A novel setup for simultaneous two-photon functional imaging and precise spectral and spatial visual stimulation in Drosophila.

Motion vision has been extensively characterised in Drosophila melanogaster, but substantially less is known about how flies process colour, or how spectral information affects other visual modalities. To accurately dissect the components of the early visual system responsible for processing colour, we developed a versatile visual stimulation setup to probe combined spatial, temporal and spectral response properties. Using flies expressing neural activity indicators, we tracked visual responses in the medulla, the second visual neuropil, to a projected colour stimulus. The introduction of custom bandpass optical filters enables simultaneous two-photon imaging and visual stimulation over a large range of wavelengths without compromising the temporal stimulation rate. With monochromator-produced light, any spectral bandwidth and centre wavelength from 390 to 730 nm can be selected to produce a narrow spectral hue. A specialised screen material scatters each band of light across the visible spectrum equally at all locations of the screen, thus enabling presentation of spatially structured stimuli. We show layer-specific shifts of spectral response properties in the medulla correlating with projection regions of photoreceptor terminals

Visual approach computation in feeding hoverflies.

On warm sunny days, female hoverflies are often observed feeding from a wide range of wild and cultivated flowers. In doing so, hoverflies serve a vital role as alternative pollinators, and are suggested to be the most important pollinators after bees and bumblebees. Unless the flower hoverflies are feeding from is large, they do not readily share the space with other insects, but instead opt to leave if another insect approaches. We used high-speed videography followed by 3D reconstruction of flight trajectories to quantify how female Eristalis hoverflies respond to approaching bees, wasps and two different hoverfly species. We found that, in 94% of the interactions, the occupant female left the flower when approached by another insect. We found that compared with spontaneous take-offs, the occupant hoverfly's escape response was performed at ∼3 times higher speed (spontaneous take-off at 0.2±0.05 m s-1 compared with 0.55±0.08 m s-1 when approached by another Eristalis). The hoverflies tended to take off upward and forward, while taking the incomer's approach angle into account. Intriguingly, we found that, when approached by wasps, the occupant Eristalis took off at a higher speed and when the wasp was further away. This suggests that feeding hoverflies may be able to distinguish these predators, demanding impressive visual capabilities. Our results, including quantification of the visual information available before occupant take-off, provide important insight into how freely behaving hoverflies perform escape responses from competitors and predators (e.g. wasps) in the wild.This work was funded by the Air Force Office of Scientific Research (FA9550-15-1-0188 to P.T. Gonzalez-Bellido and K. Nordström), the Biotechnology and Biological Sciences Research Council (BB/L024667/1 David Phillips Fellowship to T. Wardill), Australian Research Council (DP170100008), Stiftelsen Olle Engkvist Byggmästare (2016/348) and Stiftelsen Längmanska Kulturfonden (BA17-0812)

An Unexpected Diversity of Photoreceptor Classes in the Longfin Squid, Doryteuthis pealeii.

Cephalopods are famous for their ability to change color and pattern rapidly for signaling and camouflage. They have keen eyes and remarkable vision, made possible by photoreceptors in their retinas. External to the eyes, photoreceptors also exist in parolfactory vesicles and some light organs, where they function using a rhodopsin protein that is identical to that expressed in the retina. Furthermore, dermal chromatophore organs contain rhodopsin and other components of phototransduction (including retinochrome, a photoisomerase first found in the retina), suggesting that they are photoreceptive. In this study, we used a modified whole-mount immunohistochemical technique to explore rhodopsin and retinochrome expression in a number of tissues and organs in the longfin squid, Doryteuthis pealeii. We found that fin central muscles, hair cells (epithelial primary sensory neurons), arm axial ganglia, and sucker peduncle nerves all express rhodopsin and retinochrome proteins. Our findings indicate that these animals possess an unexpected diversity of extraocular photoreceptors and suggest that extraocular photoreception using visual opsins and visual phototransduction machinery is far more widespread throughout cephalopod tissues than previously recognized.This research was supported by the Office of Naval Research Basic Research Challenge grant number N00014-10-0989 to T.W.C and R.T.H and a Biotechnology and Biological Sciences Research Council (BBSRC) David Phillips Fellowship BB/L024667/1 to T.J.W. We gratefully acknowledge support from the Air Force Office of Scientific Research via grants numbered FA9550-09-0346 to R.T.H. and FA9550-12-1-0321 to T.W.C.This is the final version of the article. It first appeared from PLoS via http://dx.doi.org/10.1371/journal.pone.013538

Neural control of tuneable skin iridescence in squid

In addition to the Introduction readme document, find also the Materials and Methods readme document that describes the methods used to collect the data for this paper. The final readme, File Descriptions, describes how the files are arranged in various Zip files. The data within these zip files should be considered the gold standard data, although considerably more data exists than is reported in this repository. Please contact the authors directly ([email protected] and [email protected]) for any additional data.Fast dynamic control of skin coloration is rare in the animal kingdom, whether it be pigmentary or structural. Iridescent structural coloration results when nanoscale structures disrupt incident light and selectively reflect specific colours. Unlike animals with fixed iridescent coloration (e.g. butterflies), squid iridophores (i.e. aggregations of iridescent cells in the skin), produce dynamically tuneable structural coloration, as exogenous application of acetylcholine (ACh) changes the colour and brightness output. Previous efforts to stimulate iridophores neurally or to identify the source of endogenous ACh were unsuccessful, leaving researchers to question the activation mechanism. We developed a novel neurophysiological preparation in the squid Doryteuthis pealeii and demonstrated that electrical stimulation of neurons in the skin shifts the spectral peak of the reflected light to shorter wavelengths (>145 nm) and increases the peak reflectance (>245 %) of innervated iridophores. We show ACh is released within the iridophore layer and that extensive nerve branching is seen within the iridophore. The dynamic colour shift is significantly faster (17 s) than the peak reflectance increase (32 s) revealing two distinct mechanisms. Responses from a structurally altered preparation indicate that the reflectin protein condensation mechanism explains peak reflectance change, while an undiscovered mechanism causes the fast colour shift

Endoplasmic Reticulum Lumenal Indicators in Drosophila Reveal Effects of HSP-Related Mutations on Endoplasmic Reticulum Calcium Dynamics

Genes for endoplasmic reticulum (ER)-shaping proteins are among the most commonly mutated in hereditary spastic paraplegia (HSP). Mutation of these genes in model organisms can lead to disruption of the ER network. To investigate how the physiological roles of the ER might be affected by such disruption, we developed tools to interrogate its Ca2+ signaling function. We generated GAL4-driven Ca2+ sensors targeted to the ER lumen, to record ER Ca2+ fluxes in identified Drosophila neurons. Using GAL4 lines specific for Type Ib or Type Is larval motor neurons, we compared the responses of different lumenal indicators to electrical stimulation, in axons and presynaptic terminals. The most effective sensor, ER-GCaMP6-210, had a Ca2+ affinity close to the expected ER lumenal concentration. Repetitive nerve stimulation generally showed a transient increase of lumenal Ca2+ in both the axon and presynaptic terminals. Mutants lacking neuronal reticulon and REEP proteins, homologs of human HSP proteins, showed a larger ER lumenal evoked response compared to wild type; we propose mechanisms by which this phenotype could lead to neuronal dysfunction or degeneration. Our lines are useful additions to a Drosophila Ca2+ imaging toolkit, to explore the physiological roles of ER, and its pathophysiological roles in HSP and in axon degeneration more broadly

Cuttlefish use stereopsis to strike at prey.

The camera-type eyes of vertebrates and cephalopods exhibit remarkable convergence, but it is currently unknown whether the mechanisms for visual information processing in these brains, which exhibit wildly disparate architecture, are also shared. To investigate stereopsis in a cephalopod species, we affixed "anaglyph" glasses to cuttlefish and used a three-dimensional perception paradigm. We show that (i) cuttlefish have also evolved stereopsis (i.e., the ability to extract depth information from the disparity between left and right visual fields); (ii) when stereopsis information is intact, the time and distance covered before striking at a target are shorter; (iii) stereopsis in cuttlefish works differently to vertebrates, as cuttlefish can extract stereopsis cues from anticorrelated stimuli. These findings demonstrate that although there is convergent evolution in depth computation, cuttlefish stereopsis is likely afforded by a different algorithm than in humans, and not just a different implementation

Expression of squid iridescence depends on environmental luminance and peripheral ganglion control

Author Posting. © The Author(s), 2013. This is the author's version of the work. It is posted here by permission of Company of Biologists for personal use, not for redistribution. The definitive version was published in Journal of Experimental Biology 217 (2014):850-858, doi:10.1242/​jeb.091884.Squids display impressive changes in body coloration that are afforded by two types of dynamic skin elements: structural iridophores (which produce iridescence) and pigmented chromatophores. Both color elements are neurally controlled, but nothing is known about the iridescence circuit, or the environmental cues, that elicit iridescence expression. To tackle this knowledge gap, we performed denervation, electrical stimulation and behavioral experiments using the long-fin squid, Doryteuthis pealeii. We show that while the pigmentary and iridescence circuits originate in the brain, they are wired differently in the periphery: (i) the iridescence signals are routed through a peripheral center called the stellate ganglion and (ii) the iridescence motorneurons likely originate within this ganglion (as revealed by nerve fluorescence dye fills). Cutting the inputs to the stellate ganglion that descend from the brain shifts highly reflective iridophores into a transparent state. Taken together, these findings suggest that although brain commands are necessary for expression of iridescence, integration with peripheral information in the stellate ganglion could modulate the final output. We also demonstrate that squids change their iridescence brightness in response to environmental luminance; such changes are robust but slow (minutes to hours). The squid's ability to alter its iridescence levels may improve camouflage under different lighting intensities.This research was supported by the ONR Basic Research Challenge grant no. N00014-10-1-0989 and by the AFOSR grant FA9950090346.2015-03-1

The killer fly hunger games : target size and speed predict decision to pursuit

© The Author(s), 2015. This article is distributed under the terms of the Creative Commons Attribution License. The definitive version was published in Brain, Behavior and Evolution 86 (2015): 28-27, doi:10.1159/000435944.Predatory animals have evolved to optimally detect their prey using exquisite sensory systems such as vision, olfaction and hearing. It may not be so surprising that vertebrates, with large central nervous systems, excel at predatory behaviors. More striking is the fact that many tiny insects, with their miniscule brains and scaled down nerve cords, are also ferocious, highly successful predators. For predation, it is important to determine whether a prey is suitable before initiating pursuit. This is paramount since pursuing a prey that is too large to capture, subdue or dispatch will generate a substantial metabolic cost (in the form of muscle output) without any chance of metabolic gain (in the form of food). In addition, during all pursuits, the predator breaks its potential camouflage and thus runs the risk of becoming prey itself. Many insects use their eyes to initially detect and subsequently pursue prey. Dragonflies, which are extremely efficient predators, therefore have huge eyes with relatively high spatial resolution that allow efficient prey size estimation before initiating pursuit. However, much smaller insects, such as killer flies, also visualize and successfully pursue prey. This is an impressive behavior since the small size of the killer fly naturally limits the neural capacity and also the spatial resolution provided by the compound eye. Despite this, we here show that killer flies efficiently pursue natural (Drosophila melanogaster) and artificial (beads) prey. The natural pursuits are initiated at a distance of 7.9 ± 2.9 cm, which we show is too far away to allow for distance estimation using binocular disparities. Moreover, we show that rather than estimating absolute prey size prior to launching the attack, as dragonflies do, killer flies attack with high probability when the ratio of the prey's subtended retinal velocity and retinal size is 0.37. We also show that killer flies will respond to a stimulus of an angular size that is smaller than that of the photoreceptor acceptance angle, and that the predatory response is strongly modulated by the metabolic state. Our data thus provide an exciting example of a loosely designed matched filter to Drosophila, but one which will still generate successful pursuits of other suitable prey.This work was funded by the Air Force Office of Scientific Research (FA9550-10-0472 to R.M. Olberg and FA9550-15-1-0188 to P.T. Gonzalez-Bellido and K. Nordström), an Isaac Newton Trust/Wellcome Trust ISSF/University of Cambridge Joint Research Grant to Paloma T. Gonzalez-Bellido, a Biotechnology and Biological Sciences Research Council David Phillips Fellowship (BBSRC, BB/L024667/1) to Trevor J. Wardill, the Swedish Research Council (2012-4740) to Karin Nordström and a Shared Equipment Grant from the School of Biological Sciences (University of Cambridge)

The Killer Fly Hunger Games: Target Size and Speed Predict Decision to Pursuit.

Predatory animals have evolved to optimally detect their prey using exquisite sensory systems such as vision, olfaction and hearing. It may not be so surprising that vertebrates, with large central nervous systems, excel at predatory behaviors. More striking is the fact that many tiny insects, with their miniscule brains and scaled down nerve cords, are also ferocious, highly successful predators. For predation, it is important to determine whether a prey is suitable before initiating pursuit. This is paramount since pursuing a prey that is too large to capture, subdue or dispatch will generate a substantial metabolic cost (in the form of muscle output) without any chance of metabolic gain (in the form of food). In addition, during all pursuits, the predator breaks its potential camouflage and thus runs the risk of becoming prey itself. Many insects use their eyes to initially detect and subsequently pursue prey. Dragonflies, which are extremely efficient predators, therefore have huge eyes with relatively high spatial resolution that allow efficient prey size estimation before initiating pursuit. However, much smaller insects, such as killer flies, also visualize and successfully pursue prey. This is an impressive behavior since the small size of the killer fly naturally limits the neural capacity and also the spatial resolution provided by the compound eye. Despite this, we here show that killer flies efficiently pursue natural (Drosophila melanogaster) and artificial (beads) prey. The natural pursuits are initiated at a distance of 7.9 ± 2.9 cm, which we show is too far away to allow for distance estimation using binocular disparities. Moreover, we show that rather than estimating absolute prey size prior to launching the attack, as dragonflies do, killer flies attack with high probability when the ratio of the prey's subtended retinal velocity and retinal size is 0.37. We also show that killer flies will respond to a stimulus of an angular size that is smaller than that of the photoreceptor acceptance angle, and that the predatory response is strongly modulated by the metabolic state. Our data thus provide an exciting example of a loosely designed matched filter to Drosophila, but one which will still generate successful pursuits of other suitable prey.This work was funded the Air force Office of Scientific Research (FA9550-10-0472 to Prof. Robert Olberg). An Isaac Newton Trust / Wellcome Trust ISSF / University of Cambridge Joint Research Grant to Gonzalez-Bellido. BBSRC TO TREVOR WARDILL The Swedish Research Council (2012-4740) to Nordström and a Shared Equipment Grant from the School of Biological Sciences (U. of Cambridge).This is the final version of the article. It first appeared from Karger via http://dx.doi.org/10.1159/00043594

Long-Wavelength Reflecting Filters Found in the Larval Retinas of One Mantis Shrimp Family (Nannosquillidae).

Both vertebrates and invertebrates commonly exploit photonic structures adjacent to their photoreceptors for visual benefits. For example, use of a reflecting structure (tapetum) behind the retina increases photon capture, enhancing vision in dim light [1-5]. Colored filters positioned lateral or distal to a photoreceptive unit may also be used to tune spectral sensitivity by selective transmission of wavelengths not absorbed or scattered by the filters [6-8]. Here we describe a new category of biological optical filter that acts simultaneously as both a transmissive spectral filter and narrowband reflector. Discovered in the larval eyes of only one family of mantis shrimp (stomatopod) crustaceans (Nannosquillidae), each crystalline structure bisects the photoreceptive rhabdom into two tiers and contains an ordered array of membrane-bound vesicles with sub-wavelength diameters of 153 ± 5 nm. Axial illumination of the intrarhabdomal structural reflector (ISR) in vivo produces a narrow band of yellow reflectance (mean peak reflectivity, 572 ± 18 nm). The ISR is similar to several synthetic devices, such as bandgap filters, laser mirrors, and (in particular) fiber Bragg gratings used in optical sensors for a wide range of industries. To our knowledge, the stomatopod larval ISR is the first example of a naturally occurring analog to these human-made devices. Considering what is known about these animals' visual ecology, we propose that these reflecting filters may help improve the detection of pelagic bioluminescence in shallow water at night. VIDEO ABSTRACT