Through the looking glass: attempting to predict future opportunities and challenges in experimental biology.

To celebrate its centenary year, Journal of Experimental Biology (JEB) commissioned a collection of articles examining the past, present and future of experimental biology. This Commentary closes the collection by considering the important research opportunities and challenges that await us in the future. We expect that researchers will harness the power of technological advances, such as '-omics' and gene editing, to probe resistance and resilience to environmental change as well as other organismal responses. The capacity to handle large data sets will allow high-resolution data to be collected for individual animals and to understand population, species and community responses. The availability of large data sets will also place greater emphasis on approaches such as modeling and simulations. Finally, the increasing sophistication of biologgers will allow more comprehensive data to be collected for individual animals in the wild. Collectively, these approaches will provide an unprecedented understanding of 'how animals work' as well as keys to safeguarding animals at a time when anthropogenic activities are degrading the natural environment.

Binocular field configuration in owls: the role of foraging ecology.

The binocular field of vision differs widely in birds depending on ecological traits such as foraging. Owls (Strigiformes) have been considered to have a unique binocular field, but whether it is related to foraging has remained unknown. While taking into account allometry and phylogeny, we hypothesized that both daily activity cycle and diet determine the size and shape of the binocular field in owls. Here, we compared the binocular field configuration of 23 species of owls. While we found no effect of allometry and phylogeny, ecological traits strongly influence the binocular field shape and size. Binocular field shape of owls significantly differed from that of diurnal raptors. Among owls, binocular field shape was relatively conserved, but binocular field size differed among species depending on ecological traits, with larger binocular fields in species living in dense habitat and foraging on invertebrates. Our results suggest that (i) binocular field shape is associated with the time of foraging in the daily cycle (owls versus diurnal raptors) and (ii) that binocular field size differs between closely related owl species even though the general shape is conserved, possibly because the field of view is partially restricted by feathers, in a trade-off with auditory localization.

Dim-light colour vision in the facultatively nocturnal Asian giant honeybee, Apis dorsata.

We discovered nocturnal colour vision in the Asian giant honeybee Apis dorsata-a facultatively nocturnal species-at mesopic light intensities, down to half-moon light levels (approx. 10-2 cd m-2). The visual threshold of nocturnality aligns with their reported nocturnal activity down to the same light levels. Nocturnal colour vision in A. dorsata is interesting because, despite being primarily diurnal, its colour vision capabilities extend into dim light, while the 'model' European honeybee Apis mellifera is reported to be colour-blind at twilight. By employing behavioural experiments with naturally nesting A. dorsata colonies, we show discrimination of the trained colour from other stimuli during the day, and significantly, even at night. Nocturnal colour vision in bees has so far only been reported in the obligately nocturnal carpenter bee Xylocopa tranquebarica. The discovery of colour vision in these two bee species, despite differences in the extent of their nocturnality and the limitations of their apposition compound eye optics, opens avenues for future studies on visual adaptations for dim-light colour vision, their role in pollination of flowers at night, and the effect of light pollution on nocturnal activity in A. dorsata, a ubiquitous pollinator in natural, agricultural and urban habitats in the Asian tropics and sub-tropics.

Fast visual adaptation to dim light in a cavity-nesting bird.

Many birds move fast into dark nest cavities forcing the visual system to adapt to low light intensities. Their visual system takes between 15 and 60 min for complete dark adaptation, but little is known about the visual performance of birds during the first seconds in low light intensities. In a forced two-choice behavioural experiment we studied how well budgerigars can discriminate stimuli of different luminance directly after entering a darker environment. The birds made their choices within about 1 s and did not wait to adapt their visual system to the low light intensities. When moving from a bright facility into an environment with 0.5 log unit lower illuminance, the budgerigars detected targets with a luminance of 0.825 cd m-2 on a black background. When moving into an environment with 1.7 or 3.5 log units lower illuminance, they detected targets with luminances between 0.106 and 0.136 cd m-2. In tests with two simultaneously displayed targets, the birds discriminated similar luminance differences between the targets (Weber fraction of 0.41-0.54) in all light levels. Our results support the notion that partial adaptation of bird eyes to the lower illumination occurring within 1 s allows them to safely detect and feed their chicks.

Home ranges, directionality and the influence of moon phases on the movement ecology of Indian flying fox males in southern India.

Flying foxes of the genus Pteropus are amongst the largest fruit bats and potential long-range pollinators and seed dispersers in the paleotropics. Pteropus giganteus (currently P. medius) is the only flying fox that is distributed throughout the Indian mainland, including in urban and rural areas. Using GPS telemetry, we mapped the home ranges and examined flight patterns in P. giganteus males across moon phases in a semi-urban landscape in southern India. Home range differed between the tracked males (n=4), likely due to differences in their experience in the landscape. We found that nightly time spent outside the roost, distance commuted and the number of sites visited by tracked individuals did not differ significantly between moon phases. In 61% of total tracked nights across bats, the first foraging site was within 45˚ of the emergence direction. At the colony-level, scan-based observations showed emergence flights were mostly in the northeast (27%), west (22%) and southwest (19%) directions that could potentially be related to the distribution of foraging resources. The movement ecology of fruit bats in relation to the pollination and seed dispersal services they provide requires to be investigated in future studies. This article has an associated First Person interview with the first author of the paper.

Retinal Ganglion Cell Topography and Spatial Resolution in Three Indian Pteropodid Bats.

Pteropodidae is the only phytophagous bat family that predominantly depends on visual and olfactory cues for orientation and foraging. During daytime, pteropodids of different species roost in sites with varying light exposure. Pteropodids have larger eyes relative to body size than insectivorous bats. Retinal topography has been studied in less than 10% of the approximately 200 pteropodid species, a behavioural estimation of spatial resolution is available only for Pteropus giganteus, and little is known about the relationship between their roost site preference and visual ecology. We present retinal ganglion cell topographic maps and anatomical estimates of spatial resolution in three southern Indian pteropodid species with different roosting preferences. Ganglion cell densities are between 1,000 and 2,000 cells/mm2 in the central retina and lower in the dorsal and ventral periphery. All three species have a temporal area in the retina with peak ganglion cell densities of 4,600-6,600 cells/mm2. As a result, the foliage-roosting Cynopterus sphinx and the cave-roosting Rousettus leschenaultii have similar anatomical resolution (2.7 and 2.8 cycles/degree, respectively). The anatomical estimate for the larger tree-roosting P. giganteus (4.0 cycles/degree) is higher than the spatial resolution determined earlier in behavioural tests. Like other pteropodids and unlike other vertebrates, all three species have choroidal papillae. Based on 15 pteropodid species studied to date, we find no relationship between roost type and eye size or visual acuity. For a general understanding of the sensory ecology of pteropodids that perform key ecosystem services in the tropics, it will be essential to study additional species.

High diversity of arthropod colour vision: from genes to ecology.

Colour vision allows animals to use the information contained in the spectrum of light to control important behavioural decisions such as selection of habitats, food or mates. Among arthropods, the largest animal phylum, we find completely colour-blind species as well as species with up to 40 different opsin genes or more than 10 spectral types of photoreceptors, we find a large diversity of optical methods shaping spectral sensitivity, we find eyes with different colour vision systems looking into the dorsal and ventral hemisphere, and species in which males and females see the world in different colours. The behavioural use of colour vision shows an equally astonishing diversity. Only the neural mechanisms underlying this sensory ability seems surprisingly conserved-not only within the phylum, but even between arthropods and the other well-studied phylum, chordates. The papers in this special issue allow a glimpse into the colourful world of arthropod colour vision, and besides giving an overview this introduction highlights how much more research is needed to fill in the many missing pieces of this large puzzle. This article is part of the theme issue 'Understanding colour vision: molecular, physiological, neuronal and behavioural studies in arthropods'.

Allometric scaling of a superposition eye optimizes sensitivity and acuity in large and small hawkmoths.

Animals vary widely in body size within and across species. This has consequences for the function of organs and body parts in both large and small individuals. How these scale, in relation to body size, reveals evolutionary investment strategies, often resulting in trade-offs between functions. Eyes exemplify these trade-offs, as they are limited by their absolute size in two key performance features: sensitivity and spatial acuity. Due to their size polymorphism, insect compound eyes are ideal models for studying the allometric scaling of eye performance. Previous work on apposition compound eyes revealed that allometric scaling led to poorer spatial resolution and visual sensitivity in small individuals, across a range of insect species. Here, we used X-ray microtomography to investigate allometric scaling in superposition compound eyes-the second most common eye type in insects-for the first time. Our results reveal a novel strategy to cope with the trade-off between sensitivity and spatial acuity, as we show that the eyes of the hummingbird hawkmoth retain an optimal balance between these performance measures across all body sizes.

Spatial resolution and sensitivity of the eyes of the stingless bee, Tetragonula iridipennis.

Stingless bees are important pollinators in the tropics. The tremendous variation in body size makes them an excellent group to study how miniaturization affects vision and visual behaviours. Using direct measurements and micro-CT, we reconstructed the eye structure, estimated anatomical spatial resolution and optical sensitivity of the stingless bee Tetragonula iridipennis. T. iridipennis is similar in size to the Australian stingless bee Tetragonula carbonaria and is smaller than honeybees. It has correspondingly small eyes (area = 0.56 mm2), few ommatidia (2451 ± 127), large inter-facet (3.0 ± 0.6°) and acceptance angles (2.8°). Theoretical estimates suggest that T. iridipennis has poorer spatial resolution (0.17 cycles degree-1) than honeybees, bumblebees, and T. carbonaria. Its optical sensitivity (0.08 µm2 sr), though higher than expected, is within the range of diurnal bees. This may provide them with greater contrast sensitivity, which is likely more relevant than the absolute sensitivity in this diurnal bee. Behaviourally determined detection thresholds for single targets using y-maze experiments were 11.5° for targets that provide chromatic contrast alone and 9.1° for targets providing chromatic and achromatic contrast. Further studies into microhabitat preferences and behaviour are required to understand how miniaturization influences its visual ecology.

Seeing the world through the eyes of a butterfly: visual ecology of the territorial males of Pararge aegeria (Lepidoptera: Nymphalidae).

Combining studies of animal visual systems with exact imaging of their visual environment can get us a step closer to understand how animals see their "Umwelt". Here, we have combined both methods to better understand how males of the speckled wood butterfly, Pararge aegeria, see the surroundings of their perches. These males are well known to sit and wait for a chance to mate with a passing females, in sunspot territories in European forests. We provide a detailed description of the males' body and head posture, viewing direction, visual field and spatial resolution, as well as the visual environment. Pararge aegeria has sexually dimorphic eyes, the smallest interommatidial angles of males are around 1°, those of females 1.5°. Perching males face the antisolar direction with their retinal region of the highest resolution pointing at an angle of about 45° above the horizon; thus, looking at a rather even and dark background in front of which they likely have the best chance to detect a sunlit female passing through the sunspot.

Lens and cornea limit UV vision of birds - a phylogenetic perspective.

Most vertebrates have UV-sensitive vision, but the UV sensitivity of their eyes is limited by the transmittance of the ocular media, and the specific contribution of the different media (cornea, lens) has remained unclear. Here, we describe the transmittance of all ocular media (OMT), as well as that of lenses and corneas of birds. For 66 species belonging to 18 orders, the wavelength at which 50% of light is transmitted through the ocular media to the retina (λT0.5) ranges from 310 to 398 nm. Low λT0.5 corresponds to more UV light transmitted. Corneal λT0.5 varies only between 300 and 345 nm, whereas lens λT0.5 values are more variable (between 315 and 400 nm) and tend to be the limiting factor, determining OMT in the majority of species. OMT λT0.5 is positively correlated with eye size, but λT0.5 of corneas and lenses are not correlated with their thickness when controlled for phylogeny. Corneal and lens transmittances do not differ between birds with UV- and violet-sensitive SWS1 opsin when controlling for eye size and phylogeny. Phylogenetic relatedness is a strong predictor of OMT, and ancestral state reconstructions suggest that from ancestral intermediate OMT, highly UV-transparent ocular media (low λT0.5) evolved at least five times in our sample of birds. Some birds have evolved in the opposite direction towards a more UV-opaque lens, possibly owing to pigmentation, likely to mitigate UV damage or reduce chromatic aberration.

Visual acuity of budgerigars for moving targets.

For a bird, it is often vital to visually detect food items, predators, or individuals from the same flock, i.e. moving stimuli of various shapes. Yet, behavioural tests of visual spatial acuity traditionally use stationary gratings as stimuli. We have behaviourally tested the ability of budgerigars (Melopsittacus undulatus) to detect a black circular target, moving semi-randomly at 1.69 degrees s-1 against a brighter background. We found a detection threshold of 0.107±0.007 degrees of the visual field for a target size corresponding to a resolution of a grating with a spatial frequency of 4.68 cycles degree-1. This detection threshold is lower than the resolution limit for gratings but similar to the threshold for stationary single objects of the same shape. We conclude that the target acuity of budgerigars for moving single targets, just as for stationary single targets, is lower than their acuity for gratings.

Light, flight and the night: effect of ambient light and moon phase on flight activity of pteropodid bats.

Fruit-feeding pteropodid bats roost under varying light conditions. Some roost in trees with high exposure to daylight (> 1000 lx), while others roost in dark caves (< 0.1 lx). To understand the effect of ambient light intensity and moon phase on flight activity, we examined flight times across five lunar cycles in three pteropodid species whose roosts differ in daylight exposure. We found significant interspecific differences in flight emergence and termination times. All species initiated flights after sunset but Rousettus leschenaultii, which typically roosts in caves, delayed emergence (40 ± 11 min) more than the two tree-roosting species Pteropus giganteus (16 ± 6 min) and Cynopterus sphinx (19 ± 7 min). R. leschenaultii terminated flights earlier (30 ± 7 min before sunrise) than P. giganteus (11 ± 11 min) and C. sphinx (16 ± 10 min). All individuals from P. giganteus and C. sphinx roosts emerged within less than an hour, while emergence times were more spread out in the R. leschenaultii colony. Peak emergence times differed across moon phases in the cave-roosting R. leschenaultii but not in the other species. Flight activity in R. leschenaultii is restricted to comparatively lower light levels than the tree-roosting species. The observed interspecific differences suggest that bat species, sharing same landscapes may respond differently to light pollution.

Evolution of Insect Color Vision: From Spectral Sensitivity to Visual Ecology.

Color vision is widespread among insects but varies among species, depending on the spectral sensitivities and interplay of the participating photoreceptors. The spectral sensitivity of a photoreceptor is principally determined by the absorption spectrum of the expressed visual pigment, but it can be modified by various optical and electrophysiological factors. For example, screening and filtering pigments, rhabdom waveguide properties, retinal structure, and neural processing all influence the perceived color signal. We review the diversity in compound eye structure, visual pigments, photoreceptor physiology, and visual ecology of insects. Based on an overview of the current information about the spectral sensitivities of insect photoreceptors, covering 221 species in 13 insect orders, we discuss the evolution of color vision and highlight present knowledge gaps and promising future research directions in the field.

Chicken colour discrimination depends on background colour.

How well can a bird discriminate between two red berries on a green background? The absolute threshold of colour discrimination is set by photoreceptor noise, but animals do not perform at this threshold; their performance can depend on additional factors. In humans and zebra finches, discrimination thresholds for colour stimuli depend on background colour, and thus the adaptive state of the visual system. We have tested how well chickens can discriminate shades of orange or green presented on orange or green backgrounds. Chickens discriminated slightly smaller colour differences between two stimuli presented on a similarly coloured background, compared with a background of very different colour. The slope of the psychometric function was steeper when stimulus and background colours were similar but shallower when they differed markedly, indicating that background colour affects the certainty with which the animals discriminate the colours. The effect we find for chickens is smaller than that shown for zebra finches. We modelled the response to stimuli using Bayesian and maximum likelihood estimation and implemented the psychometric function to estimate the effect size. We found that the result is independent of the psychophysical method used to evaluate the effect of experimental conditions on choice performance.

The Pupillary Response of the Common Octopus (Octopus vulgaris).

Cephalopods have very conspicuous eyes that are often compared to fish eyes. However, in contrast to many fish, the eyes of cephalopods possess mobile pupils. To increase the knowledge of pupillary and thus visual function in cephalopods, the dynamics of the pupil of one of the model species among cephalopods, the common octopus (Octopus vulgaris), was determined in this study. We measured pupillary area as a function of ambient luminance to document the light and dark reaction of the octopus eye. The results show that weak light (<1 cd/m2) is enough to cause a pupil constriction in octopus, and that the pupil reacts fast to changing light conditions. The t50-value defined as the time required for achieving half-maximum constriction ranged from 0.45 to 1.29 s and maximal constriction from 10 to 20% of the fully dilated pupil area, depending on the experimental condition. Axial light had a stronger influence on pupil shape than light from above, which hints at a shadow effect of the horizontal slit pupil. We observed substantial variation of the pupil area under all light conditions indicating that light-independent factors such as arousal or the need to camouflage the eye affect pupil dilation/constriction. In conclusion, the documentation of pupil dynamics provides evidence that the pupil of octopus is adapted to low ambient light levels. Nevertheless it can quickly adapt to and thus function under brighter illumination and in a very inhomogeneous light environment, an ability mediated by the dynamic pupil in combination with previously described additional processes of light/dark adaptation in octopus.

Visual adaptations of diurnal and nocturnal raptors.

Raptors have always fascinated mankind, owls for their highly sensitive vision, and eagles for their high visual acuity. We summarize what is presently known about the eyes as well as the visual abilities of these birds, and point out knowledge gaps. We discuss visual fields, eye movements, accommodation, ocular media transmittance, spectral sensitivity, retinal anatomy and what is known about visual pathways. The specific adaptations of owls to dim-light vision include large corneal diameters compared to axial (and focal) length, a rod-dominated retina and low spatial and temporal resolution of vision. Adaptations of diurnal raptors to high acuity vision in bright light include rod- and double cone-free foveae, high cone and retinal ganglion cell densities and high temporal resolution. We point out that more studies, preferably using behavioural and non-invasive methods, are desirable.

Inter-individual differences in foveal shape in a scavenging raptor, the black kite Milvus migrans.

Birds, and especially raptors, are believed to forage mainly using visual cues. Indeed, raptors (scavengers and predators) have the highest visual acuity known to date. However, scavengers and predators differ in their visual systems such as in their foveal configuration. While the function of the foveal shape remains unknown, individual variation has never been quantified in birds. In this study, we examined whether foveal shape differs among individuals in relation to eye size, sex, age, eye (left or right) and genetic proximity in a scavenging raptor, the black kite Milvus migrans. We assessed foveal shape in 47 individuals using spectral domain optical coherence tomography (OCT) and geometric morphometric analysis. We found that foveal depth was significantly related to eye size. While foveal width also increased with eye size, it was strongly related to age; younger individuals had a wider fovea with a more pronounced rim. We found no relationship between foveal shape and genetic proximity, suggesting that foveal shape is not a hereditary trait. Our study revealed that the shape of the fovea is directly linked to eye size and that the physical structure of the fovea may develop during the entire life of black kites.

Lens transmittance shapes ultraviolet sensitivity in the eyes of frogs from diverse ecological and phylogenetic backgrounds.

The amount of short wavelength (ultraviolet (UV), violet and blue) light that reaches the retina depends on the transmittance properties of the ocular media, especially the lens, and varies greatly across species in all vertebrate groups studied previously. We measured the lens transmittance in 32 anuran amphibians with different habits, geographical distributions and phylogenetic positions and used them together with eye size and pupil shape to evaluate the relationship with diel activity pattern, elevation and latitude. We found an unusually high lens UV transmittance in the most basal species, and a cut-off range that extends into the visible spectrum for the rest of the sample, with lenses even absorbing violet light in some diurnal species. However, other diurnal frogs had lenses that transmit UV light like the nocturnal species. This unclear pattern in the segregation of ocular media transmittance and diel activity is shared with other vertebrates and is consistent with the absence of significant correlations in our statistical analyses. Although we did not detect a significant phylogenetic effect, closely related species tend to have similar transmittances, irrespective of whether they share the same diel pattern or not, suggesting that anuran ocular media transmittance properties might be related to phylogeny.

How fast can raptors see?

Birds, and especially raptors, are highly visual animals. Some of them have the highest spatial resolving power known in the animal kingdom, allowing prey detection at distance. While many raptors visually track fast-moving and manoeuvrable prey, requiring high temporal resolution, this aspect of their visual system has never been studied before. In this study, we estimated how fast raptors can see, by measuring the flicker fusion frequency of three species with different lifestyles. We found that flicker fusion frequency differed among species, being at least 129 Hz in the peregrine falcon, Falco peregrinus, 102 Hz in the saker falcon, Falco cherrug, and 81 Hz in the Harris's hawk, Parabuteo unicinctus We suggest a potential link between fast vision and hunting strategy, with high temporal resolution in the fast-flying falcons that chase fast-moving, manoeuvrable prey and a lower resolution in the Harris's hawk, which flies more slowly and targets slower prey.