Live Fruit Flies Research Articles

Free radical production induced by visible light in live fruit flies

Visible light triggers free radical production in alive and intact Drosophila melanogaster. We exposed fruit flies to red (613–631 nm), green (515–535 nm), and blue (455–475 nm) light while we monitored changes in unpaired electron content with an electron spin resonance spectrometer (ESR/EPR). The immediate response to light is a rapid increase in spin content lasting approximately 10 s followed by a slower, linear increase for approximately 170 s. When the light is turned off, the spin population promptly decays with a similar time course, though never fully returning to baseline. The magnitude and time course of the spin production depends on the wavelength of the light. Initially, we surmised that eumelanin might be responsible for the spin change because of its documented ability for visible light absorption and its highly stable free radical content. To explore this, we utilized different fruit fly strains with varying eumelanin content and clarified the relation of melanin types with the spin response. Our findings revealed that flies with darker cuticle have at least three-fold more unpaired electrons than flies with yellow cuticle. However, to our surprise, the increase in unpaired electron population by light was not drastically different amongst the genotypes. This suggests that light-induced free radical production may not exclusively rely on the presence of black melanin, but may instead be dependent on light effects on quinone-based cuticular polymers.

Rapid volumetric imaging with Bessel-Beam three-photon microscopy.

Owing to its tissue-penetration ability, multi-photon fluorescence microscopy allows for the high-resolution, non-invasive imaging of deep tissue in vivo; the recently developed three-photon microscopy (3PM) has extended the depth of high-resolution, non-invasive functional imaging of mouse brains to beyond 1.0 mm. However, the low repetition rate of femtosecond lasers that are normally used in 3PM limits the temporal resolution of point-scanning three-photon microscopy. To increase the volumetric imaging speed of 3PM, we propose a combination of an axially elongated needle-like Bessel-beam with three-photon excitation (3PE) to image biological samples with an extended depth of focus. We demonstrate the higher signal-to-background ratio (SBR) of the Bessel-beam 3PM compared to the two-photon version both theoretically and experimentally. Finally, we perform simultaneous calcium imaging of brain regions at different axial locations in live fruit flies and rapid volumetric imaging of neuronal structures in live mouse brains. These results highlight the unique advantage of conducting rapid volumetric imaging with a high SBR in the deep brain in vivo using scanning Bessel-3PM.

Rapid adaptive remote focusing microscope for sensing of volumetric neural activity.

The ability to record neural activity in the brain of a living organism at cellular resolution is of great importance for defining the neural circuit mechanisms that direct behavior. Here we present an adaptive two-photon microscope optimized for extraction of neural signals over volumes in intact Drosophila brains, even in the presence of specimen motion. High speed volume imaging was made possible through reduction of spatial resolution while maintaining the light collection efficiency of a high resolution, high numerical aperture microscope. This enabled simultaneous recording of odor-evoked calcium transients in a defined volume of mushroom body Kenyon cell bodies in a live fruit fly.

The FlyCatwalk: a high-throughput feature-based sorting system for artificial selection in Drosophila.

Experimental evolution is a powerful tool for investigating complex traits. Artificial selection can be applied for a specific trait and the resulting phenotypically divergent populations pool-sequenced to identify alleles that occur at substantially different frequencies in the extreme populations. To maximize the proportion of loci that are causal to the phenotype among all enriched loci, population size and number of replicates need to be high. These requirements have, in fact, limited evolution studies in higher organisms, where the time investment required for phenotyping is often prohibitive for large-scale studies. Animal size is a highly multigenic trait that remains poorly understood, and an experimental evolution approach may thus aid in gaining new insights into the genetic basis of this trait. To this end, we developed the FlyCatwalk, a fully automated, high-throughput system to sort live fruit flies (Drosophila melanogaster) based on morphometric traits. With the FlyCatwalk, we can detect gender and quantify body and wing morphology parameters at a four-old higher throughput compared with manual processing. The phenotyping results acquired using the FlyCatwalk correlate well with those obtained using the standard manual procedure. We demonstrate that an automated, high-throughput, feature-based sorting system is able to avoid previous limitations in population size and replicate numbers. Our approach can likewise be applied for a variety of traits and experimental settings that require high-throughput phenotyping.

Magnetic Resonance Spectroscopy of live <em>Drosophila melanogaster</em> using Magic Angle Spinning

High-Resolution Magic Angle Spinning (HRMAS) proton magnetic resonance spectroscopy ((1)H-MRS) is a novel non-destructive technique that improves spectral line-widths and allows high-resolution spectra to be obtained from extracts, intact cells, cell cultures, and more importantly intact tissue to investigate relationships between metabolites and cellular processes. In vivo HRMAS (1)H-MRS studies have yet to be reported in the live fruit fly Drosophila melanogaster. Drosophila, as a simpler genetic organism, allows the multiple biological functions and various evolutionarily conserved signaling pathways to be examined at the whole organism level and it is a useful model for investigating genetics and physiology. To this end, we developed and implemented an in vivo HRMAS (1)H-MRS method to investigate live Drosophila at 14.1 T. Here, we outline an HRMAS (1)H-MRS protocol for the molecular characterization of Drosophila with a conventional MR spectrometer equipped with an HRMAS probe. This technique is a novel, in vivo, non-destructive Drosophila metabolite measurement approach, which enables the identification of disease biomarkers and thus may contribute to novel therapeutic development.

Light Sheet Microscopy Optimized for Depth Penetration to Study Embryogenesis

Fluorescence light sheet microscopy (FLSM) has gained widespread recognition in recent years, due to its distinct advantages for the 3-dimensional (3D) imaging of living biological samples. FLSM uses a planar sheet of light to illuminate a sample, generating fluorescence over an optical section of the sample that is collected by a wide-field microscope camera oriented orthogonal to the light sheet. The orthogonal geometry between the illumination and detection pathways enables massive parallelization in both illumination and detection; futhermore, it permits optical and physical access to samples (3D cell cultures or whole embryos) in ways that are impossible in the collinear geometry of standard microscopes. Because of these features, FLSM significantly outperforms standard laser-scanning confocal microscopy in imaging speed, phototoxicity, and signal to noise in many imaging applications. An important aspect of any 3D imaging technique is its imaging depth limit (how deep into a sample useful information can be collected). In this respect, standard FLSM fares only slightly better than confocal microscopy. To overcome this hurdle, we have optimized FLSM for imaging of live thick samples by minimizing the degradation of the light sheet due to scattering, while preserving acceptable axial resolution. Using this approach we have imaged whole, live fruit fly embryos and zebrafish embryos. We achieve higher depth penetration than standard FLSM, while maintaining sub-cellular resolution, at imaging speed of about ten times faster than standard confocal microscopy.

Lifespan Extension by the Antioxidant Curcumin in Drosophila Melanogaster

The interest in health benefits associated with consumption of anti-oxidants has led to investigations examining the possibility that diets rich in anti-oxidants promote lifespan extension. Studies using the standard fruit fly (Drosophila melanogaster) model of longevity have shown that the antioxidants vitamin E and N-acetyl cysteine prolong lifespan. Turmeric is a spice which has been consumed and used for medicinal purposes for many centuries in Asia. Interestingly, turmeric contains the powerful antioxidant, curcumin. To test the hypothesis that dietary curcumin prolongs lifespan, groups of 30 male D. melanogaster were cultured on media containing 1) no additive; 2) 0.5 mg of curcumin/gram of media; 3) 1.0 mg of curumin/gram of media; 4) 1.0 μg of of the superoxide dismutase inhibitor, disulfiram/gram of media; 5) 10 g of disulfiram/gram of media; 6) 0.5 mg curcumin and 1.0 g disulfiram/ gram of media; 7) 1.0 mg curcumin and 1.0 g disulfiram/ gram of media; 8) 0.5 mg curcumin and 10 g disulfiram/gram of media; or 9) 1.0 mg curcumin and 10 g disulfiram/gram of media. The number of live fruitflies was noted daily and mean lifespan determined for each treatment group. A significant (P≤0.05) increase in mean lifespan was noted only for the fruitflies maintained on 1.0 mg of curcumin/gram of media; this effect was reversed by addition of disulfiram. These results demonstrate that dietary curcumin prolongs lifespan and that this effect is associated with enhanced superoxide dismutase activity.

Side‐effects of insecticides on two erigonid spider species

AbstractThe current rearing technique for Erigone atra (Blackwall) and Oedothorax apicatus (Blackwall) (Araneae, Erigonidae) was improved. To reduce time spent rearing on live fruit flies the spiders were kept on a culture of the Collembola species Lepidocyrtus lanuginosus (Gmelin) (Entomobryidae). Side‐effects on spiders of two pyrethroid insecticides (fenvalerate and lambda‐cyhalothrin) and one carbamate insecticide (pirimicarb) were tested. Sensitivity of adults of both sexes and juveniles to insecticides and their influence on the rate of emergence of spiderlings from cocoons were investigated using topical application, spraying or residual contact. LD50 values for adults ranged from 0.49 to 2.52 ng a.i./spider for lambda‐cyhalothrin and from 5.75 to 98.20 ng a.i./spider for fenvalerate. Topical application also resulted in up to a week's delay of web‐building. A moving laboratory spraying equipment was used to spray spiders with different insecticide dosages and water volumes. Pyrethroids sprayed onto adults in webs had stronger effects than pyrethroids sprayed onto sitting or walking spiders on the soil surface. Residual contamination caused higher mortality of spiders after contact with lambda‐cyhalothrin than fenvalerate. In all tests, males were more susceptible to pyrethroids than females; this difference was related to body weight. Mortality rate was higher for E. atra than for O. apicatus. Both pyrethroids were also toxic to spiderlings. Lambda‐cyhalothrin inhibited emergence of E. atra spiderlings from cocoons. Pirimicarb was harmless to both spider species.

The essentiality of nectar and arthropods in the diet of the Anna's hummingbird ( Calypte anna)

1. 1. Three adult male Anna's hummingbirds ( Calyptae anna) were fed each of the following three diets over time: live fruit flies and a 22% sucrose in water solution (control diet), a 22% sucrose solution only, or live fruit flies and water only, and the birds' body mass changes and behavior were recorded. 2. 2. The hummingbirds were able to subsist for 10 days on the 22% sucrose solution with body mass loss but no obvious behavior changes. 3. 3. In the absence of the sucrose solution the hummingbirds made no attempt to catch fruit flies and became listless or torpid within a few hours. 4. 4. The results suggest that hummingbirds have a remarkable ability to survive with no protein in their diets as long as nectar is available.