The effect of gut microbiota elimination in Drosophila melanogaster: A how‐to guide for host–microbiota studies

In recent years, there has been a surge in interest in the effects of the microbiota on the host. Increasingly, we are coming to understand the importance of the gut microbiota in modulating host physiology, ecology, behavior, and evolution. One method utilized to evaluate the effect of the microbiota is to suppress or eliminate it, and compare the effect on the host with that of untreated individuals. In this study, we evaluate some of these commonly used methods in the model organism, Drosophila melanogaster. We test the efficacy of a low‐dose streptomycin diet, egg dechorionation, and an axenic or sterile diet, in the removal of gut bacteria within this species in a fully factorial design. We further determine potential side effects of these methods on host physiology by performing a series of standard physiological assays. Our results showed that individuals from all treatments took significantly longer to develop, and weighed less, compared to normal flies. Males and females that had undergone egg dechorionation weighed significantly less than streptomycin reared individuals. Similarly, axenic female flies, but not males, were much less active when analyzed in a locomotion assay. All methods decreased the egg to adult survival, with egg dechorionation inducing significantly higher mortality. We conclude that low‐dose streptomycin added to the dietary media is more effective at removing the gut bacteria than egg dechorionation and has somewhat less detrimental effects to host physiology. More importantly, this method is the most practical and reliable for use in behavioral research. Our study raises the important issue that the efficacy of and impacts on the host of these methods require investigation in a case‐by‐case manner, rather than assuming homogeneity across species and laboratories.     

Tetracycline‐exposed Drosophila melanogaster males produce fewer offspring but a relative excess of sons

A large diversity of species possesses endosymbionts; these endosymbionts can exhibit mutualistic, parasitic, and commensal relationships with their hosts. Previous work has consistently revealed that depleting endosymbiont titer with antibiotic treatment can significantly alter host fitness and function, particularly with respect to reproductive phenotypes. Although these findings are often interpreted as resulting from the breakdown of highly coevolved symbioses, it is possible that antibiotic treatment itself rather than endosymbiont removal contributes to the observed perturbations in reproductive phenotypes. Here, we investigate the effect of tetracycline treatment on sex ratio and male reproductive fitness using Drosophila melanogaster as a model system. Our results indicate that tetracycline‐treated males produce a relative excess of sons. We also find that tetracycline treatment reduces the number of progeny produced by treated males but not treated females. These findings are independent of the effects of tetracycline on Wolbachia titer and implicate the antibiotic itself as mediating these changes. It is yet unclear whether the sex ratio shift and reduction in male reproductive fitness are direct or indirect consequences of tetracycline exposure, and more work is needed to determine the molecular mechanisms by which these disturbances in reproductive phenotypes arise. Our data highlight the importance of considering the potentially confounding effects of antibiotic treatment when investigating the effects of endosymbiont depletion on host phenotypes.     

Sexual dimorphism in the nutritional requirement for adult lifespan in Drosophila melanogaster

The nutritional requirements of Drosophila have mostly been studied for development and reproduction, but the minimal requirements for adult male and female flies for lifespan have not been established. Following development on a complete diet, we find substantial sex difference in the basic nutritional requirement of adult flies for full length of life. Relative to females, males require less of each nutrient, and for some nutrients that are essential for development, adult males have no requirement at all for lifespan. The most extreme (and surprising) sex differences were that chronic cholesterol and vitamin deficiencies had no effect on the lifespan of adult males, but they greatly decreased lifespan in females. Female oogenesis rather than chromosomal karyotype and mating status is the key cause of this gender difference in life‐sustaining nutritional requirements. These data are important to the way we understand the mechanisms by which diet modifies lifespan.     

The role of species‐specific sensory cues in male responses to mating rivals in Drosophila melanogaster fruitflies

Complex sets of cues can be important in recognizing and responding to conspecific mating competitors and avoiding potentially costly heterospecific competitive interactions. Within Drosophila melanogaster, males can detect sensory inputs from conspecifics to assess the level of competition. They respond to rivals by significantly extending mating duration and gain significant fitness benefits from doing so. Here, we tested the idea that the multiple sensory cues used by D. melanogaster males to detect conspecifics also function to minimize “off‐target” responses to heterospecific males that they might encounter (Drosophila simulans, Drosophila yakuba, Drosophila pseudoobscura, or Drosophila virilis). Focal D. melanogaster males exposed to D. simulans or D. pseudoobscura subsequently increased mating duration, but to a lesser extent than following exposure to conspecific rivals. The magnitude of rivals’ responses expressed by D. melanogaster males did not align with genetic distance between species, and none of the sensory manipulations caused D. melanogaster to respond to males of all other species tested. However, when we removed or provided “false” sensory cues, D. melanogaster males became more likely to show increased mating duration responses to heterospecific males. We suggest that benefits of avoiding inaccurate assessment of the competitive environment may shape the evolution of recognition cues.     

Dynamics of the continent‐wide spread of a Drosophila defensive symbiont

Facultative symbionts can represent important sources of adaptation for their insect hosts and thus have the potential for rapid spread. Drosophila neotestacea harbours a heritable symbiont, Spiroplasma, that confers protection against parasitic nematodes. We previously found a cline in Spiroplasma prevalence across central Canada, ending abruptly at the Rocky Mountains. Resampling these populations 9 years later revealed that Spiroplasma had increased substantially across the region, resembling a Fisherian wave of advance. Associations between Spiroplasma infection and host mitochondrial DNA indicate that the increase was due to local increase of Spiroplasma‐infected flies. Finally, we detected Spiroplasma west of the Rocky Mountains for the first time and showed that defence against nematodes occurs in flies with a western genetic background. Because nematode infection is common throughout D. neotestacea's range, we expect Spiroplasma to spread to the Pacific coast.     

Microscale Laser Surgery Demonstrates the Grasping Function of the Male Sex Combs in Drosophila melanogaster and Drosophila bipectinata

Male secondary sexual traits of animals are richly diversified in form and complexity, yet there are many species in which their precise function remains unknown. Within the genus Drosophila, species belonging to the melanogaster and obscura species groups have evolved a remarkable variety of sex combs, male‐limited secondary sexual traits located on the tarsi of both front legs. Information concerning sex comb function is minimal or absent, except for D. melanogaster, where previous studies indicate that the sex combs are used for grasping the female prior to copulation. These studies, however, do not unambiguously demonstrate comb function, because it has not been possible to ascribe observed behavioral outcomes of the various comb manipulations to changes in the combs per se. We used microscale laser surgery to manipulate comb size in D. melanogaster and D. bipectinata, and tested the hypothesis that the sex combs function as grasping devices in courtship, making them essential for copulation to ensue. Results of high‐resolution behavioral analysis in small observation arenas demonstrated that in both species in which sex combs were surgically eliminated, males were unable to grasp, mount or copulate. The combless foretarsi of these altered males slipped off the end (D. melanogaster) and sides (D. bipectinata) of the female abdomen when courting males attempted to grasp. In most cases, males whose sex combs were reduced but not completely removed exhibited similar copulation probabilities as surgical control males, a result we demonstrated in observation chambers as well as under more ecologically realistic conditions inside population cages where males and females interacted on the surface of fruit substrates. Thus, the sex combs in D. melanogaster and D. bipectinata are grasping devices, essential for mounting and copulation.     

Comparative population genomics of latitudinal variation in Drosophila simulans and Drosophila melanogaster

Examples of clinal variation in phenotypes and genotypes across latitudinal transects have served as important models for understanding how spatially varying selection and demographic forces shape variation within species. Here, we examine the selective and demographic contributions to latitudinal variation through the largest comparative genomic study to date of Drosophila simulans and Drosophila melanogaster, with genomic sequence data from 382 individual fruit flies, collected across a spatial transect of 19 degrees latitude and at multiple time points over 2 years. Consistent with phenotypic studies, we find less clinal variation in D. simulans than D. melanogaster, particularly for the autosomes. Moreover, we find that clinally varying loci in D. simulans are less stable over multiple years than comparable clines in D. melanogaster. D. simulans shows a significantly weaker pattern of isolation by distance than D. melanogaster and we find evidence for a stronger contribution of migration to D. simulans population genetic structure. While population bottlenecks and migration can plausibly explain the differences in stability of clinal variation between the two species, we also observe a significant enrichment of shared clinal genes, suggesting that the selective forces associated with climate are acting on the same genes and phenotypes in D. simulans and D. melanogaster.     

Body‐colour variation in an orb‐web spider and its effect on predation success

Animal body coloration serves several functions such as thermoregulation, camouflage, aposematism, and intraspecific communication. In some orb‐web spiders, bright and conspicuous body colours are used to attract prey. On the other hand, there are other species whose body colour does not attract prey. Using a spider species showing individual body‐colour variation, the present study aimed to determine whether or not the variation in body colour shows a correlation with predation rates. We studied the orb‐web spider (Cyclosa argenteoalba) using both field observations and T‐maze experiments, in which the prey were exposed to differently coloured spiders. Cyclosa argenteoalba has silver‐ and black‐coloured areas on its dorsal abdomen, with the ratio of these two colours varying continuously among individuals. The bright and conspicuous silver area reflects ultraviolet light. Results of both field observations and colour choice experiments using Drosophila flies as prey showed that darker spiders have a greater chance of capturing prey than silver spiders. This indicates that body‐colour variation affects predation success among individuals and that the bright silver colour does not function to attract prey in C. argenteoalba.     

A cryptic rock‐paper‐scissors game between Drosophila males

Explaining the maintenance of genetic variation in characters associated with Darwinian fitness is a preoccupation of evolutionary biologists. Spatial or temporal variation in the environment can certainly promote polymorphism, yet even populations of ‘model organisms’, like fruit flies, kept on invariant protocols for hundreds of generations in the laboratory often show fitness variation that exceeds what would be expected from the input of new mutations alone. Such observations suggest either complexities of selection or of genetic architecture, and offer a powerful tool for the study of mechanisms that promote stable polymorphism. In this issue of Molecular Ecology, Zhang et al. ( 2013 ) report examples of nontransitivity in the outcome of postcopulatory sexual selection in the fruit fly, Drosophila, that follow the rules of the popular stalemate‐breaking game roshambo – or rock, paper, scissors (RPS). The important feature of RPS is that while each strategy beats one other, it in turn is beaten by the third. Using chromosome extraction lines, the authors confirm earlier findings that the outcome of postcopulatory sexual selection via sperm competition for a male depends, in part, upon the competitor male's genotype. But taking it one step further, they demonstrate the nontransitivities between males required for circular RPS cycles in sperm competition between males, and are able to identify at least four associated loci. Because the postmating phenotype involves hundreds of potentially interacting peptides and receptors, this is an important step to understanding the persistence of variation in a critical component of male fitness.     

The effects of age and dietary restriction on the tissue‐specific metabolome of Drosophila

Dietary restriction (DR) is a robust intervention that extends lifespan and slows the onset of age‐related diseases in diverse organisms. While significant progress has been made in attempts to uncover the genetic mechanisms of DR, there are few studies on the effects of DR on the metabolome. In recent years, metabolomic profiling has emerged as a powerful technology to understand the molecular causes and consequences of natural aging and disease‐associated phenotypes. Here, we use high‐resolution mass spectroscopy and novel computational approaches to examine changes in the metabolome from the head, thorax, abdomen, and whole body at multiple ages in Drosophila fed either a nutrient‐rich ad libitum (AL) or nutrient‐restricted (DR) diet. Multivariate analysis clearly separates the metabolome by diet in different tissues and different ages. DR significantly altered the metabolome and, in particular, slowed age‐related changes in the metabolome. Interestingly, we observed interacting metabolites whose correlation coefficients, but not mean levels, differed significantly between AL and DR. The number and magnitude of positively correlated metabolites was greater under a DR diet. Furthermore, there was a decrease in positive metabolite correlations as flies aged on an AL diet. Conversely, DR enhanced these correlations with age. Metabolic set enrichment analysis identified several known (e.g., amino acid and NAD metabolism) and novel metabolic pathways that may affect how DR effects aging. Our results suggest that network structure of metabolites is altered upon DR and may play an important role in preventing the decline of homeostasis with age.     

Starvation tolerance associated with prolonged sleep bouts upon starvation in a single natural population of Drosophila melanogaster

Large genetic variations in starvation tolerance in animals indicate that there are multiple strategies to cope with low‐nutrient conditions. Fruit flies (Drosophila melanogaster) typically respond to starvation by suppressing sleep and enhancing locomotor activity presumably to search for food. However, we hypothesized that in a natural population, there are costs and benefits to sleep suppression under low‐nutrient conditions and that conserving energy through sleep could be a better strategy depending on food availability. In this study, we quantified the variation in sleep‐related traits in 21 wild‐derived inbred lines from Katsunuma, Japan, under fed and starved conditions and analysed the relationship between those traits and starvation tolerance. Although most of the lines responded to starvation by suppressing the total time in sleep, there were indeed two lines that responded by significantly increasing the sleep‐bout durations and thus not reducing the total time in sleep. These genotypes survived longer in acute starvation conditions compared to genotypes that responded by the immediate suppression of sleep, which could be due to the reduced metabolic rate during the long uninterrupted sleep bouts. The coexistence of the enhanced foraging and resting strategies upon starvation within a single population is consistent with the presence of a behavioural trade‐off between food search and energy conservation due to unpredictable food availability in nature. These results provide insights into the evolutionary mechanisms that contribute to the maintenance of genetic variations underlying environmental stress resistance.     

Massive excretion of calcium oxalate from late prepupal salivary glands of Drosophila melanogaster demonstrates active nephridial‐like anion transport

The Drosophila salivary glands (SGs) were well known for the puffing patterns of their polytene chromosomes and so became a tissue of choice to study sequential gene activation by the steroid hormone ecdysone. One well‐documented function of these glands is to produce a secretory glue, which is released during pupariation to fix the freshly formed puparia to the substrate. Over the past two decades SGs have been used to address specific aspects of developmentally‐regulated programmed cell death (PCD) as it was thought that they are doomed for histolysis and after pupariation are just awaiting their fate. More recently, however, we have shown that for the first 3–4 h after pupariation SGs undergo tremendous endocytosis and vacuolation followed by vacuole neutralization and membrane consolidation. Furthermore, from 8 to 10 h after puparium formation (APF) SGs display massive apocrine secretion of a diverse set of cellular proteins. Here, we show that during the period from 11 to 12 h APF, the prepupal glands are very active in calcium oxalate (CaOx) extrusion that resembles renal or nephridial excretory activity. We provide genetic evidence that Prestin, a Drosophila homologue of the mammalian electrogenic anion exchange carrier SLC26A5, is responsible for the instantaneous production of CaOx by the late prepupal SGs. Its positive regulation by the protein kinases encoded by fray and wnk lead to increased production of CaOx. The formation of CaOx appears to be dependent on the cooperation between Prestin and the vATPase complex as treatment with bafilomycin A₁ or concanamycin A abolishes the production of detectable CaOx. These data demonstrate that prepupal SGs remain fully viable, physiologically active and engaged in various cellular activities at least until early pupal period, that is, until moments prior to the execution of PCD.     

UCP4C mediates uncoupled respiration in larvae of Drosophila melanogaster

Larvae of Drosophila melanogaster reared at 23°C and switched to 14°C for 1 h are 0.5°C warmer than the surrounding medium. In keeping with dissipation of energy, respiration of Drosophila melanogaster larvae cannot be decreased by the F‐ATPase inhibitor oligomycin or stimulated by protonophore. Silencing of Ucp4C conferred sensitivity of respiration to oligomycin and uncoupler, and prevented larva‐to‐adult progression at 15°C but not 23°C. Uncoupled respiration of larval mitochondria required palmitate, was dependent on Ucp4C and was inhibited by guanosine diphosphate. UCP4C is required for development through the prepupal stages at low temperatures and may be an uncoupling protein.     

Symbiont strain is the main determinant of variation in Wolbachia‐mediated protection against viruses across Drosophila species

Wolbachia is a common heritable bacterial symbiont in insects. Its evolutionary success lies in the diverse phenotypic effects it has on its hosts coupled to its propensity to move between host species over evolutionary timescales. In a survey of natural host–symbiont associations in a range of Drosophila species, we found that 10 of 16 Wolbachia strains protected their hosts against viral infection. By moving Wolbachia strains between host species, we found that the symbiont genome had a much greater influence on the level of antiviral protection than the host genome. The reason for this was that the level of protection depended on the density of the symbiont in host tissues, and Wolbachia rather than the host‐controlled density. The finding that virus resistance and symbiont density are largely under the control of symbiont genes in this system has important implications both for the evolution of these traits and for public health programmes using Wolbachia to prevent mosquitoes from transmitting disease.     

Developmental Deformity Due to scalloped Non‐Function in Drosophila Brain Leads to Cognitive Impairment

Neural identity and wiring specificity are fundamental to brain function. Factors affecting proliferation of the progenitor cells leading to an expansion or regression of specific neuronal clusters are expected to challenge the process of formation of precise synaptic connections with their partners and their further integration to result in proper functional neural circuitry. We have investigated the role of scalloped, a Hippo pathway gene in Drosophila brain development and have shown that its function is critical to regulate proliferation of Mushroom Body Neuroblasts and to limit the neuronal cluster size to normal in the fly brain. Here we investigate the consequent effect of the anatomical phenotype of mutant flies on the brain function, as exemplified by their cognitive performance. We demonstrate that the neural expansion in important neural clusters of the olfactory pathway, caused due to Scalloped inactivation, imparts severe disabilities in learning, short‐term memory and long‐term memory. Scalloped knockdown in αβ Kenyon Cell clusters drastically reduces long‐term memory performance. Scalloped deficiency induced neural expansion in antennal lobe and ellipsoid body neurons bring down short‐term memory performance significantly. We also demonstrate that the cognitive impairments observed here are not due to a problem in memory formation or execution in the adult, but are due to the developmental deformities caused in the respective class of neurons. Our results strongly indicate that the additional neurons generated by Scalloped inactivation are not synergistically integrated into, but rather perturb the formation of precise functional circuitry.     

β‐Guanidinopropionic acid extends the lifespan of Drosophila melanogaster via an AMP‐activated protein kinase‐dependent increase in autophagy

Previous studies have demonstrated that AMP‐activated protein kinase (AMPK) controls autophagy through the mammalian target of rapamycin (mTOR) and Unc‐51 like kinase 1 (ULK1/Atg1) signaling, which augments the quality of cellular housekeeping, and that β‐guanidinopropionic acid (β‐GPA), a creatine analog, leads to a chronic activation of AMPK. However, the relationship between β‐GPA and aging remains elusive. In this study, we hypothesized that feeding β‐GPA to adult Drosophila produces the lifespan extension via activation of AMPK‐dependent autophagy. It was found that dietary administration of β‐GPA at a concentration higher than 900 mm induced a significant extension of the lifespan of Drosophila melanogaster in repeated experiments. Furthermore, we found that Atg8 protein, the homolog of microtubule‐associated protein 1A/1B‐light chain 3 (LC3) and a biomarker of autophagy in Drosophila, was significantly upregulated by β‐GPA treatment, indicating that autophagic activity plays a role in the effect of β‐GPA. On the other hand, when the expression of Atg5 protein, an essential protein for autophagy, was reduced by RNA interference (RNAi), the effect of β‐GPA on lifespan extension was abolished. Moreover, we found that AMPK was also involved in this process. β‐GPA treatment significantly elevated the expression of phospho‐T172‐AMPK levels, while inhibition of AMPK by either AMPK‐RNAi or compound C significantly attenuated the expression of autophagy‐related proteins and lifespan extension in Drosophila. Taken together, our results suggest that β‐GPA can induce an extension of the lifespan of Drosophila via AMPK‐Atg1‐autophagy signaling pathway.