Drosulfakinin activates CCKLR-17D1 and promotes larval locomotion and escape response in Drosophila

Neuropeptides are ubiquitous in both mammals and invertebrates and play essential roles in regulation and modulation of many developmental and physiological processes through activation of G-protein-coupled-receptors (GPCRs). However, the mechanisms by which many of the neuropeptides regulate specific neural function and behaviors remain undefined. Here we investigate the functions of Drosulfakinin (DSK), the Drosophila homolog of vertebrate neuropeptide cholecystokinin (CCK), which is the most abundant neuropeptide in the central nervous system. We provide biochemical evidence that sulfated DSK-1 and DSK-2 activate the CCKLR-17D1 receptor in a cell culture assay. We further examine the role of DSK and CCKLR-17D1 in the regulation of larval locomotion, both in a semi-intact larval preparation and in intact larvae under intense light exposure. Our results suggest that DSK/CCKLR-17D1 signaling promote larval body wall muscle contraction and is necessary for mediating locomotor behavior in stress-induced escape response.     

Silencing synaptic communication between random interneurons during Drosophila larval locomotion

Genetic manipulation of individual neurons provides a powerful approach toward understanding their contribution to stereotypic behaviors. We describe and evaluate a method for identifying candidate interneurons and associated neuropile compartments that mediate Drosophila larval locomotion. We created Drosophila larvae that express green fluorescent protein (GFP) and a shibire(ts1) (shi(ts1)) transgene (a temperature-sensitive neuronal silencer) in small numbers of randomly selected cholinergic neurons. These larvae were screened for aberrant behavior at an elevated temperature (31-32°C). Among larvae with abnormal locomotion or sensory-motor responses, some had very small numbers of GFP-labeled temperature-sensitive interneurons. Labeled ascending interneurons projecting from the abdominal ganglia to specific brain neuropile compartments emerged as candidates for mediation of larval locomotion. Random targeting of small sets of neurons for functional evaluation, together with anatomical mapping of their processes, provides a tool for identifying the regions of the central nervous system that are required for normal locomotion. We discuss the limitations and advantages of this approach to discovery of interneurons that regulate motor behavior.     

A neuropeptidergic circuit gates selective escape behavior of Drosophila larvae

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The different effects of structurally related sulfakinins on Drosophila melanogaster odor preference and locomotion suggest involvement of distinct mechanisms

Sulfakinins are myoactive peptides and antifeedant factors. Naturally occurring drosulfakinin I (DSK I; FDDYGHMRFNH₂) and drosulfakinin II (DSK II; GGDDQFDDYGHMRFNH₂) contain sulfated or nonsulfated tyrosine. We discovered sDSK II and nsDSK II influenced Drosophila melanogaster larval odor preference. However, sDSK I, nsDSK I, MRFNH₂, and saline did not influence odor preference. We discovered sDSK I and nsDSK I influenced larval locomotion. However, sDSK II, nsDSK II, MRFNH₂, and saline did not influence locomotion. Our novel data suggest distinct mechanisms underlie the effects of DSK I and DSK II peptides on odor preference and locomotion, parameters important to many facets of animal survival.     

Social effects for locomotion vary between environments in Drosophila melanogaster females

Despite strong purifying or directional selection, variation is ubiquitous in populations. One mechanism for the maintenance of variation is indirect genetic effects (IGEs), as the fitness of a given genotype will depend somewhat on the genes of its social partners. IGEs describe the effect of genes in social partners on the expression of the phenotype of a focal individual. Here, we ask what effect IGEs, and variation in IGEs between abiotic environments, has on locomotion in Drosophila. This trait is known to be subject to intralocus sexually antagonistic selection. We estimate the coefficient of interaction, Ψ, using six inbred lines of Drosophila. We found that Ψ varied between abiotic environments, and that it may vary across among male genotypes in an abiotic environment specific manner. We also found evidence that social effects of males alter the value of a sexually dimorphic trait in females, highlighting an interesting avenue for future research into sexual antagonism. We conclude that IGEs are an important component of social and sexual interactions and that they vary between individuals and abiotic environments in complex ways, with the potential to promote the maintenance of phenotypic variation.     

L-glutamate activates excitatory and inhibitory channels in Drosophila larval muscle

Muscle fibers from Drosophila larvae show an L-glutamate-sensitive membrane potential. Bath-applied L-glutamate depolarizes the muscle in the range from 0.5 to 20 microM. Greater concentrations of the agonist repolarize the fibers. The repolarizing effect disappears if chloride is replaced by sulfate in the external medium. Intracellular recordings show the occurrence of depolarizing and hyperpolarizing spontaneous miniature postsynaptic potentials (smpp). Patch-clamp studies indicate the presence of two types of receptor channels: (i) an anion-selective channel activated by both L-glutamate and GABA. In outside out-patches, bathed in symmetrical 140 mM Cl- and 200 microM GABA, the channel displays conductance substates of 40, 80 and 110 pS. In the presence of 200 microM L-glutamate only the 40 and 80 pS substates are observed; (ii) a cation-selective channel activated only by L-glutamate that has a conductance of 104 pS in cell-attached patches (128 mM Na+ outside). The presence of these two types of receptor channels in Drosophila muscle may explain the effect of bath-applied L-glutamate on membrane potential and the presence of inhibitory and excitatory smpp.     

Identifying neuropeptide and protein hormone receptors in Drosophila melanogaster by exploiting genomic data.

Most neuropeptide and protein hormone receptors belong to the large superfamily of G-protein-coupled receptors (GPCRs). These cell membrane proteins steer many important processes such as development, reproduction, homeostasis and behaviour when activated by their corresponding ligands. The first insect genome, that of the fruitfly Drosophila melanogaster, was sequenced in 2000, and about 200 GPCRs have been annnotated in this model insect. About 50 of these receptors were predicted to have neuropeptides or protein hormones as their ligands. Since 2000, the cDNAs of most of these candidate receptors have been cloned and for many receptors the endogenous ligand has been identified. In this review, we will give an update about the current knowledge of all Drosophila neuropeptide and protein hormone receptors, and discuss their phylogenetic relationships.     

Temperature and clinal variation in larval growth efficiency in Drosophila melanogaster

Geographic clines in ectotherm species including Drosophila melanogaster have been found throughout the world, with genetically larger body size and shorter development time occurring at high latitudes. Temperature is thought to play a major role in the evolution of this clinal variation. Laboratory thermal selection has effects similar to those seen in geographical clines. Evolution at low temperatures results in more rapid development to larger adult flies. This study investigated the effects of geographical origin and experimental temperature on larval growth efficiency in D. melanogaster. Larvae from populations that had evolved at high latitudes were found to use limited food more efficiently, so that the overall adult body size achieved was larger. Larvae reared at a lower experimental temperature (18 °C) used food more efficiently than those reared at a higher temperature (25 °C). The increases in growth efficiency found in populations from high latitudes could explain their increased body size and more rapid development.     

Olfactory stimuli and moonwalker SEZ neurons can drive backward locomotion in Drosophila

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Consequences of adaptation to larval crowding on sexual and fecundity selection in Drosophila melanogaster

Sexual selection is a major force influencing the evolution of sexually reproducing species. Environmental factors such as larval density can manipulate adult condition and influence the direction and strength of sexual selection. While most studies on the influence of larval crowding on sexual selection are either correlational or single-generation manipulations, it is unclear how evolution under chronic larval crowding affects sexual selection. To answer this, we measured the strength of sexual selection on male and female Drosophila melanogaster that had evolved under chronic larval crowding for over 250 generations in the laboratory, along with their controls which had never experienced crowding, in a common garden high-density environment. We measured selection coefficients on male mating success and sex-specific reproductive success, as separate estimates allowed dissection of sex-specific effects. We show that experimental evolution under chronic larval crowding decreases the strength of sexual and fecundity selection in males but not in females, relative to populations experiencing crowding for the first time. The effect of larval crowding in reducing reproductive success is almost twice in females than in males. Our study highlights the importance of studying how evolution in a novel, stressful environment can shape adult fitness in organisms.     

Patterns of growth and tract formation during the early development of secondary lineages in the Drosophila larval brain

The Drosophila brain consists of a relatively small number of invariant, genetically determined lineages which provide a model to study the relationship between gene function and neuronal architecture. In following this long‐term goal, we reconstruct the morphology (projection pattern and connectivity) and gene expression patterns of brain lineages throughout development. In this article, we focus on the secondary phase of lineage morphogenesis, from the reactivation of neuroblast proliferation in the first larval instar to the time when proliferation ends and secondary axon tracts have fully extended in the late third larval instar. We have reconstructed the location and projection of secondary lineages at close (4 h) intervals and produced a detailed map in the form of confocal z‐projections and digital three‐dimensional models of all lineages at successive larval stages. Based on these reconstructions, we could compare the spatio‐temporal pattern of axon formation and morphogenetic movements of different lineages in normal brain development. In addition to wild type, we reconstructed lineage morphology in two mutant conditions. (1) Expressing the construct UAS‐p35 which rescues programmed cell death we could systematically determine which lineages normally lose hemilineages to apoptosis. (2) so‐Gal4‐driven expression of dominant‐negative EGFR ablated the optic lobe, which allowed us to conclude that the global centrifugal movement normally affecting the cell bodies of lateral lineages in the late larva is causally related to the expansion of the optic lobe, and that the central pattern of axonal projections of these lineages is independent of the presence or absence of the optic lobe. © 2015 Wiley Periodicals, Inc. Develop Neurobiol 76: 434–451, 2016     

Evolution of behavior and neural control of the fast-start escape response

The fast-start startle behavior is the primary mechanism of rapid escape in fishes and is a model system for examining neural circuit design and musculoskeletal function. To develop a dataset for evolutionary analysis of the startle response, the kinematics and muscle activity patterns of the fast-start were analyzed for four fish species at key branches in the phylogeny of vertebrates. Three of these species (Polypterus palmas, Lepisosteus osseus, and Amia calva) represent the base of the actinopterygian radiation. A fourth species (Oncorhynchus mykiss) provided data for a species in the central region of the teleost phylogeny. Using these data, we explored the evolution of this behavior within the phylogeny of vertebrates. To test the hypothesis that startle features are evolutionarily conservative, the variability of motor patterns and kinematics in fast-starts was described. Results show that the evolution of the startle behavior in fishes, and more broadly among vertebrates, is not conservative. The fast-start has undergone substantial change in suites of kinematics and electromyogram features, including the presence of either a one- or a two-stage kinematic response and change in the extent of bilateral muscle activity. Comparative methods were used to test the evolutionary hypothesis that changes in motor control are correlated with key differences in the kinematics and behavior of the fast-start. Significant evolutionary correlations were found between several motor pattern and behavioral characters. These results suggest that the startle neural circuit itself is not conservative. By tracing the evolution of motor pattern and kinematics on a phylogeny, it is shown that major changes in the neural circuit of the startle behavior occur at several levels in the phylogeny of vertebrates.     

Covariation of larval gene expression and adult body size in natural populations of Drosophila melanogaster

Understanding adaptive phenotypic variation is one of the most fundamental problems in evolutionary biology. Genes involved in adaptation are most likely those that affect traits most intimately connected to fitness: life-history traits. The genetics of quantitative trait variation (including life histories) is still poorly understood, but several studies suggest that (1) quantitative variation might be the result of variation in gene expression, rather than protein evolution, and (2) natural variation in gene expression underlies adaptation. The next step in studying the genetics of adaptive phenotypic variation is therefore an analysis of naturally occuring covariation of global gene expression and a life-history trait. Here, we report a microarray study addressing the covariation in larval gene expression and adult body weight, a life-history trait involved in adaptation. Natural populations of Drosophila melanogaster show adaptive geographic variation in adult body size, with larger animals at higher latitudes. Conditions during larval development also affect adult size with larger flies emerging at lower temperatures. We found statistically significant differences in normalized larval gene expression between geographic populations at one temperature (genetic variation) and within geographic populations between temperatures (developmental plasticity). Moreover, larval gene expression correlated highly with adult weight, explaining 81% of its natural variation. Of the genes that show a correlation of gene expression with adult weight, most are involved in cell growth or cell maintenance or are associated with growth pathways.     

Evolution of increased adult longevity in Drosophila melanogaster populations selected for adaptation to larval crowding

In holometabolous animals such as Drosophila melanogaster, larval crowding can affect a wide range of larval and adult traits. Adults emerging from high larval density cultures have smaller body size and increased mean life span compared to flies emerging from low larval density cultures. Therefore, adaptation to larval crowding could potentially affect adult longevity as a correlated response. We addressed this issue by studying a set of large, outbred populations of D. melanogaster, experimentally evolved for adaptation to larval crowding for 83 generations. We assayed longevity of adult flies from both selected (MCUs) and control populations (MBs) after growing them at different larval densities. We found that MCUs have evolved increased mean longevity compared to MBs at all larval densities. The interaction between selection regime and larval density was not significant, indicating that the density dependence of mean longevity had not evolved in the MCU populations. The increase in longevity in MCUs can be partially attributed to their lower rates of ageing. It is also noteworthy that reaction norm of dry body weight, a trait probably under direct selection in our populations, has indeed evolved in MCU populations. To the best of our knowledge, this is the first report of the evolution of adult longevity as a correlated response of adaptation to larval crowding.     

Allatostatin-C/AstC-R2 Is a Novel Pathway to Modulate the Circadian Activity Pattern in Drosophila

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Erythritol ingestion impairs adult reproduction and causes larval mortality in Drosophila melanogaster fruit flies (Diptera: Drosophilidae)

Previous feeding studies showed the polyalcohol erythritol was toxic when ingested by adult laboratory fruit flies (Drosophila melanogaster). We asked whether erythritol could additionally affect fly population growth either through larval toxicity or through effects on adult reproduction. Females did not avoid laying on food substrates with 1M erythritol; laying rate on 1M erythritol food was similar to control food when females were given free‐choice access. Eggs laid or placed on 0.5 M to 2.5 M erythritol foods hatched at normal rates, suggesting erythritol was not toxic to eggs upon contact. Drosophila melanogaster larvae readily consumed food containing 1 M erythritol, but none of these larvae reached pupation. Longevity of larvae feeding on in 1 M erythritol food was significantly reduced relative to controls, and mean ± SE larval lifespan on erythritol was 1.54 ± 0.10 days (max. = 3 days). Exposing cohorts of second‐instar larvae to food with varying concentrations of erythritol showed the LD50 (at 24 hr) concentration was approximately 0.6 M. Taken together, these results suggest erythritol could be employed in effective larval‐sink baits. Adults flies fed with erythritol produced significantly fewer eggs on days when they fed on 1 M erythritol, and egg production was significantly reduced for one additional day after the adults were moved to control food. These findings suggest erythritol is rapid and effective at temporarily suppressing D. melanogaster reproduction, increasing its potential for use in effective insect population control.