Dopamine in Drosophila: setting arousal thresholds in a miniature brain

In mammals, the neurotransmitter dopamine (DA) modulates a variety of behaviours, although DA function is mostly associated with motor control and reward. In insects such as the fruitfly, Drosophila melanogaster, DA also modulates a wide array of behaviours, ranging from sleep and locomotion to courtship and learning. How can a single molecule play so many different roles? Adaptive changes within the DA system, anatomical specificity of action and effects on a variety of behaviours highlight the remarkable versatility of this neurotransmitter. Recent genetic and pharmacological manipulations of DA signalling in Drosophila have launched a surfeit of stories—each arguing for modulation of some aspect of the fly''s waking (and sleeping) life. Although these stories often seem distinct and unrelated, there are some unifying themes underlying DA function and arousal states in this insect model. One of the central roles played by DA may involve perceptual suppression, a necessary component of both sleep and selective attention.     

Temperature-mediated Behavioral Relationships in Bursaphelenchus xylophilus, B. mucronatus,and Their Hybrids

The influence of temperature on reproduction and movement was examined for seven geographic isolates of Bursaphelenchus xylophilus, three of B. mucronatus, and two of their interspecific hybrids. All nematode isolates tended to be more active and fecund the higher the temperature, with the isolates of B. xylophilus reaching a reproductive peak at higher temperatures than isolates of B. mucronatus. Most isolates of B. xylophilus and B. mucronatus did not produce significantly more progeny at higher male-to-female ratios. The interspecific hybrids appear to possess temperature-related characteristics of either B. xylophilus or both of the parents.     

Interaction between Sleep and Metabolism in Drosophila with Altered Octopamine Signaling

Sleep length and metabolic dysfunction are correlated, but the causal relationship between these processes is unclear. Octopamine promotes wakefulness in the fly by acting through the insulin-producing cells (IPCs) in the fly brain. To determine if insulin signaling mediates the effects of octopamine on sleep:wake behavior, we assayed flies in which insulin signaling activity was genetically altered. We found that increasing insulin signaling does not promote wake, nor does insulin appear to mediate the wake-promoting effects of octopamine. Octopamine also affects metabolism in invertebrate species, including, as we show here, Drosophila melanogaster. Triglycerides are decreased in mutants with compromised octopamine signaling and elevated in flies with increased activity of octopaminergic neurons. Interestingly, this effect is mediated at least partially by insulin, suggesting that effects of octopamine on metabolism are independent of its effects on sleep. We further investigated the relative contribution of metabolic and sleep phenotypes to the starvation response of flies with altered octopamine signaling. Hyperactivity (indicative of foraging) induced by starvation was elevated in octopamine receptor mutants, despite their high propensity for sleep, indicating that their metabolic state dictates their behavioral response under these conditions. Moreover, flies with increased octopamine signaling do not suppress sleep in response to starvation, even though they are normally hyper-aroused, most likely because of their high triglyceride levels. Together, these data suggest that observed correlations between sleep and metabolic phenotypes can result from shared molecular pathways rather than causality, and environmental conditions can lead to the dominance of one phenotype over the other.     

An Inexpensive,Scalable Behavioral Assay for Measuring Ethanol Sedation Sensitivity and Rapid Tolerance in Drosophila

Alcohol use disorder (AUD) is a serious health challenge. Despite a large hereditary component to AUD, few genes have been unambiguously implicated in their etiology. The fruit fly, Drosophila melanogaster, is a powerful model for exploring molecular-genetic mechanisms underlying alcohol-related behaviors and therefore holds great promise for identifying and understanding the function of genes that influence AUD. The use of the Drosophila model for these types of studies depends on the availability of assays that reliably measure behavioral responses to ethanol. This report describes an assay suitable for assessing ethanol sensitivity and rapid tolerance in flies. Ethanol sensitivity measured in this assay is influenced by the volume and concentration of ethanol used, a variety of previously reported genetic manipulations, and also the length of time the flies are housed without food immediately prior to testing. In contrast, ethanol sensitivity measured in this assay is not affected by the vigor of fly handling, sex of the flies, and supplementation of growth medium with antibiotics or live yeast. Three different methods for quantitating ethanol sensitivity are described, all leading to essentially indistinguishable ethanol sensitivity results. The scalable nature of this assay, combined with its overall simplicity to set-up and relatively low expense, make it suitable for small and large scale genetic analysis of ethanol sensitivity and rapid tolerance in Drosophila.     

A Novel Gene Controlling the Timing of Courtship Initiation in Drosophila melanogaster

Over the past 35 years, developmental geneticists have made impressive progress toward an understanding of how genes specify morphology and function, particularly as they relate to the specification of each physical component of an organism. In the last 20 years, male courtship behavior in Drosophila melanogaster has emerged as a robust model system for the study of genetic specification of behavior. Courtship behavior is both complex and innate, and a single gene, fruitless (fru), is both necessary and sufficient for all aspects of the courtship ritual. Typically, loss of male-specific Fruitless protein function results in male flies that perform the courtship ritual incorrectly, slowly, or not at all. Here we describe a novel requirement for fru: we have identified a group of cells in which male Fru proteins are required to reduce the speed of courtship initiation. In addition, we have identified a gene, Trapped in endoderm 1 (Tre1), which is required in these cells for normal courtship and mating behavior. Tre1 encodes a G-protein-coupled receptor required for establishment of cell polarity and cell migration and has previously not been shown to be involved in courtship behavior. We describe the results of feminization of the Tre1-expressing neurons, as well as the effects on courtship behavior of mutation of Tre1. In addition, we show that Tre1 is expressed in a sexually dimorphic pattern in the central and peripheral nervous systems and investigate the role of the Tre1 cells in mate identification.     

Disruption of Endocytosis with the Dynamin Mutant shibirets1 Suppresses Seizures in Drosophila

One challenge in modern medicine is to control epilepsies that do not respond to currently available medications. Since seizures consist of coordinated and high-frequency neural activity, our goal was to disrupt neurotransmission with a synaptic transmission mutant and evaluate its ability to suppress seizures. We found that the mutant shibire, encoding dynamin, suppresses seizure-like activity in multiple seizure–sensitive Drosophila genotypes, one of which resembles human intractable epilepsy in several aspects. Because of the requirement of dynamin in endocytosis, increased temperature in the shi^ts1 mutant causes impairment of synaptic vesicle recycling and is associated with suppression of the seizure-like activity. Additionally, we identified the giant fiber neuron as critical in the seizure circuit and sufficient to suppress seizures. Overall, our results implicate mutant dynamin as an effective seizure suppressor, suggesting that targeting or limiting the availability of synaptic vesicles could be an effective and general method of controlling epilepsy disorders.     

Genetic Analysis of Esterase Polymorphism in the Soybean Cyst Nematode, Heterodera glycines

The genetic basis of esterase polymorphism in Heterodera glycines was investigated through controlled matings and analysis of F₁ and F₂ progeny. Three nematode lines, each fixed for a different esterase phenotype, were isolated and purified through repeated directional selection and inbreeding. Each phenotype was characterized by its distinct pair of closely spaced bands of esterase activity. Single-female single-male crosses were conducted according to a modified agar-plate mating technique. F₁ progeny were homogeneous, exhibiting both parental esterase phenotypes (codominant heterozygotes) but no hybrid bands. Approximately 1,500 F₂ progeny segregated in a 1:2:1 ratio for expression of the esterase phenotypes of the female parental line, the heterozygote, and the male parental line. Apparently the three esterase phenotypes correspond to three codominant alleles of a single esterase locus. Reciprocal crosses gave similar results, suggesting no maternal inheritance.     

Enhanced tethered-flight duration and locomotor activity by overexpression of the human gene SOD1 in Drosophila motorneurons

Mutation of the human gene superoxide dismutase (hSOD1) is associated with the fatal neurodegenerative disease familial amyotrophic lateral sclerosis (Lou Gehrig’s disease). Selective overexpression of hSOD1 in Drosophila motorneurons increases lifespan to 140% of normal. The current study was designed to determine resistance to lifespan decline and failure of sensorimotor functions by overexpressing hSOD1 in Drosophila‘s motorneurons. First, we measured the ability to maintain continuous flight and wingbeat frequency (WBF) as a function of age (5 to 50 days). Flies overexpressing hSOD1 under the D42-GAL4 activator were able to sustain flight significantly longer than controls, with the largest effect observed in the middle stages of life. The hSOD1-expressed line also had, on average, slower wingbeat frequencies in late, but not early life relative to age-matched controls. Second, we examined locomotor (exploratory walking) behavior in late life when flies had lost the ability to fly (age ≥ 60 d). hSOD1-expressed flies showed significantly more robust walking activity relative to controls. Findings show patterns of functional decline dissimilar to those reported for other life-extended lines, and suggest that the hSOD1 gene not only delays death but enhances sensorimotor abilities critical to survival even in late life.     

Energy-Dependent Modulation of Glucagon-Like Signaling in Drosophila via the AMP-Activated Protein Kinase

Adipokinetic hormone (AKH) is the equivalent of mammalian glucagon, as it is the primary insect hormone that causes energy mobilization. In Drosophila, current knowledge of the mechanisms regulating AKH signaling is limited. Here, we report that AMP-activated protein kinase (AMPK) is critical for normal AKH secretion during periods of metabolic challenges. Reduction of AMPK in AKH cells causes a suite of behavioral and physiological phenotypes resembling AKH cell ablations. Specifically, reduced AMPK function increases life span during starvation and delays starvation-induced hyperactivity. Neither AKH cell survival nor gene expression is significantly impacted by reduced AMPK function. AKH immunolabeling was significantly higher in animals with reduced AMPK function; this result is paralleled by genetic inhibition of synaptic release, suggesting that AMPK promotes AKH secretion. We observed reduced secretion in AKH cells bearing AMPK mutations employing a specific secretion reporter, confirming that AMPK functions in AKH secretion. Live-cell imaging of wild-type AKH neuroendocrine cells shows heightened excitability under reduced sugar levels, and this response was delayed and reduced in AMPK-deficient backgrounds. Furthermore, AMPK activation in AKH cells increases intracellular calcium levels in constant high sugar levels, suggesting that the underlying mechanism of AMPK action is modification of ionic currents. These results demonstrate that AMPK signaling is a critical feature that regulates AKH secretion, and, ultimately, metabolic homeostasis. The significance of these findings is that AMPK is important in the regulation of glucagon signaling, suggesting that the organization of metabolic networks is highly conserved and that AMPK plays a prominent role in these networks.     

Microevolutionary Patterns and Molecular Markers: The Genetics of Geographic Variation in Ascaris suum

Molecular markers have been used only rarely to characterize the population genetic structure of nematodes. Published studies have suggested that different taxa may show distinct genetic architectures. Isoenzyme and RAPD markers have been used to investigate geographic variation of Ascaris suum at the level of infrapopulations (nematodes within individual hosts), within localities, and among geographic regions. Independent estimates of genetic differentiation among population samples based on isoenzyme and RAPD data showed similar patterns and substantial correlation. Heterozygote deficiencies within infrapopulations and large values for inbreeding coefficients among infrapopulations suggested that the composition of these populations was not consistent with a model of random recruitment from a large panmictic pool of life-cycle stages. Both isoenzyme and RAPD markers revealed moderate levels of genetic differentiation among samples representing infrapopulations and localities. Of total gene diversity, 9.4% (isoenzyme) and 9.2% (RAPD) was partitioned among infrapopulations. Geographic localities accounted for 7.8% (isoenzyme) and 6.2% (RAPD) of total diversity. Only infrapopulations from the same farm had low levels of differentiation.     

Clustering and Protein Dynamics of Drosophila melanogaster Telomeres

Telomeres are obligatory chromosomal landmarks that demarcate the ends of linear chromosomes to distinguish them from broken ends and can also serve to organize the genome. In both budding and fission yeast, they cluster at the periphery of the nucleus, potentially to establish a compartment of silent chromatin. To gain insight into telomere organization in higher organisms, we investigated their distribution in interphase nuclei of Drosophila melanogaster. We focused on the syncytial blastoderm, an excellent developmental stage for live imaging due to the synchronous division of the nuclei at this time. We followed the EGFP-labeled telomeric protein HOAP in vivo and found that the 16 telomeres yield four to six foci per nucleus, indicative of clustering. Furthermore, we confirmed clustering in other somatic tissues. Importantly, we observed that HOAP signal intensity in the clusters increases in interphase, potentially due to loading of HOAP to newly replicated telomeres. To determine the rules governing clustering, we used in vivo imaging and fluorescence in situ hybridization to test several predictions. First, we inspected mutant embryos that develop as haploids and found that clustering is not mediated by associations between homologs. Second, we probed specifically for a telomere of novel sequence and found strong evidence against DNA sequence identity and homology as critical factors. Third, we ruled out predominance of intrachromosomal interactions by marking both ends of a chromosome. Based on these results, we propose that clustering is independent of sequence and is likely maintained by an as yet undetermined factor.     

Identification of Genes That Promote or Inhibit Olfactory Memory Formation in Drosophila

Genetic screens in Drosophila melanogaster and other organisms have been pursued to filter the genome for genetic functions important for memory formation. Such screens have employed primarily chemical or transposon-mediated mutagenesis and have identified numerous mutants including classical memory mutants, dunce and rutabaga. Here, we report the results of a large screen using panneuronal RNAi expression to identify additional genes critical for memory formation. We identified >500 genes that compromise memory when inhibited (low hits), either by disrupting the development and normal function of the adult animal or by participating in the neurophysiological mechanisms underlying memory formation. We also identified >40 genes that enhance memory when inhibited (high hits). The dunce gene was identified as one of the low hits and further experiments were performed to map the effects of the dunce RNAi to the α/β and γ mushroom body neurons. Additional behavioral experiments suggest that dunce knockdown in the mushroom body neurons impairs memory without significantly affecting acquisition. We also characterized one high hit, sickie, to show that RNAi knockdown of this gene enhances memory through effects in dopaminergic neurons without apparent effects on acquisition. These studies further our understanding of two genes involved in memory formation, provide a valuable list of genes that impair memory that may be important for understanding the neurophysiology of memory or neurodevelopmental disorders, and offer a new resource of memory suppressor genes that will aid in understanding restraint mechanisms employed by the brain to optimize resources.     

Transmission of the Pinewood Nematode, Bursaphelenchus xylophilus,to Slash Pine Trees and Log Bolts by a Cerambycid Beetle,Monochamus titillator, in Florida

Field-collected adults of the southern pine sawyer, Monochamus titillator (F.) (Coleoptera: Cerambycidae), naturally infested with fourth-stage juveniles (dauerlarvae) of the pinewood nematode, Bursaphelenchus xylophilus (Steiner and Buhrer, 1934) Nickle, 1970, were maturation fed on excised shoots of typical slash pine, Pinus elliottii Engelm. var elliottii, for 21 days. During August 1981, a male and female adult beetle were held in a sleeve cage placed on the terminal of a side branch of each of seven replicate, healthy 10-year-old slash pine trees. All seven branch terminals showed evidence of beetle feeding on the bark after 1 week, and pinewood nematodes were present in wood samples taken near these feeding sites. Four of the seven trees showed wilt symptoms in 4-6 weeks and died about 9 weeks after beetle feeding. Pinewood nematodes were recovered from the roots and trunks of the dead trees. Each of seven replicate slash pine log bolts was enclosed in a jar with a pair of the same beetles used in the sleeve cages. After 1 week, wood underlying beetle oviposition sites in the bark of all replicate log bolts was infested with the pinewood nematode.     

Meloidogyne incognita and Tomato Response to Thiamine,Ascorbic Acid, L-arginine,and L-glutamic Acid

The influence of solutions of ascorbic acid, thiamine, L-arginine, and L-gtutamic acid on egg hatch, juvenile survival, and development and reproduction of Meloidogyne incognita in susceptible and resistant tomatoes was studied. Maximum inhibition of egg hatch occurred at 2,000, 4,000, and 2,000 ppm for ascorbic acid, L-arginine, and L-glutamic acid, respectively. Larval survival was significantly reduced by concentrations of 2,000 ppm ascorbic acid and 1,000 ppm of L-arginine. Maximum inhibition of egg hatch and mortality of juveniles was achieved at a concentration of 4,000 ppm of ascorbic acid and L-arginine. L-glutamic acid and thiamine had respective moderate and minimal toxic effects. Foliar sprays of ascorbic acid, L-arginine, or L-glutamic acid suppressed the numbers of root galls, females, and egg masses on the susceptible tomato cultivar Tropic. Ascorbic acid and L-arginine had highly significant effects when applied to foliage before inoculation with nematodes. Thiamine had little effect. All sprays suppressed the numbers of root galls and females in roots of the resistant cultivar VFN8 when treatments were applied before inoculation. They were not, however, effective as post-inoculation treatments. Growth of a susceptible cultivar was improved by post-inoculation and pre-inoculation treatments when compared with the control plants which had neither nematode infection nor chemical treatment. No positive growth response to chemical treatment was seen in resistant control plants.     

Taxonomic revision of the genus Callimerus Gorham s. l. (Coleoptera,Cleridae). Part I. latifrons species-group

The latifrons species-group (=Brachycallimerus sensu Chapin 1924, Corporaal 1950; = flavofasciatus-group sensu Kolibáč 1998) of Callimerus Gorham is redefined and revised. Five species are recognized including one new species Callimerus cacuminis Yang & Yang sp. n. (type locality: Yunnan, China). Callimerus flavofasciatus Schenkling, 1902 is newly synonymized with Callimerus latifrons Gorham, 1876. Callimerus trifasciatus Schenkling, 1899a is transferred to the genus Corynommadius Schenkling, 1899a. Callimerus gorhami Corporaal, 1949 and Callimerus pallidus Gorham, 1892 are excluded from the latifrons species-group (their assignment to a species-group will be dealt with in a subsequent paper). A key to species of the latifrons species-group is given and habitus of each type specimen, male terminalia, and other diagnostic characters are illustrated.     

Neprilysins: An Evolutionarily Conserved Family of Metalloproteases That Play Important Roles in Reproduction in Drosophila

Members of the M13 class of metalloproteases have been implicated in diseases and in reproductive fitness. Nevertheless, their physiological role remains poorly understood. To obtain a tractable model with which to analyze this protein family’s function, we characterized the gene family in Drosophila melanogaster and focused on reproductive phenotypes. The D. melanogaster genome contains 24 M13 class protease homologs, some of which are orthologs of human proteases, including neprilysin. Many are expressed in the reproductive tracts of either sex. Using RNAi we individually targeted the five Nep genes most closely related to vertebrate neprilysin, Nep1-5, to investigate their roles in reproduction. A reduction in Nep1, Nep2, or Nep4 expression in females reduced egg laying. Nep1 and Nep2 are required in the CNS and the spermathecae for wild-type fecundity. Females that are null for Nep2 also show defects as hosts of sperm competition as well as an increased rate of depletion for stored sperm. Furthermore, eggs laid by Nep2 mutant females are fertilized normally, but arrest early in embryonic development. In the male, only Nep1 was required to induce normal patterns of female egg laying. Reduction in the expression of Nep2-5 in the male did not cause any dramatic effects on reproductive fitness, which suggests that these genes are either nonessential for male fertility or perform redundant functions. Our results suggest that, consistent with the functions of neprilysins in mammals, these proteins are also required for reproduction in Drosophila, opening up this model system for further functional analysis of this protein class and their substrates.     

Genetic Architecture and Functional Characterization of Genes Underlying the Rapid Diversification of Male External Genitalia Between Drosophila simulans and Drosophila mauritiana

Male sexual characters are often among the first traits to diverge between closely related species and identifying the genetic basis of such changes can contribute to our understanding of their evolutionary history. However, little is known about the genetic architecture or the specific genes underlying the evolution of male genitalia. The morphology of the claspers, posterior lobes, and anal plates exhibit striking differences between Drosophila mauritiana and D. simulans. Using QTL and introgression-based high-resolution mapping, we identified several small regions on chromosome arms 3L and 3R that contribute to differences in these traits. However, we found that the loci underlying the evolution of clasper differences between these two species are independent from those that contribute to posterior lobe and anal plate divergence. Furthermore, while most of the loci affect each trait in the same direction and act additively, we also found evidence for epistasis between loci for clasper bristle number. In addition, we conducted an RNAi screen in D. melanogaster to investigate if positional and expression candidate genes located on chromosome 3L, are also involved in genital development. We found that six of these genes, including components of Wnt signaling and male-specific lethal 3 (msl3), regulate the development of genital traits consistent with the effects of the introgressed regions where they are located and that thus represent promising candidate genes for the evolution these traits.