Osmosis as nature's method for establishing optical alignment.

For eyes to maintain optimal focus, precise coordination is required between lens optics and retina position, a mechanism that in vertebrates is governed by genetics, visual feedback, and possibly intraocular pressure (IOP).1 While the underlying processes have been intensely studied in vertebrates, they remain elusive in arthropods, though visual feedback may be unimportant.2 How do arthropod eyes remain functional while undergoing substantial growth? Here, we test whether a common physiological process, osmoregulation,3 could regulate growth in the sophisticated camera-type eyes of the predatory larvae of Thermonectus marmoratus diving beetles. Upon molting, their eye tubes elongate in less than an hour, and osmotic pressure measurements reveal that this growth is preceded by a transient increase in hemolymph osmotic pressure. Histological evaluation of support cells that determine the lens-to-retina spacing reveals swelling rather than the addition of new cells. In addition, as expected, treating larvae with hyperosmotic media post-molt leads to far-sighted (hyperopic) eyes due to a failure of proper lengthening of the eye tube and results in impaired hunting success. This study suggests that osmoregulation could be of ubiquitous importance for properly focused eyes.

Host plant shift differentially alters olfactory sensitivity in female and male Drosophila mojavensis.

Animals may vary in their utilization of plants depending on plant availability, and also on the sex of the animal. Evolutionary adaptations may arise, particularly in specialist animals to the chemistry of the host plants, and these adaptations may differ between the sexes due to differences in their interactions with the plants. Drosophila mojavensis uses different host cacti across its range, and volatile chemicals emitted by the host are the primary cue for host plant identification. In this study, we measured responses of individual olfactory sensory neurons to a large suite of odorants across males and females of the two southern D. mojavensis populations. We show that a switch in host plant is accompanied by changes in the olfactory system, but the effect of this switch is minor compared to that of sex. That is, we observe differences in olfactory receptor neuron specificity and sensitivity to odorants between sexes, and to a lesser extent between populations. The majority of sensory differences are restricted to only three of the 17 sensory neurons measured. Further, we found numerous differences between sexes that only occur within one population, i.e., sex-by-population interactions.

Behavioral and Transcriptional Response to Selection for Olfactory Behavior in Drosophila.

The detection, discrimination, and behavioral responses to chemical cues in the environment can have marked effects on organismal survival and reproduction, eliciting attractive or aversive behavior. To gain insight into mechanisms mediating this hedonic valence, we applied thirty generations of divergent artificial selection for Drosophila melanogaster olfactory behavior. We independently selected for positive and negative behavioral responses to two ecologically relevant chemical compounds: 2,3-butanedione and cyclohexanone. We also tested the correlated responses to selection by testing behavioral responses to other odorants and life history traits. Measurements of behavioral responses of the selected lines and unselected controls to additional odorants showed that the mechanisms underlying responses to these odorants are, in some cases, differentially affected by selection regime and generalization of the response to other odorants was only detected in the 2,3-butanedione selection lines. Food consumption and lifespan varied with selection regime and, at times, sex. An analysis of gene expression of both selection regimes identified multiple differentially expressed genes. New genes and genes previously identified in mediating olfactory behavior were identified. In particular, we found functional enrichment of several gene ontology terms, including cell-cell adhesion and sulfur compound metabolic process, the latter including genes belonging to the glutathione S-transferase family. These findings highlight a potential role for glutathione S-transferases in the evolution of hedonic valence to ecologically relevant volatile compounds and set the stage for a detailed investigation into mechanisms by which these genes mediate attraction and aversion.

Evolution of coeloconic sensilla in the peripheral olfactory system of Drosophila mojavensis.

Populations inhabiting habitats with different environmental conditions, such as climate, resource availability, predation, competition, can undergo selection for traits that are adaptive in one habitat and not the other, leading to divergence between populations. Changes in the olfactory systems of insects that rely on different host plants, for example, can occur in response to differences in sensory stimuli between habitats. In this study, we investigate the evolution of host preference by characterizing the coeloconic sensilla in Drosophila mojavensis, a species that breeds on different necrotic cacti across its geographic range. These cactus species differ in the volatile chemicals they emit, a primary sensory cue for host plant discrimination. Analysis of odor-evoked responses identified four coeloconic sensilla that were qualitatively similar to those of Drosophila melanogaster, but varied in the breadth and strength of their olfactory sensory neuron responses to some acids and amines. Variation in responses to certain odorants among D. mojavensis populations was also observed. Compared to D. melanogaster, there was a lack of sensitivity of antennal coeloconic type 3 (ac3) sensilla to primary ligands of OR35a across all populations. Consistent with this result was a lack of detectable Or35a gene expression. Using a comparative approach, we then examined odor specificity of ac3 sensilla for seven additional Drosophila species, and found that OR35a-like sensitivity may be limited to the melanogaster subgroup. The variation in specificity that was observed among species is not clearly attributable to the degree of ecological specialization, nor to the ecological niche.

Population differences in olfaction accompany host shift in Drosophila mojavensis.

Evolutionary shifts in plant-herbivore interactions provide a model for understanding the link among the evolution of behaviour, ecological specialization and incipient speciation. Drosophila mojavensis uses different host cacti across its range, and volatile chemicals emitted by the host are the primary cue for host plant identification. In this study, we show that changes in host plant use between distinct D. mojavensis populations are accompanied by changes in the olfactory system. Specifically, we observe differences in olfactory receptor neuron specificity and sensitivity, as well as changes in sensillar subtype abundance, between populations. Additionally, RNA-seq analyses reveal differential gene expression between populations for members of the odorant receptor gene family. Hence, alterations in host preference are associated with changes in development, regulation and function at the olfactory periphery.

Spectral sensitivity of the principal eyes of sunburst diving beetle, Thermonectus marmoratus (Coleoptera: Dytiscidae), larvae.

The principal eyes of sunburst diving beetle, Thermonectus marmoratus, larvae are among the most unusual eyes in the animal kingdom. They are composed of long tubes connecting bifocal lenses with two retinas: a distal retina situated a few hundred micrometers behind the lens, and a proximal retina that is situated directly beneath. A recent molecular study on first instar larvae suggests that the distal retina expresses a long-wavelength-sensitive opsin (TmLW), whereas the proximal retina predominantly expresses an ultraviolet-sensitive opsin (TmUV II). Using cloning and in situ hybridization we here confirm that this opsin distribution is, for the most part, maintained in third instar larvae (with the exception of the TmUV I that is weakly expressed only in proximal retinas of first instar larvae). We furthermore use intracellular electrophysiological recordings and neurobiotin injections to determine the spectral sensitivity of individual photoreceptor cells. We find that photoreceptors of the proximal retina have a sensitivity curve that peaks at 374-375 nm. The shape of the curve is consistent with the predicted absorbance of a single-opsin template. The spectral response of photoreceptors from the distal retina confirms their maximum sensitivity to green light with the dominant λ-peak between 520 and 540 nm, and the secondary ß-peak between 340 and 360 nm. These physiological measurements support molecular predictions and represent important steps towards understanding the functional organization of the unusual stemmata of T. marmoratus larvae.

Molecular evidence for color discrimination in the Atlantic sand fiddler crab, Uca pugilator.

Fiddler crabs are intertidal brachyuran crabs that belong to the genus Uca. Approximately 97 different species have been identified, and several of these live sympatrically. Many have species-specific body color patterns that may act as signals for intra- and interspecific communication. To understand the behavioral and ecological role of this coloration we must know whether fiddler crabs have the physiological capacity to perceive color cues. Using a molecular approach, we identified the opsin-encoding genes and determined their expression patterns across the eye of the sand fiddler crab, Uca pugilator. We identified three different opsin-encoding genes (UpRh1, UpRh2 and UpRh3). UpRh1 and UpRh2 are highly related and have similarities in their amino acid sequences to other arthropod long- and medium-wavelength-sensitive opsins, whereas UpRh3 is similar to other arthropod UV-sensitive opsins. All three opsins are expressed in each ommatidium, in an opsin-specific pattern. UpRh3 is present only in the R8 photoreceptor cell, whereas UpRh1 and UpRh2 are present in the R1-7 cells, with UpRh1 expression restricted to five cells and UpRh2 expression present in three cells. Thus, one photoreceptor in every ommatidium expresses both UpRh1 and UpRh2, providing another example of sensory receptor coexpression. These results show that U. pugilator has the basic molecular machinery for color perception, perhaps even trichromatic vision.

Biological bifocal lenses with image separation.

Almost all animal eyes follow a few, relatively well-understood functional plans. Only rarely do researchers discover an eye that diverges fundamentally from known types. The principal eye E2 of sunburst diving beetle (Thermonectus marmoratus) larvae clearly falls into the rarer category. On the basis of two different tests, we here report that it has truly bifocal lenses, something that has been previously suggested only for certain trilobites. Our evidence comes from (1) the relative contrast in images of a square wave grating and (2) the refraction of a narrow laser beam projected through the lens. T. marmoratus larvae have two retinas at different depths behind the lens, and these are situated so that each can receive its own focused image. This is consistent with a novel eye organization that possibly comprises "two eyes in one." Moreover, we find that in contrast to most commercial bifocal lenses, the lens of E2 exhibits asymmetry, which results in separation of the images both dorsoventrally and rostrocaudally within the layered retina. Visual contrast might thus be improved over conventional bifocal lenses because the unfocused version of one image is shifted away from the focused version of the other, an organization which could potentially be exploited in optical engineering.

Fiddler crabs accurately measure two-dimensional distance over three-dimensional terrain.

Foraging fiddler crabs (Uca spp.) monitor the location of, and are able to return to, their burrows by employing path integration. This requires them to accurately measure both the directions and distances of their locomotory movements. Even though most fiddler crabs inhabit relatively flat terrain, they must cope with vertical features of their environment, such as sloping beaches, mounds and shells, which may represent significant obstacles. To determine whether fiddler crabs can successfully perform path integration among such three-dimensional obstacles, we tested their ability to measure distance while we imposed a vertical detour. By inserting a large hill in the homeward path of foraging crabs we show that fiddler crabs can cope with vertical detours: they accurately travel the correct horizontal distance, despite the fact that the shape of the hill forces them to change their gait from what would be used on flat ground. Our results demonstrate a flexible path integrator capable of measuring, and either integrating or discarding, the vertical dimension.

Direct evidence for distance measurement via flexible stride integration in the fiddler crab.

While on foraging excursions, fiddler crabs track their burrow location despite having no visual contact with it . They do this by path integration, a common navigational process in which motion vectors (the direction and distance of animals' movements) are summed to form a single "home vector" linking the current location with the point of origin. Here, we identify the mechanism by which the integrator measures distance, by decoupling motor output from both inertial and visual feedback. Fiddler crabs were passively translated to a position such that the home vector lay across an acetate sheet on the ground. After being frightened, crabs tried to escape but slipped as they did so. Detailed high-speed video analysis reveals that crabs measure distance by integrating strides, rather than linear acceleration or optic flow: the number of steps they took depended on both the length of the home vector and how large their steps were, whether they slipped and fell short or not. This is the most direct evidence to date of a stride integrator that is flexible enough to account for significant variation in stride length and frequency.

Behavioral evidence for within-eyelet resolution in twisted-winged insects (Strepsiptera).

Compound eyes are typically composed of hundreds to thousands of ommatidia, each containing 8-10 receptors. The maximal spatial frequency at which a compound eye can sample the environment is determined by the inter-ommatidial angle. Males of the insect order Strepsiptera are different: their eyes are composed of a smaller number of relatively large units (eyelets), each with an extended retina. Building on a study of Xenos vesparum, we use a behavioral paradigm based on the optomotor response to investigate the possibility that the eyelets of the Strepsiptera Xenos peckii are image-forming units. From anatomical evidence, we hypothesize that spatial sampling in the strepsipteran eye is determined not only by the interactions of widely spaced photoreceptors in different eyelets, but also by the angular separation between groups of closely spaced photoreceptors within eyelets. We compared X. peckii's optomotor response with the predictions of an elementary motion detector (EMD) model consisting of two distinctly different sampling bases. The best match between our empirical results and the model shows that the optomotor response in X. peckii males is determined by both the small (intra-eyelet) and large (possibly inter-eyelet) separations. Our results indicate that the X. peckii eye has sampling bases around 10 degrees and 20 degrees , and that each eyelet could be composed of up to 13 sampling points, which is consistent with previous anatomical findings. This study is the first to use the EMD model explicitly to investigate the possibility that strepsipteran eyes combine motion detection features from both camera and compound eyes.

The role of target elevation in prey selection by tiger beetles (Carabidae: Cicindela spp.).

The elevation of objects in the visual field has long been recognized as a potential distance cue, but it has been demonstrated to a reasonable extent in only four species: humans, frogs, fiddler crabs and backswimmers. Many tiger beetles hunt in flat, sandy areas, and their eyes show "flat-world" adaptations, such as an extended visual streak of higher acuity that corresponds to the horizon. They are therefore possible candidates for the use of elevation as a cue for distance. We tested this empirically and with simulation. In a behavioral prey selection paradigm, in which starved beetles were presented moving prey-targets having different size, speed and elevation, the beetles showed a strong preference for large targets when these were low in the visual field and a weaker preference for small targets when these were near the horizon. Striking of targets above the horizon was reduced compared to sub-horizontal targets, and lacked the size-elevation interaction. We simulated these empirical results with a model that converted elevation to distance, and used distance to estimate the absolute size of the targets. Simulated strike probability was then determined by the similarity between this absolute size and an independently confirmed preferred prey size. The results of the simulation model matched the empirical data as well as the best statistical model of the behavioral results. While some aspects of the model, and the beetles' behavior, differ from the strict geometry of the "elevation hypothesis", our results nevertheless indicate that tiger beetles use elevation to estimate distance to prey, and that it is therefore one of the determinants of prey selection.

Mechanisms of homing in the fiddler crab Uca rapax. 2. Information sources and frame of reference for a path integration system

Fiddler crabs Uca rapax are central-place foragers, making feeding excursions of up to several meters from their burrows. This study investigates the sources of directional and distance information used by these crabs when returning to their burrows. We tested the spatial frame of reference (egocentric or exocentric), and the source of spatial information (idiothetic or allothetic) used during homing. We also tested which components of their locomotion they integrated (only voluntary, or voluntary plus reflexive). Fiddler crabs in their natural mudflat habitat were passively rotated during normal foraging behavior using experimenter-controlled disks, before they returned home. Crabs resisted passive rotations on the disk by counter-rotating when the disk turned, which was a compensatory response to unintended movement. Crabs were usually situated eccentrically on the disk, and therefore were also subjected to a translation when the disk rotated. No crab actively compensated for this translation. Crabs that fully compensated for disk rotation made no directional homing error. Crabs that did not fully compensate homed in a direction that reflected their new body orientation. In other words, if we succeeded in reorienting a crab (i.e. it undercompensated for disk rotation), its homing error was equal to the angle by which it had been reoriented, regardless of the magnitude of the optomotor compensation. Computer-modelled crabs, each equipped with a path integrator utilizing different combinations of external (allothetic) and path-related (idiothetic) input, traversed the digitized paths of the real crabs. The home vector computed by the model crab was then compared to the homing direction observed in the real crab. The model home vector that most closely matched that of the real crab was taken to comprise the path integration mechanism employed by fiddler crabs. The model that best matched the real crab gained direction and distance idiothetically (from internal sources such as proprioceptors), and integrated only voluntary locomotory information. Crabs were also made to run home across a patch of wet acetate, on which they slipped and were thus forced to take more steps on the homeward path than theoretically required by the home vector. Crabs whose running velocity across the patch was unusually low also stopped short of their burrow before finding it. Crabs whose running velocity was not impeded by the patch did not stop short, but ran straight to the burrow entrance, as did control crabs that ran home with no slippery patch. We interpret this to mean that the velocity of some crabs was impeded because of slipping, and these therefore stopped short of their burrow after having run out their homing vector. This is positive evidence in support of the hypothesis that path integration is mediated either by leg proprioceptors or by efferent commands, but our data do not allow us to distinguish between these two possibilities.

Mechanisms of homing in the fiddler crab Uca rapax. 1. Spatial and temporal characteristics of a system of small-scale navigation

Fiddler crabs Uca rapax are central-place foragers, making feeding excursions of up to 2 m from their burrows. We describe the natural feeding excursions of path-integrating fiddler crabs and analyze their paths for signs of significant systematic or random navigation errors. No signs of any systematic errors are evident. Random errors are small, probably due to a combination of the short length and low sinuosity of the foraging paths, as well as the fiddler crabs' unique method of locomotion that allows them to remain oriented to their burrows throughout the foraging path and to minimize large body turns. We further examined the extent to which their body orientation during foraging (transverse body axis pointing more or less towards home) accurately represented their stored home vector. By examining sequences of fast escape, we have shown that crabs can correct for deviations of their transverse body axis from home during their escape path. Thus their stored home vector is independent of their moment-to-moment body orientation. Crabs were subjected to passive translational displacements and barrier obstructions. Responses to translational displacements were identical to those observed by previous authors, namely that crabs returned in the correct egocentric direction and distance as though no displacement had occurred. Covering the burrow entrance resulted in crabs returning to the correct position of the burrow, and then beginning to search. When a barrier was placed between foraging crabs and their burrow, crabs oriented their bodies toward the burrow as accurately as with no barrier.

Mechanisms of homing in the fiddler crab Uca rapax. 1. Spatial and temporal characteristics of a system of small-scale navigation.

Fiddler crabs Uca rapax are central-place foragers, making feeding excursions of up to 2 m from their burrows. We describe the natural feeding excursions of path-integrating fiddler crabs and analyze their paths for signs of significant systematic or random navigation errors. No signs of any systematic errors are evident. Random errors are small, probably due to a combination of the short length and low sinuosity of the foraging paths, as well as the fiddler crabs' unique method of locomotion that allows them to remain oriented to their burrows throughout the foraging path and to minimize large body turns. We further examined the extent to which their body orientation during foraging (transverse body axis pointing more or less towards home) accurately represented their stored home vector. By examining sequences of fast escape, we have shown that crabs can correct for deviations of their transverse body axis from home during their escape path. Thus their stored home vector is independent of their moment-to-moment body orientation. Crabs were subjected to passive translational displacements and barrier obstructions. Responses to translational displacements were identical to those observed by previous authors, namely that crabs returned in the correct egocentric direction and distance as though no displacement had occurred. Covering the burrow entrance resulted in crabs returning to the correct position of the burrow, and then beginning to search. When a barrier was placed between foraging crabs and their burrow, crabs oriented their bodies toward the burrow as accurately as with no barrier.

Mechanisms of homing in the fiddler crab Uca rapax. 2. Information sources and frame of reference for a path integration system.

Fiddler crabs Uca rapax are central-place foragers, making feeding excursions of up to several meters from their burrows. This study investigates the sources of directional and distance information used by these crabs when returning to their burrows. We tested the spatial frame of reference (egocentric or exocentric), and the source of spatial information (idiothetic or allothetic) used during homing. We also tested which components of their locomotion they integrated (only voluntary, or voluntary plus reflexive). Fiddler crabs in their natural mudflat habitat were passively rotated during normal foraging behavior using experimenter-controlled disks, before they returned home. Crabs resisted passive rotations on the disk by counter-rotating when the disk turned, which was a compensatory response to unintended movement. Crabs were usually situated eccentrically on the disk, and therefore were also subjected to a translation when the disk rotated. No crab actively compensated for this translation. Crabs that fully compensated for disk rotation made no directional homing error. Crabs that did not fully compensate homed in a direction that reflected their new body orientation. In other words, if we succeeded in reorienting a crab (i.e. it undercompensated for disk rotation), its homing error was equal to the angle by which it had been reoriented, regardless of the magnitude of the optomotor compensation. Computer-modelled crabs, each equipped with a path integrator utilizing different combinations of external (allothetic) and path-related (idiothetic) input, traversed the digitized paths of the real crabs. The home vector computed by the model crab was then compared to the homing direction observed in the real crab. The model home vector that most closely matched that of the real crab was taken to comprise the path integration mechanism employed by fiddler crabs. The model that best matched the real crab gained direction and distance idiothetically (from internal sources such as proprioceptors), and integrated only voluntary locomotory information. Crabs were also made to run home across a patch of wet acetate, on which they slipped and were thus forced to take more steps on the homeward path than theoretically required by the home vector. Crabs whose running velocity across the patch was unusually low also stopped short of their burrow before finding it. Crabs whose running velocity was not impeded by the patch did not stop short, but ran straight to the burrow entrance, as did control crabs that ran home with no slippery patch. We interpret this to mean that the velocity of some crabs was impeded because of slipping, and these therefore stopped short of their burrow after having run out their homing vector. This is positive evidence in support of the hypothesis that path integration is mediated either by leg proprioceptors or by efferent commands, but our data do not allow us to distinguish between these two possibilities.