Pleiotropic genes are genes that affect more than one trait. For example, many genes required for pigmentation in the fruit fly also affect traits such as circadian rhythms, vision, and mating behavior. Here, we present evidence that two pigmentation genes, and , which encode enzymes catalyzing reciprocal reactions in the melanin biosynthesis pathway, also affect cuticular hydrocarbon (CHC) composition in females. More specifically, we report that loss-of-function mutants have a CHC profile that is biased toward long (>25C) chain CHCs, whereas loss-of-function mutants have a CHC profile that is biased toward short (<25C) chain CHCs. Moreover, pharmacological inhibition of dopamine synthesis, a key step in the melanin synthesis pathway, reversed the changes in CHC composition seen in mutants, making the CHC profiles similar to those seen in mutants. These observations suggest that genetic variation affecting and/or activity might cause correlated changes in pigmentation and CHC composition in natural populations. We tested this possibility using the Genetic Reference Panel (DGRP) and found that CHC composition covaried with pigmentation as well as levels of and expression in newly eclosed adults in a manner consistent with the and mutant phenotypes. These data suggest that the pleiotropic effects of and might contribute to covariation of pigmentation and CHC profiles in .

Developmental genes can have complex cis-regulatory regions, with multiple enhancers scattered across stretches of DNA spanning tens or hundreds of kilobases. Early work revealed remarkable modularity of enhancers, where distinct regions of DNA, bound by combinations of transcription factors, drive gene expression in defined spatio-temporal domains. Nevertheless, a few reports have shown that enhancer function may be required in multiple developmental stages, implying that regulatory elements can be pleiotropic. In these cases, it is not clear whether the pleiotropic enhancers employ the same transcription factor binding sites to drive expression at multiple developmental stages or whether enhancers function as chromatin scaffolds, where independent sets of transcription factor binding sites act at different stages. In this work we have studied the activity of the enhancers of the shavenbaby gene throughout D. melanogaster development. We found that all seven shavenbaby enhancers drive gene expression in multiple tissues and developmental stages at varying levels of redundancy. We have explored how this pleiotropy is encoded in two of these enhancers. In one enhancer, the same transcription factor binding sites contribute to embryonic and pupal expression, whereas for a second enhancer, these roles are largely encoded by distinct transcription factor binding sites. Our data suggest that enhancer pleiotropy might be a common feature of cis-regulatory regions of developmental genes and that this pleiotropy can be encoded through multiple genetic architectures.

Holometabolous insects pass through a sedentary pupal stage and often choose a location for pupation that is different from the site of larval feeding. We have characterized a difference in pupariation site choice within and between sibling species of Drosophila. We found that, in nature, Drosophila sechellia pupariate within their host fruit, Morinda citrifolia, and that they perform this behavior in laboratory assays. In contrast, in the laboratory, geographically diverse strains of Drosophila simulans vary in their pupariation site preference; D. simulans lines from the ancestral range in southeast Africa pupariate on fruit, or a fruit substitute, whereas populations from Europe or the New World select sites off of fruit. We explored the genetic basis for the evolved preference in puariation site preference by performing quantitative trait locus mapping within and between species. We found that the interspecific difference is controlled largely by loci on chromosomes X and II. In contrast, variation between two strains of D. simulans appears to be highly polygenic, with the majority of phenotypic effects due to loci on chromosome III. These data address the genetic basis of how new traits arise as species diverge and populations disperse.

Genes are regulated by transcription factors that bind to regions of genomic DNA called enhancers. Considerable effort is focused on identifying transcription factor binding sites, with the goal of predicting gene expression from DNA sequence. Despite this effort, general, predictive models of enhancer function are currently lacking. Here we combine quantitative models of enhancer function with manipulations using engineered transcription factors to examine the extent to which enhancer function can be controlled in a quantitatively predictable manner. Our models, which incorporate few free parameters, can accurately predict the contributions of ectopic transcription factor inputs. These models allow the predictable 'tuning' of enhancers, providing a framework for the quantitative control of enhancers with engineered transcription factors.

Sexually dimorphic courtship behaviors in Drosophila melanogaster develop from the activity of the sexual differentiation genes, doublesex (dsx) and fruitless (fru), functioning with other regulatory factors that have received little attention. The dissatisfaction (dsf) gene encodes an orphan nuclear receptor homologous to vertebrate Tlx and Drosophila tailless that is critical for the development of several aspects of female- and male-specific sexual behaviors. Here, we report the pattern of dsf expression in the central nervous system and show that the activity of sexually dimorphic abdominal interneurons that co-express dsf and dsx is necessary and sufficient for vaginal plate opening in virgin females, ovipositor extrusion in mated females, and abdominal curling in males during courtship. We find that dsf activity results in different neuroanatomical outcomes in females and males, promoting and suppressing, respectively, female development and function of these neurons depending upon the sexual state of dsx expression. We posit that dsf and dsx interact to specify sex differences in the neural circuitry for dimorphic abdominal behaviors.

BACKGROUND: In a series of landmark papers, Kyriacou, Hall, and colleagues reported that the average inter-pulse interval of Drosophila melanogaster male courtship song varies rhythmically (KH cycles), that the period gene controls this rhythm, and that evolution of the period gene determines species differences in the rhythm's frequency. Several groups failed to recover KH cycles, but this may have resulted from differences in recording chamber size.

RESULTS: Here, using recording chambers of the same dimensions as used by Kyriacou and Hall, I found no compelling evidence for KH cycles at any frequency. By replicating the data analysis procedures employed by Kyriacou and Hall, I found that two factors--data binned into 10-second intervals and short recordings--imposed non-significant periodicity in the frequency range reported for KH cycles. Randomized data showed similar patterns.

CONCLUSIONS: All of the results related to KH cycles are likely to be artifacts of binning data from short songs. Reported genotypic differences in KH cycles cannot be explained by this artifact and may have resulted from the use of small sample sizes and/or from the exclusion of samples that did not exhibit song rhythms.

The evolutionary expansion of sensory neuron populations detecting important environmental cues is widespread, but functionally enigmatic. We investigated this phenomenon through comparison of homologous neural pathways of Drosophila melanogaster and its close relative Drosophila sechellia, an extreme specialist for Morinda citrifolia noni fruit. D. sechellia has evolved species-specific expansions in select, noni-detecting olfactory sensory neuron (OSN) populations, through multigenic changes. Activation and inhibition of defined proportions of neurons demonstrate that OSN population increases contribute to stronger, more persistent, noni-odor tracking behavior. These sensory neuron expansions result in increased synaptic connections with their projection neuron (PN) partners, which are conserved in number between species. Surprisingly, having more OSNs does not lead to greater odor-evoked PN sensitivity or reliability. Rather, pathways with increased sensory pooling exhibit reduced PN adaptation, likely through weakened lateral inhibition. Our work reveals an unexpected functional impact of sensory neuron expansions to explain ecologically-relevant, species-specific behavior.

From the star-nosed mole’s eponymous mechanosensory organ to the platypus’ electroreceptive bill, the expansion of sensory neuron populations detecting important environmental cues is a widespread evolutionary phenomenon in animals1–6. How such neuron increases contribute to improved sensory detection and behaviour remain largely unexplained. Here we address this question through comparative analysis of olfactory pathways in Drosophila melanogaster and its close relative Drosophila sechellia, which feeds and breeds exclusively on Morinda citrifolia noni fruit7–9. We show that D. sechellia displays selective, large expansions of noni-detecting olfactory sensory neuron (OSN) populations, and that this trait has a multigenic basis. These expansions are accompanied by an increase in synaptic connections between OSNs and their projection neuron (PN) partners that transmit information to higher brain centres. Quantification of odour-evoked responses of partner OSNs and PNs reveals that OSN population expansions do not lead to heightened PN sensitivity, beyond that due to sensory receptor tuning differences. Rather, these pathways – but not those with conserved OSN numbers – exhibit non-adapting PN activity upon odour stimulation. In noni odour plume-tracking assays, D. sechellia exhibits enhanced performance compared to D. melanogaster. Through activation and inhibition of defined proportions of a noni-sensing OSN population, we establish that increased neuron numbers contribute to this behavioural persistence. Our work reveals an unexpected functional impact of sensory neuron expansions that can synergise with peripheral receptor tuning changes to explain ecologically-relevant, species-specific behaviour.

To perform most behaviors, animals must send commands from higher-order processing centers in the brain to premotor circuits that reside in ganglia distinct from the brain, such as the mammalian spinal cord or insect ventral nerve cord. How these circuits are functionally organized to generate the great diversity of animal behavior remains unclear. An important first step in unraveling the organization of premotor circuits is to identify their constituent cell types and create tools to monitor and manipulate these with high specificity to assess their function. This is possible in the tractable ventral nerve cord of the fly. To generate such a toolkit, we used a combinatorial genetic technique (split-GAL4) to create 195 sparse driver lines targeting 198 individual cell types in the ventral nerve cord. These included wing and haltere motoneurons, modulatory neurons, and interneurons. Using a combination of behavioral, developmental, and anatomical analyses, we systematically characterized the cell types targeted in our collection. Taken together, the resources and results presented here form a powerful toolkit for future investigations of neural circuits and connectivity of premotor circuits while linking them to behavioral outputs.

Background

• Many Drosophila species use acoustic communication during courtship and studies of these communication systems have provided insight into neurobiology, behavioral ecology, ethology, and evolution.

• Recording Drosophila courtship sounds and associated behavior is challenging, especially at high throughput, and previously designed devices are relatively expensive and complex to assemble.

Results

• We present construction plans for a modular system utilizing mostly off-the-shelf, relatively inexpensive components that provides simultaneous high-resolution audio and video recording of 96 isolated or paired Drosophila individuals.

• We provide open-source control software to record audio and video.

• We designed high intensity LED arrays that can be used to perform optogenetic activation and inactivation of labelled neurons.

• The basic design can be modified to facilitate novel study designs or to record insects larger than Drosophila.

• Fewer than 96 microphones can be used in the system if the full array is not required or to reduce costs.

Implications

• Our hardware design and software provide an improved platform for reliable and comparatively inexpensive high-throughput recording of Drosophila courtship acoustic and visual behavior and perhaps for recording acoustic signals of other small animals.