis established a correlation between aggression and decreased whole-brain oxidative phosphorylation activity in the honey bee (Apis mellifera), suggesting that brain metabolic plasticity may modulate this naturally occurring behavior. Here we demonstrate that the relationship between brain metabolism and aggression is causal, conserved over evolutionary time, cell type-specific, and modulated by the social environment. Pharmacologically treating honey bees to inhibit complexes I or V in the oxidative phosphorylation pathway resulted in increased aggression. In addition, transgenic RNAi lines and genetic manipulation to knock down gene expression in complex I in fruit fly (Drosophila melanogaster) neurons resulted in increased aggression, but knockdown in glia had no effect. Finally, honey bee colony-level social manipulations that decrease individual aggression attenuated the effects of oxidative phosphorylation inhibition on aggression, demonstrating a specific effect of the social environment on brain function. Because decreased neuronal oxidative phosphorylation is usually associated with brain disease, these findings provide a powerful context for understanding brain metabolic plasticity and naturally occurring behavioral plasticity.

Highlights
Winner clones do not need draper, wasp, or psr to preserve growth advantage
Winner cells do not need draper, wasp, or psr to kill loser cells
Hemocytes are required for removal of the resulting apoptotic debris
Summary

Cell competition is a mechanism that eliminates slow dividing cells from a growing population. It is believed that the genes wasp, psr, and draper are active in the cells that win the competition (“winner cells”) and that they are essential in the winner cells for the induction of apoptosis and for the elimination of the “loser cells.” Here, we show that lack of those genes in winner cells appears to be dispensable for cell-competition-induced apoptosis and during dmyc-induced supercompetition. Moreover, winner clones do not need those genes in order to preserve their growth advantage. Finally, we find that most of the clearance of the apoptotic debris is not performed by winners but by recruited hemocytes, which are required for the removal of the apoptotic corpses at the very end. Therefore, engulfment is a consequence—not a cause—of loser cells' death.

Metal-induced toxicity in fruit fly (Drosophila melanogaster) is one of the established models for studying neurotoxicity and neurodegenerative diseases. Phytochemicals, especially alkaloids, have been reported to exhibit neuroprotection. Here, we assessed the protective effect of alkaloid extract from African Jointfir (Gnetum africanum) leaf on manganese-(Mn-) induced toxicity in wild type fruit fly. Flies were exposed to 10 mM Mn, the alkaloid extract and cotreatment of Mn plus extract, respectively. The survival rate and locomotor performance of the flies were assessed 5 days posttreatment, at which point the flies were homogenized and assayed for acetylcholinesterase (AChE) activity, nitric oxide (NO), and reactive oxygen species (ROS) levels. Results showed that the extract significantly reverted Mn-induced reduction in the survival rate and locomotor performance of the flies. Furthermore, the extract counteracted the Mn-induced elevation in AChE activity, NO, and ROS levels. The alkaloid extract of the African Jointfir leaf may hence be a source of useful phytochemicals for the development of novel therapies for the management of neurodegeneration.

The interaction of proteins with chromatin is fundamental for several essential cellular processes. During the development of an organism, genes must to be tightly regulated both temporally and spatially. This is achieved through the action of chromatin-binding proteins such as transcription factors, histone modifiers, nucleosome remodelers, and lamins. Furthermore, protein–DNA interactions are important in the adult, where their perturbation can lead to disruption of homeo-stasis, metabolic dysregulation, and diseases such as cancer. Understanding the nature of these interactions is of paramount importance in almost all areas of molecular biological research. In recent years, DNA adenine methyltransferase identification (DamID) has emerged as one of the most comprehensive and versatile methods available for profiling protein–DNA interactions on a genomic scale. DamID has been used to map a variety of chromatin-binding proteins in several model organisms and has the potential for continued adaptation and application in the field of genomic biology.

Although Drosophila larval neuroblasts are routinely used to define mutations affecting mitosis, the dynamics of karyokinesis in this system remain to be described. Here we outline a simple method for the short-term culturing of neuroblasts, from Drosophila third instar larvae, that allows mitosis to be followed by high-resolution multi-mode light microscopy. At 24°C, spindle formation takes 7±0.5 minutes. Analysis of neuroblasts containing various GFPtagged proteins (e.g. histone, fizzy, fizzy-related and a- tubulin) reveals that attaching kinetochores exhibit sudden, rapid pole-directed motions and that congressing and metaphase chromosomes do not undergo oscillations. By metaphase, the arms of longer chromosomes can be resolved as two chromatids, and they often extend towards a pole. Anaphase A and B occur concurrently, and during anaphase A chromatids move poleward at 3.2±0.1 mm/minute, whereas during anaphase B the spindle poles separate at 1.6±01 mm/minute. In larger neuroblasts, the spindle undergoes a sudden shift in position during midanaphase, after which the centrally located centrosome preferentially generates a robust aster and stops moving, even while the spindle continues to elongate. Together these two processes contribute to an asymmetric positioning of the spindle midzone, which, in turn, results in an asymmetric cytokinesis. Bipolar spindles form predominately (83%) in association with the separating centrosomes. However, in 17% of the cells, secondary spindles form around chromosomes without respect to centrosome position: in most cases these spindles coalesce with the primary spindle by anaphase, but in a few they remain separate and define additional ectopic poles.

The interaction of proteins and RNA with chromatin underlies the regulation of gene expression. The ability to profile easily these interactions is fundamental for understanding chromatin biology in vivo. DNA adenine methyltransferase identification (DamID) profiles genome-wide protein-DNA interactions without antibodies, fixation or protein pull-downs. Recently, DamID has been adapted for applications beyond simple assaying of protein-DNA interactions, such as for studying RNA-chromatin interactions, chromatin accessibility and long-range chromosome interactions. Here, we provide an overview of DamID and introduce improvements to the technology, discuss their applications and compare alternative methodologies.

The Drosophila serrata species complex from Australia and New Guinea has been widely used in evolutionary studies of speciation and climatic adaptation.  It is believed to consist of D. serrata, D. birchii and D. dominicana, although knowledge of the latter is limited. Here we present evidence for a previously undescribed cryptic member of the D. serrata species complex. This new cryptic species is widespread in far north Queensland, Australia and is likely to have been previously mistaken for D. serrata. It shows complete reproductive isolation when crossed with both D. serrata and D. birchii. The cryptic species can be easily distinguished from D. serrata and D. birchii using either microsatellite loci or visual techniques. Although it occurs sympatrically with both D. serrata and D. birchii, it differs from these species in development time, viability, wing size and wing morphology. Its discovery explains patterns of recently described mitochondrial DNA divergence within D. serrata, and may also help to clarify some ambiguities evident in early evolutionary literature on reproductive incompatibility within the D. serrata species complex.

We examined levels and patterns of nucleotide variation in 21 strains of Drosophila kikkawai from Miyako island, Japan for the partial regions of the following seven nuclear genes: Adh , Ddc , esc , ksr , Pgi , su(f) , and Tpi. The nucleotide variation at total sites (π t) ranged from 0.0013 in the ksr , to 0.0173 in the Adh. The nucleotide divergence at total sites (K t) between D. kikkawai and D. lini ranged from 0.0286 in the Tpi to 0.0687 in the su(f). The levels of nucleotide polymorphism and divergence were heterogeneous among the investigated gene regions. The HKA test, which tests imbalance between the intra and interspecific nucleotide variation, showed that the intraspecific nucleotide variation in the Pgi region was much lower than the interspecific variation, while intraspecific variation in the Tpi region was only slightly lower than interspecific variation. The MK test showed an excess of low frequency replacement polymorphic changes in the Adh region, suggesting that most replacement mutations are deleterious. Fay and Wu's test detected an excess of newly arisen variants in the Ddc region. In total, four of the seven gene regions showed significant deviation from the neutrality.

d espite the various roles of regulator of G protein signaling (rGs) protein in the G protein signaling pathway that have been defined, the function of rGs has not been characterized in longevity signaling pathways. we found that reduced expression of loco, a drosophila rGs protein, resulted in a longer lifespan of flies with stronger resistance to stress, higher mnsod activity and increased fat content. in contrast, overexpression of the loco gene shortened the fly lifespan significantly, lowered stress resistance and reduced fat content, also indicating that the rGs domain containing Gtpase-activating protein (Gap) activity is related to the regulation of longevity. interestingly, expressional changes of yeast rGs2 and rat rGs14, homologs to the fly loco, also affected oxidative stress resistance and longevity in the respective species. it is known that loco inactivates inhibi-tory Gαi • GTP protein to reduce activity of adenylate cyclase (ac) and rGs14 interacts with activated h-ras and raf-1 kinases, which subsequently inhibits ErK phosphorylation. we propose that loco/rGs14 protein may regulate stress resistance and longevity as an activator in ac-camp-pKa pathway and/or as a molecular scaffold that sequesters active Ras and Raf from Ras • GTP-Raf-MEK-ErK signaling pathway. consistently, our data showed that downregulation of loco significantly diminishes camp amounts and increases pErK levels with higher resistance to the oxidative stress.

Drosophila Cuticular Hydrocarbons (CH) influence courtship behaviour, mating, aggregation, oviposition, and resistance to desiccation. We measured levels of 24 different CH compounds of individual male D. melanogaster hourly under a variety of environmental (LD/DD) conditions. Using a model-based analysis of CH variation, we developed an improved normalization method for CH data, and show that CH compounds have reproducible cyclic within-day temporal patterns of expression which differ between LD and DD conditions. Multivariate clustering of expression patterns identified 5 clusters of co-expressed compounds with common chemical characteristics. Turnover rate estimates suggest CH production may be a significant metabolic cost. Male cuticular hydrocarbon expression is a dynamic trait influenced by light and time of day; since abundant hydrocarbons affect male sexual behavior, males may present different pheromonal profiles at different times and under different conditions.

We have isolated phage clones from Drosophila melanogaster genomic and cDNA libraries containing a sequence homologous to the murine Int-1 protooncogene. The Drosophila gene is represented by a single locus at position 28A1-2 on chromosome 2. The gene is expressed as a 2.9-kilobase-long polyadenylylated mRNA in embryo, larval, and pupal stages. It is hardly detectable in adult flies. The longest open reading frame of the cDNA clone corresponds to a protein 469 amino acids long. Alignment of the predicted amino acid sequences shows that the Drosophila protein is 86 amino acids longer than its murine counterpart. In spite of the difference in length, the two proteins are highly conserved with an overall sequence homology of 54%. Both Drosophila and murine Int-1 proteins begin with a hydrophobic leader sequence and contain cysteine residues and sites for glycosylation (four in the murine protein and one in the Drosophila protein) in conserved positions, suggesting that they play important functional roles.

Reproduction in individual animals of sexual species depends largely upon their ability to detect and distinguish specific signal(s) among those produced by various potential sexual partners. In Drosophila melanogaster males, there is a natural polymorphism for discrimination of female and male principal pheromones that segregates with chromosome 3. We have mapped two loci on chromosome 3 that change sex-pheromone discrimination in males. We successively exploited meiotic recombination, deficiencies and enhancer-trap strains ; excision of the transposon in two selected enhancer-trap strains clearly reverted the discrimination phenotype. These results indicate that pheromonal discrimination is a character that can be genetically manipulated, and provide further insights into the evolution of the specific mate recognition system. * Corresponding author. Tel : j33 (0) 3 80 39 37 82 (office), j33 (0) 3 80 39 62

Kaji demonstrated that several organic compounds, especially lactamide, increased eye facet number in the mutant Bar. To determine if this effect was specific to the Bar mutant, we tested the effect of lactamide on eyeless-2 (ey2), a fourth chromosome, recessive mutant which also reduced eye facet number. Under conditions in which Bar-eyed flies showed up to a four-fold increase in facet number, ey2 had reduced facet number (approximately a 20% reduction).

he central nervous system (CNS) consists of an enormous number of cells, and large cellular variance, integrated into an elaborate network. The CNS is the most complex animal organ, and therefore its establishment must be controlled by many different genetic programs. Considering the high level of complexity in the human CNS, addressing issues related to human neurodevelopment represents a major challenge. Since comparative studies have revealed that neurodevelopmental programs are well conserved through evolution, on both the genetic and functional levels, studies on invertebrate neurodevelopmental programs are often translatable to vertebrates. Indeed, the basis of our current knowledge about vertebrate CNS development has been greatly aided by studies on invertebrates, and in particular on the Drosophila melanogaster (fruit fly) model system.
This thesis attempted to identify novel genes regulating neural cell specification and proliferation in the CNS, using the Drosophila model system. Moreover, I aimed to address how those genes govern neural progenitor cells (neuroblasts; NBs) to obtain/maintain their stemness identity and proliferation capacity, and how they drive NBs through temporal windows and series of programmed asymmetric division, which gradually reduces their stemness identity in favor of neural differentiation, resulting in appropriate lineage progression. In the first project, we conducted a forward genetic screen in Drosophila embryos, aimed at isolating genes involved in regulation of neural proliferation and specification, at the single cell resolution. By taking advantage of the restricted expression of the neuropeptide FMRFa in the last-born cell of the NB lineage 5-6T, the Ap4 neuron, we could monitor the entire lineage progression. This screen succeeded in identifying 43 novel genes controlling different aspects of CNS development. One of the genes isolated, Ctr9, displayed extra Ap4/FMRFa neurons. Ctr9 encodes a component of the RNA polymerase II complex Paf1, which is involved in a number of transcriptional processes. The Paf1C, including Ctr9, is highly conserved from yeast to human, and in the past couple of years, its importance for transcription has become increasingly appreciated. However, studies in the Drosophila system have been limited. In the screen, we isolated the first mutant of Drosophila Ctr9 and conducted the first detailed phenotypic study on its function in the Drosophila embryonic CNS. Loss of function of Ctr9 leads to extra NB numbers, higher proliferation ratio and lower expression of neuropeptides. Gene expression analysis identified several other genes regulated by Ctr9, which may explain the Ctr9 mutant phenotypes. In summary, we identified Ctr9 as an essential gene for proper CNS development in Drosophila, and this provides a platform for future study on the Drosophila Paf1C. Another interesting gene isolated in the screen was worniou (wor), a member of the Snail family of transcription factors. In contrast to Ctr9, which displayed additional Ap4/FMRFa neurons, wor mutants displayed a loss of these neurons. Previous studies in our group have identified many genes acting to stop NB lineage progression, but how NBs are pushed to proliferate and generate their lineages was not well known. Since wor may constitute a “driver” of proliferation, we decided to study it further. Also, we identified five other transcription factors acting together with Wor as pro-proliferative in both NBs and their daughter cells. These “drivers” are gradually replaced by the previously identified late-acting “stoppers.” Early and late factors regulate each other and the cell cycle, and thereby orchestrate proper neural lineage progression.