The Effect of Stress on Motor Function in Drosophila

Exposure to unpredictable and uncontrollable conditions causes animals to perceive stress and change their behavior. It is unclear how the perception of stress modifies the motor components of behavior and which molecular pathways affect the behavioral change. In order to understand how stress affects motor function, we developed an experimental platform that quantifies walking motions in Drosophila. We found that stress induction using electrical shock results in backwards motions of the forelegs at the end of walking strides. These leg retrogressions persisted during repeated stimulation, although they habituated substantially. The motions also continued for several strides after the end of the shock, indicating that stress induces a behavioral aftereffect. Such aftereffect could also be induced by restricting the motion of the flies via wing suspension. Further, the long-term effects could be amplified by combining either immobilization or electric shock with additional stressors. Thus, retrogression is a lingering form of response to a broad range of stressful conditions, which cause the fly to search for a foothold when it faces extreme and unexpected challenges. Mutants in the cAMP signaling pathway enhanced the stress response, indicating that this pathway regulates the behavioral response to stress. Our findings identify the effect of stress on a specific motor component of behavior and define the role of cAMP signaling in this stress response.     

A Single Pair of Neurons Modulates Egg-Laying Decisions in Drosophila

Animals have to judge environmental cues and choose the most suitable option for them from many different options. Female fruit flies selecting an optimum site to deposit their eggs is a biologically important reproductive behavior. When given the direct choice between ovipositing their eggs in a sucrose-containing medium or a caffeine-containing medium, female flies prefer the latter. However, the neural circuits and molecules that regulate this decision-making processes during egg-laying site selection remain poorly understood. In the present study, we found that amnesiac (amn) mutant flies show significant defects in egg-laying decisions, and such defects can be reversed by expressing the wild-type amn transgene in two dorsal paired medial (DPM) neurons in the brain. Silencing neuronal activity with an inward rectifier potassium channel (Kir2.1) in DPM neurons also impairs egg-laying decisions. Finally, the activity in mushroom body αβ neurons is required for the egg-laying behavior, suggesting a possible “DPM-αβ neurons” brain circuit modulating egg-laying decisions. Our results highlight the brain circuits and molecular mechanisms of egg-laying decisions in Drosophila.     

Differences in Hematopoietic Stem Cells Contribute to Sexually Dimorphic Inflammatory Responses to High Fat Diet-induced Obesity

Women of reproductive age are protected from metabolic disease relative to postmenopausal women and men. Most preclinical rodent studies are skewed toward the use of male mice to study obesity-induced metabolic dysfunction because of a similar protection observed in female mice. How sex differences in obesity-induced inflammatory responses contribute to these observations is unknown. We have compared and contrasted the effects of high fat diet-induced obesity on glucose metabolism and leukocyte activation in multiple depots in male and female C57Bl/6 mice. With both short term and long term high fat diet, male mice demonstrated increased weight gain and CD11c⁺ adipose tissue macrophage content compared with female mice despite similar degrees of adipocyte hypertrophy. Competitive bone marrow transplant studies demonstrated that obesity induced a preferential contribution of male hematopoietic cells to circulating leukocytes and adipose tissue macrophages compared with female cells independent of the sex of the recipient. Sex differences in macrophage and hematopoietic cell in vitro activation in response to obesogenic cues were observed to explain these results. In summary, this report demonstrates that male and female leukocytes and hematopoietic stem cells have cell-autonomous differences in their response to obesity that contribute to an amplified response in males compared with females.     

The Imd Pathway Is Involved in Antiviral Immune Responses in Drosophila

Cricket Paralysis virus (CrPV) is a member of the Dicistroviridae family of RNA viruses, which infect a broad range of insect hosts, including the fruit fly Drosophila melanogaster. Drosophila has emerged as an effective system for studying innate immunity because of its powerful genetic techniques and the high degree of gene and pathway conservation. Intra-abdominal injection of CrPV into adult flies causes a lethal infection that provides a robust assay for the identification of mutants with altered sensitivity to viral infection. To gain insight into the interactions between viruses and the innate immune system, we injected wild type flies with CrPV and observed that antimicrobial peptides (AMPs) were not induced and hemocytes were depleted in the course of infection. To investigate the contribution of conserved immune signaling pathways to antiviral innate immune responses, CrPV was injected into isogenic mutants of the Immune Deficiency (Imd) pathway, which resembles the mammalian Tumor Necrosis Factor Receptor (TNFR) pathway. Loss-of-function mutations in several Imd pathway genes displayed increased sensitivity to CrPV infection and higher CrPV loads. Our data show that antiviral innate immune responses in flies infected with CrPV depend upon hemocytes and signaling through the Imd pathway.     

A Small Subset of Fruitless Subesophageal Neurons Modulate Early Courtship in Drosophila

We show that a small subset of two to six subesophageal neurons, expressing the male products of the male courtship master regulator gene products fruitless^Male (fru^M), are required in the early stages of the Drosophila melanogaster male courtship behavioral program. Loss of fru^M expression or inhibition of synaptic transmission in these fru^M(+) neurons results in delayed courtship initiation and a failure to progress to copulation primarily under visually-deficient conditions. We identify a fru^M-dependent sexually dimorphic arborization in the tritocerebrum made by two of these neurons. Furthermore, these SOG neurons extend descending projections to the thorax and abdominal ganglia. These anatomical and functional characteristics place these neurons in the position to integrate gustatory and higher-order signals in order to properly initiate and progress through early courtship.     

Caspase Inhibition in Select Olfactory Neurons Restores Innate Attraction Behavior in Aged Drosophila

Sensory and cognitive performance decline with age. Neural dysfunction caused by nerve death in senile dementia and neurodegenerative disease has been intensively studied; however, functional changes in neural circuits during the normal aging process are not well understood. Caspases are key regulators of cell death, a hallmark of age-related neurodegeneration. Using a genetic probe for caspase-3-like activity (DEVDase activity), we have mapped age-dependent neuronal changes in the adult brain throughout the lifespan of Drosophila. Spatio-temporally restricted caspase activation was observed in the antennal lobe and ellipsoid body, brain structures required for olfaction and visual place memory, respectively. We also found that caspase was activated in an age-dependent manner in specific subsets of Drosophila olfactory receptor neurons (ORNs), Or42b and Or92a neurons. These neurons are essential for mediating innate attraction to food-related odors. Furthermore, age-induced impairments of neural transmission and attraction behavior could be reversed by specific inhibition of caspase in these ORNs, indicating that caspase activation in Or42b and Or92a neurons is responsible for altering animal behavior during normal aging.     

Fluorescence Behavioral Imaging (FBI) Tracks Identity in Heterogeneous Groups of Drosophila

Distinguishing subpopulations in group behavioral experiments can reveal the impact of differences in genetic, pharmacological and life-histories on social interactions and decision-making. Here we describe Fluorescence Behavioral Imaging (FBI), a toolkit that uses transgenic fluorescence to discriminate subpopulations, imaging hardware that simultaneously records behavior and fluorescence expression, and open-source software for automated, high-accuracy determination of genetic identity. Using FBI, we measure courtship partner choice in genetically mixed groups of Drosophila.     

Advantage of the Highly Restricted Odorant Receptor Expression Pattern in Chemosensory Neurons of Drosophila

A fundamental molecular feature of olfactory systems is that individual neurons express only one receptor from a large odorant receptor gene family. While numerous theories have been proposed, the functional significance and evolutionary advantage of generating a sophisticated one-receptor-per neuron expression pattern is not well understood. Using the genetically tractable Drosophila melanogaster as a model, we demonstrate that the breakdown of this highly restricted expression pattern of an odorant receptor in neurons leads to a deficit in the ability to exploit new food sources. We show that animals with ectopic co-expression of odorant receptors also have a competitive disadvantage in a complex environment with limiting food sources. At the level of the olfactory system, we find changes in both the behavioral and electrophysiological responses to odorants that are detected by endogenous receptors when an olfactory receptor is broadly misexpressed in chemosensory neurons. Taken together these results indicate that restrictive expression patterns and segregation of odorant receptors to individual neuron classes are important for sensitive odor-detection and appropriate olfactory behaviors.     

Limbic Neurons Shape Sex Recognition and Social Behavior in Sexually Naive Males

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Quantification of Food Intake in Drosophila

Measurement of food intake in the fruit fly Drosophila melanogaster is often necessary for studies of behaviour, nutrition and drug administration. There is no reliable and agreed method for measuring food intake of flies in undisturbed, steady state, and normal culture conditions. We report such a method, based on measurement of feeding frequency by proboscis-extension, validated by short-term measurements of food dye intake. We used the method to demonstrate that (a) female flies feed more frequently than males, (b) flies feed more often when housed in larger groups and (c) fly feeding varies at different times of the day. We also show that alterations in food intake are not induced by dietary restriction or by a null mutation of the fly insulin receptor substrate chico. In contrast, mutation of takeout increases food intake by increasing feeding frequency while mutation of ovo^D increases food intake by increasing the volume of food consumed per proboscis-extension. This approach provides a practical and reliable method for quantification of food intake in Drosophila under normal, undisturbed culture conditions.     

How Food Controls Aggression in Drosophila

How animals use sensory information to weigh the risks vs. benefits of behavioral decisions remains poorly understood. Inter-male aggression is triggered when animals perceive both the presence of an appetitive resource, such as food or females, and of competing conspecific males. How such signals are detected and integrated to control the decision to fight is not clear. For instance, it is unclear whether food increases aggression directly, or as a secondary consequence of increased social interactions caused by attraction to food. Here we use the vinegar fly, Drosophila melanogaster, to investigate the manner by which food influences aggression. We show that food promotes aggression in flies, and that it does so independently of any effect on frequency of contact between males, increase in locomotor activity or general enhancement of social interactions. Importantly, the level of aggression depends on the absolute amount of food, rather than on its surface area or concentration. When food resources exceed a certain level, aggression is diminished, suggestive of reduced competition. Finally, we show that detection of sugar via Gr5a⁺ gustatory receptor neurons (GRNs) is necessary for food-promoted aggression. These data demonstrate that food exerts a specific effect to promote aggression in male flies, and that this effect is mediated, at least in part, by sweet-sensing GRNs.     

Reinforcement of Gametic Isolation in Drosophila

Reinforcement, a process by which natural selection increases reproductive isolation between populations, has been suggested to be an important force in the formation of new species. However, all existing cases of reinforcement involve an increase in mate discrimination between species. Here, I report the first case of reinforcement of postmating prezygotic isolation (i.e., barriers that act after mating but before fertilization) in animals. On the slopes of the African island of São Tomé, Drosophila yakuba and its endemic sister species D. santomea hybridize within a well-demarcated hybrid zone. I find that D. yakuba females from within this zone, but not from outside it, show an increase in gametic isolation from males of D. santomea, an apparent result of natural selection acting to reduce maladaptive hybridization between species. To determine whether such a barrier could evolve under laboratory conditions, I exposed D. yakuba lines derived from allopatric populations to experimental sympatry with D. santomea, and found that both behavioral and gametic isolation become stronger after only four generations. Reinforcement thus appears to be the best explanation for the heightened gametic isolation seen in sympatry. This appears to be the first example in animals in which natural selection has promoted the evolution of stronger interspecific genetic barriers that act after mating but before fertilization. This suggests that many other genetic barriers between species have been increased by natural selection but have been overlooked because they are difficult to study.