Polarization sensitivity and decentralized visual processing in an animal with a distributed visual system.

The marine mollusc Acanthopleura granulata (Mollusca; Polyplacophora) has a distributed visual array composed of hundreds of small image-forming eyes embedded within its eight dorsal shell plates. As in other animals with distributed visual systems, we still have a poor understanding of the visual capabilities of A. granulata and we have yet to learn where and how it processes visual information. Using behavioral trials involving isoluminant looming visual stimuli, we found that A. granulata demonstrates spatial vision with an angular resolution of 6 deg. We also found that A. granulata responds to looming stimuli defined by contrasting angles of linear polarization. To learn where and how A. granulata processes visual information, we traced optic nerves using fluorescent lipophilic dyes. We found that the optic nerves innervate the underlying lateral neuropil, a neural tissue layer that circumnavigates the body. Adjacent optic nerves innervate the lateral neuropil with highly overlapping arborizations, suggesting it is the site of an integrated visuotopic map. Using immunohistochemistry, we found that the lateral neuropil of A. granulata is subdivided into two separate layers. In comparison, we found that a chiton with eyespots (Chiton tuberculatus) and two eyeless chitons (Ischnochiton papillosus and Chaetopleura apiculata) have lateral neuropil that is a singular circular layer without subdivision, findings consistent with previous work on chiton neuroanatomy. Overall, our results suggest that A. granulata effectuates its visually mediated behaviors using a unique processing scheme: it extracts spatial and polarization information using a distributed visual system, and then integrates and processes that information using decentralized neural circuits.

Two sides of the same wing: ventral scales enhance dorsal wing color in the butterfly Speyeria mormonia.

Biological visual signals are often produced by complex interactions between light-absorbing and light-scattering structures, but for many signals, potential interactions between different light-interacting components have yet to be tested. Butterfly wings, for example, are thin enough that their two sides may not be optically isolated. We tested whether ventral wing scales of the Mormon fritillary, Speyeria mormonia, affect the appearance of dorsal orange patches, which are thought to be involved in sexual signaling. Using reflectance spectroscopy, we found that ventral scales, either silvered or non-silvered, make dorsal orange patches significantly brighter, with the silvered scales having the greater effect. Computational modeling indicates that both types of ventral scale enhance the chromatic perceptual signal of dorsal orange patches, with only the silvered scales also enhancing their achromatic perceptual signal. A lack of optical independence between the two sides of the wings of S. mormonia implies that the wing surfaces of butterflies have intertwined signaling functions and evolutionary histories.

The marine gastropod Conomurex luhuanus (Strombidae) has high-resolution spatial vision and eyes with complex retinas.

All species within the conch snail family Strombidae possess large camera-type eyes that are surprisingly well-developed compared with those found in most other gastropods. Although these eyes are known to be structurally complex, very little research on their visual function has been conducted. Here, we use isoluminant expanding visual stimuli to measure the spatial resolution and contrast sensitivity of a strombid, Conomurex luhuanus. Using these stimuli, we show that this species responds to objects as small as 1.06 deg in its visual field. We also show that C. luhuanus responds to Michelson contrasts of 0.07, a low contrast sensitivity between object and background. The defensive withdrawal response elicited by visual stimuli of such small angular size and low contrast suggests that conch snails may use spatial vision for the early detection of potential predators. We support these findings with morphological estimations of spatial resolution of 1.04 deg. These anatomical data therefore agree with the behavioural measures and highlight the benefits of integrating behavioural and morphological approaches in animal vision studies. Using contemporary imaging techniques [serial block-face scanning electron microscopy (SBF-SEM), in conjunction with transmission electron microscopy (TEM)], we found that C. luhuanus have more complex retinas, in terms of cell type diversity, than expected based on previous studies of the group using TEM alone. We find the C. luhuanus retina comprises six cell types, including a newly identified ganglion cell and accessory photoreceptor, rather than the previously described four cell types.

Panoramic spatial vision in the bay scallop Argopecten irradians.

We have a growing understanding of the light-sensing organs and light-influenced behaviours of animals with distributed visual systems, but we have yet to learn how these animals convert visual input into behavioural output. It has been suggested they consolidate visual information early in their sensory-motor pathways, resulting in them being able to detect visual cues (spatial resolution) without being able to locate them (spatial vision). To explore how an animal with dozens of eyes processes visual information, we analysed the responses of the bay scallop Argopecten irradians to both static and rotating visual stimuli. We found A. irradians distinguish between static visual stimuli in different locations by directing their sensory tentacles towards them and were more likely to point their extended tentacles towards larger visual stimuli. We also found that scallops track rotating stimuli with individual tentacles and with rotating waves of tentacle extension. Our results show, to our knowledge for the first time that scallops have both spatial resolution and spatial vision, indicating their sensory-motor circuits include neural representations of their visual surroundings. Exploring a wide range of animals with distributed visual systems will help us learn the different ways non-cephalized animals convert sensory input into behavioural output.

Automated methods for efficient and accurate electroretinography.

Electroretinography (ERG) is a foundational method for assessing visual system physiology, but accurate ERG can be time- and labor-intensive, often involving manual adjustment of the wavelength and intensity of light stimuli and real-time comparison of physiological responses to inform those adjustments. Furthermore, current approaches to ERG often require expertise beyond that necessary for the electrophysiological preparation itself. To improve both the efficiency and accessibility of ERG, we designed an automated system for stimulus presentation and data acquisition. Here, we test this novel system's ability to accurately assess spectral sensitivity in the well-characterized visual system of the crayfish Procambarus clarkii using three approaches: the first, based on response magnitude, maximizes efficiency; the second is a well-established method we use to further validate our efficient approach's accuracy. Third, we explore the potential benefits of extensible automation using a method assessing the interplay between temporal acuity and spectral sensitivity. Using our system, we are able to acquire accurate results in ERG experiments quickly (testing the entire visible spectrum in 8 min, 30 s using our response magnitude approach). Moreover, data collected via all three methods yielded results consistent with each other and previous work on P. clarkii.

A snapping shrimp has the fastest vision of any aquatic animal.

Animals use their sensory systems to sample information from their environments. The physiological properties of sensory systems differ, leading animals to perceive their environments in different ways. For example, eyes have different temporal sampling rates, with faster-sampling eyes able to resolve faster-moving scenes. Eyes can also have different dynamic ranges. For every eye, there is a light level below which vision is unreliable because of an insufficient signal-to-noise ratio and a light level above which the photoreceptors are saturated. Here, we report that the eyes of the snapping shrimp Alpheus heterochaelis have a temporal sampling rate of at least 160 Hz, making them the fastest-sampling eyes ever described in an aquatic animal. Fast-sampling eyes help flying animals detect objects moving across their retinas at high angular velocities. A. heterochaelis are fast-moving animals that live in turbid, structurally complex oyster reefs and their fast-sampling eyes, like those of flying animals, may help them detect objects moving rapidly across their retinas. We also report that the eyes of A. heterochaelis have a broad dynamic range that spans conditions from late twilight (approx. 1 lux) to direct sunlight (approx. 100 000 lux), a finding consistent with the circatidal activity patterns of this shallow-dwelling species.

Vision in the snapping shrimp Alpheus heterochaelis.

Snapping shrimp engage in heterospecific behavioral associations in which their partners, such as goby fish, help them avoid predators. It has been argued that snapping shrimp engage in these partnerships because their vision is impaired by their orbital hood, an extension of their carapace that covers their eyes. To examine this idea, we assessed the visual abilities of snapping shrimp. We found the big claw snapping shrimp, Alpheus heterochaelis, has spatial vision provided by compound eyes with reflecting superposition optics. These eyes view the world through an orbital hood that is 80-90% as transparent as seawater across visible wavelengths (400-700 nm). Through electroretinography and microspectrophotometry, we found the eyes of A. heterochaelis have a temporal sampling rate of >40 Hz and have at least two spectral classes of photoreceptors (λmax=500 and 519 nm). From the results of optomotor behavioral experiments, we estimate the eyes of A. heterochaelis provide spatial vision with an angular resolution of ∼8 deg. We conclude that snapping shrimp have competent visual systems, suggesting the function and evolution of their behavioral associations should be re-assessed and that these animals may communicate visually with conspecifics and heterospecific partners.

Evidence for spatial vision in Chiton tuberculatus, a chiton with eyespots.

To better understand relationships between the structures and functions of the distributed visual systems of chitons, we compare how morphological differences between the light-sensing structures of these animals relate to their visually guided behaviors. All chitons have sensory organs - termed aesthetes - embedded within their protective shell plates. In some species, the aesthetes are interspersed with small, image-forming eyes. In other species, the aesthetes are paired with pigmented eyespots. Previously, we compared the visually influenced behaviors of chitons with aesthetes to those of chitons with both aesthetes and eyes. Here, we characterize the visually influenced behaviors of chitons with aesthetes and eyespots. We find that chitons with eyespots engage in behaviors consistent with spatial vision, but appear to use spatial vision for different tasks than chitons with eyes. Unlike chitons with eyes, Chiton tuberculatus and C. marmoratus fail to distinguish between sudden appearances of overhead objects and equivalent, uniform changes in light levels. We also find that C. tuberculatus orients to static objects with angular sizes as small as 10 deg. Thus, C. tuberculatus demonstrates spatial resolution that is at least as fine as that demonstrated by chitons with eyes. The eyespots of Chiton are smaller and more numerous than the eyes found in other chitons and they are separated by angles of <0.5 deg, suggesting that the light-influenced behaviors of Chiton may be more accurately predicted by the network properties of their distributed visual system than by the structural properties of their individual light-detecting organs.

Opsins in Limulus eyes: characterization of three visible light-sensitive opsins unique to and co-expressed in median eye photoreceptors and a peropsin/RGR that is expressed in all eyes.

The eyes of the horseshoe crab Limulus polyphemus have long been used for studies of basic mechanisms of vision, and the structure and physiology of Limulus photoreceptors have been examined in detail. Less is known about the opsins Limulus photoreceptors express. We previously characterized a UV opsin (LpUVOps1) that is expressed in all three types of Limulus eyes (lateral compound eyes, median ocelli and larval eyes) and three visible light-sensitive rhabdomeric opsins (LpOps1, -2 and -5) that are expressed in Limulus lateral compound and larval eyes. Physiological studies showed that visible light-sensitive photoreceptors are also present in median ocelli, but the visible light-sensitive opsins they express were unknown. In the current study we characterize three newly identified, visible light-sensitive rhabdomeric opsins (LpOps6, -7 and -8) that are expressed in median ocelli. We show that they are ocellar specific and that all three are co-expressed in photoreceptors distinct from those expressing LpUVOps1. Our current findings show that the pattern of opsin expression in Limulus eyes is much more complex than previously thought and extend our previous observations of opsin co-expression in visible light-sensitive Limulus photoreceptors. We also characterize a Limulus peropsin/RGR (LpPerOps1). We examine the phylogenetic relationship of LpPerOps1 with other peropsins and RGRs, demonstrate that LpPerOps1 transcripts are expressed in each of the three types of Limulus eyes and show that the encoded protein is expressed in membranes of cells closely associated with photoreceptors in each eye type. These finding suggest that peropsin was in the opsin repertoire of euchelicerates.

Using phylogenetically-informed annotation (PIA) to search for light-interacting genes in transcriptomes from non-model organisms.

BACKGROUND: Tools for high throughput sequencing and de novo assembly make the analysis of transcriptomes (i.e. the suite of genes expressed in a tissue) feasible for almost any organism. Yet a challenge for biologists is that it can be difficult to assign identities to gene sequences, especially from non-model organisms. Phylogenetic analyses are one useful method for assigning identities to these sequences, but such methods tend to be time-consuming because of the need to re-calculate trees for every gene of interest and each time a new data set is analyzed. In response, we employed existing tools for phylogenetic analysis to produce a computationally efficient, tree-based approach for annotating transcriptomes or new genomes that we term Phylogenetically-Informed Annotation (PIA), which places uncharacterized genes into pre-calculated phylogenies of gene families. RESULTS: We generated maximum likelihood trees for 109 genes from a Light Interaction Toolkit (LIT), a collection of genes that underlie the function or development of light-interacting structures in metazoans. To do so, we searched protein sequences predicted from 29 fully-sequenced genomes and built trees using tools for phylogenetic analysis in the Osiris package of Galaxy (an open-source workflow management system). Next, to rapidly annotate transcriptomes from organisms that lack sequenced genomes, we repurposed a maximum likelihood-based Evolutionary Placement Algorithm (implemented in RAxML) to place sequences of potential LIT genes on to our pre-calculated gene trees. Finally, we implemented PIA in Galaxy and used it to search for LIT genes in 28 newly-sequenced transcriptomes from the light-interacting tissues of a range of cephalopod mollusks, arthropods, and cubozoan cnidarians. Our new trees for LIT genes are available on the Bitbucket public repository ( http://bitbucket.org/osiris_phylogenetics/pia/ ) and we demonstrate PIA on a publicly-accessible web server ( http://galaxy-dev.cnsi.ucsb.edu/pia/ ). CONCLUSIONS: Our new trees for LIT genes will be a valuable resource for researchers studying the evolution of eyes or other light-interacting structures. We also introduce PIA, a high throughput method for using phylogenetic relationships to identify LIT genes in transcriptomes from non-model organisms. With simple modifications, our methods may be used to search for different sets of genes or to annotate data sets from taxa outside of Metazoa.

Simplifying numerical ray tracing for characterization of optical systems.

Ray tracing, a computational method for tracing the trajectories of rays of light through matter, is often used to characterize mechanical or biological visual systems with aberrations that are larger than the effect of diffraction inherent in the system. For example, ray tracing may be used to calculate geometric point spread functions (PSFs), which describe the image of a point source after it passes through an optical system. Calculating a geometric PSF is useful because it gives an estimate of the detail and quality of the image formed by a given optical system. However, when using ray tracing to calculate a PSF, the accuracy of the estimated PSF directly depends on the number of discrete rays used in the calculation; higher accuracies may require more computational power. Furthermore, adding optical components to a modeled system will increase its complexity and require critical modifications so that the model will describe the system correctly, sometimes necessitating a completely new model. Here, we address these challenges by developing a method that represents rays of light as a continuous function that depends on the light's initial direction. By utilizing Chebyshev approximations (via the chebfun toolbox in MATLAB) for the implementation of this method, we greatly simplified the calculations for the location and direction of the rays. This method provides high precision and fast calculation speeds that allow the characterization of any symmetrical optical system (with a centered point source) in an analytical-like manner. Next, we demonstrate our methods by showing how they can easily calculate PSFs for complicated optical systems that contain multiple refractive and/or reflective interfaces.

A morphological basis for path-dependent evolution of visual systems.

Path dependence influences macroevolutionary predictability by constraining potential outcomes after critical evolutionary junctions. Although it has been demonstrated in laboratory experiments, path dependence is difficult to demonstrate in natural systems because of a lack of independent replicates. Here, we show that two types of distributed visual systems recently evolved twice within chitons, demonstrating rapid and path-dependent evolution of a complex trait. The type of visual system that a chiton lineage can evolve is constrained by the number of openings for sensory nerves in its shell plates. Lineages with more openings evolve visual systems with thousands of eyespots, whereas those with fewer openings evolve visual systems with hundreds of shell eyes. These macroevolutionary outcomes shaped by path dependence are both deterministic and stochastic because possibilities are restricted yet not entirely predictable.

Snapping shrimp have helmets that protect their brains by dampening shock waves.

Shock waves are supersonic high-amplitude pressure waves that cause barotrauma when they transfer kinetic energy to the tissues of animals.1-4 Snapping shrimp (Alpheidae) produce shock waves and are exposed to them frequently, so we asked if these animals have evolved mechanisms of physical protection against them. Snapping shrimp generate shock waves by closing their snapping claws rapidly enough to form cavitation bubbles that release energy as an audible "snap" and a shock wave when they collapse.5-8 We tested if snapping shrimp are protected from shock waves by a helmet-like extension of their exoskeleton termed the orbital hood. Using behavioral trials, we found shock wave exposure slowed shelter-seeking and caused a loss of motor control in Alpheus heterochaelis from which we had removed orbital hoods but did not significantly affect behavior in shrimp with unaltered orbital hoods. Shock waves thus have the potential to harm snapping shrimp but may not do so under natural conditions because of protection provided to shrimp by their orbital hoods. Using pressure recordings, we discovered the orbital hoods of A. heterochaelis dampen shock waves. Sealing the anterior openings of orbital hoods diminished how much they altered the magnitudes of shock waves, which suggests these helmet-like structures dampen shock waves by trapping and expelling water so that kinetic energy is redirected and released away from the heads of shrimp. Our results indicate orbital hoods mitigate blast-induced neurotrauma in snapping shrimp by dampening shock waves, making them the first biological armor system known to have such a function. VIDEO ABSTRACT.

Spectral sensitivity of the concave mirror eyes of scallops: potential influences of habitat, self-screening and longitudinal chromatic aberration.

Scallop eyes contain two retinas, one proximal and one distal. Molecular evidence suggests that each retina expresses a different visual pigment. To test whether these retinas have different spectral sensitivities, we used microspectrophotometry to measure the absorption spectra of photoreceptors from the eyes of two different scallop species. Photoreceptors from the proximal and distal retinas of the sea scallop Placopecten magellanicus had absorption peak wavelengths (λ(max)) of 488 ± 1 nm (mean ± s.e.m.; N=20) and 513 ± 3 nm (N=26), respectively. Photoreceptors from the corresponding retinas of the bay scallop Argopecten irradians had λ(max) values of 506 ± 1 nm (N=21) and 535 ± 3 nm (N=14). Assuming that the proximal and distal receptors had equal absorption coefficients (k(D)=0.0067 microm(-1)), we found that self-screening within the scallop eye caused the proximal and distal receptors in P. magellanicus to have peak absorption at 490 and 520 nm, respectively, and the corresponding receptors in A. irradians to have peak absorption at 504 and 549 nm. We conclude that environment may influence the λ(max) of scallop visual pigments: P. magellanicus, generally found in blue oceanic water, has visual pigments that are maximally sensitive to shorter wavelengths than those found in A. irradians, which lives in greener inshore water. Scallop distal retinas may be sensitive to longer wavelengths of light than scallop proximal retinas to correct for either self-screening by the retinas or longitudinal chromatic aberration of the lens.

Understanding the dermal light sense in the context of integrative photoreceptor cell biology.

While the concept of a dermal light sense has existed for over a century, little progress has been made in our understanding of the mechanisms underlying dispersed photoreception and the evolutionary histories of dispersed photoreceptor cells. These cells historically have been difficult to locate and positively identify, but modern molecular techniques, integrated with existing behavioral, morphological, and physiological data, will make cell identification easier and allow us to address questions of mechanism and evolution. With this in mind, we propose a new classification scheme for all photoreceptor cell types based on two axes, cell distribution (aggregated vs. dispersed) and position within neural networks (first order vs. high order). All photoreceptor cells fall within one of four quadrants created by these axes: aggregated/high order, dispersed/high order, aggregated/first order, or dispersed/first order. This new method of organization will help researchers make objective comparisons between different photoreceptor cell types. Using integrative data from four major phyla (Mollusca, Cnidaria, Echinodermata, and Arthropoda), we also provide evidence for three hypotheses for dispersed photoreceptor cell function and evolution. First, aside from echinoderms, we find that animals often use dispersed photoreceptor cells for tasks that do not require spatial vision. Second, although there are both echinoderm and arthropod exceptions, we find that dispersed photoreceptor cells generally lack morphological specializations that either enhance light gathering or aid in the collection of directional information about light. Third, we find that dispersed photoreceptor cells have evolved a number of times in Metazoa and that most dispersed photoreceptor cells have likely evolved through the co-option of existing phototransduction cascades. Our new classification scheme, combined with modern investigative techniques, will help us address these hypotheses in great detail and generate new hypothesis regarding the function and evolution of dispersed photoreceptor cells.

The orbital hoods of snapping shrimp have surface features that may represent tradeoffs between vision and protection.

Snapping shrimp (Alpheidae) are decapod crustaceans named for the snapping claws with which they produce cavitation bubbles. Snapping shrimp use the shock waves released by collapsing cavitation bubbles as weapons. Along with their distinctive claws, snapping shrimp have orbital hoods, extensions of their carapace that cover their heads and eyes. Snapping shrimp view the world through their orbital hoods, so we asked if the surfaces of the orbital hoods of the snapping shrimp Alpheus heterochaelis have features that minimize the scattering of light. Using SEM, we found that surface features, primarily microbial epibionts, covered less space on the surfaces of the orbital hoods of A. heterochaelis (∼18%) than they do elsewhere on the carapace (∼50%). Next, we asked if these surface features influence aerophobicity. By measuring the contact angles of air bubbles, we found the orbital hoods of A. heterochaelis are less aerophobic than other regions of the carapace. Surfaces that are less aerophobic are more likely to have cavitation bubbles adhere to them and are more likely to have shock waves cause new cavitation bubbles to nucleate upon them. Computational modeling indicates the orbital hoods of A. heterochaelis face a functional trade-off: fewer surface features, such as less extensive communities of microbial epibionts, may minimize the scattering of light at the cost of making the adhesion and nucleation of cavitation bubbles more likely.

The Iron-Responsive Genome of the Chiton Acanthopleura granulata.

Molluscs biomineralize structures that vary in composition, form, and function, prompting questions about the genetic mechanisms responsible for their production and the evolution of these mechanisms. Chitons (Mollusca, Polyplacophora) are a promising system for studies of biomineralization because they build a range of calcified structures including shell plates and spine- or scale-like sclerites. Chitons also harden the calcified teeth of their rasp-like radula with a coat of iron (as magnetite). Here we present the genome of the West Indian fuzzy chiton Acanthopleura granulata, the first from any aculiferan mollusc. The A. granulata genome contains homologs of many genes associated with biomineralization in conchiferan molluscs. We expected chitons to lack genes previously identified from pathways conchiferans use to make biominerals like calcite and nacre because chitons do not use these materials in their shells. Surprisingly, the A. granulata genome has homologs of many of these genes, suggesting that the ancestral mollusc may have had a more diverse biomineralization toolkit than expected. The A. granulata genome has features that may be specialized for iron biomineralization, including a higher proportion of genes regulated directly by iron than other molluscs. A. granulata also produces two isoforms of soma-like ferritin: one is regulated by iron and similar in sequence to the soma-like ferritins of other molluscs, and the other is constitutively translated and is not found in other molluscs. The A. granulata genome is a resource for future studies of molluscan evolution and biomineralization.

Visual Ecology: Now You See, Now You Don't.

During the day, the brittle star Ophiocoma wendtii demonstrates spatial vision due to a distributed network of extraocular photoreceptors whose fields of view are restricted by chromatophores. At night, these chromatophores contract and O. wendtii loses spatial vision.

Context-dependent evolution of ostracod morphology along the ecogeographical gradient of ocean depth.

Ecogeographical rules inform our understanding of biodiversity by seeking reliable associations between organismal phenotypes and environmental factors. Reminiscent of classic ecogeographical rules, environmental factors vary in predictable ways with ocean depth, leading to predictions about organismal phenotypes. A valuable group for studying associations between habitat depth and phenotype is cylindroleberidid ostracods (Crustacea) because of previous phylogenetic analyses and their enormous depth range. Using phylogenetic comparative methods, we asked how habitat depth relates to body size and eye morphology in 232 cylindroleberidid species measured from museum specimens and literature descriptions. For each species, we recorded maximum habitat depth, body size, absolute eye size, number of ommatidia (facets) per eye, and diameter of the largest ommatidium. We find that the relationship between morphology and habitat depth in cylindroleberidids depends on pelagic zone: as depth increases in the photic zone, body size increases and eyes have fewer ommatidia; as depth increases in the disphotic zone, body size does not change and eyes have more ommatidia. We did not find a relationship between absolute eye size and depth in either pelagic zone. Overall, we find that associations between phenotypes and ecogeographical gradients depend on interactions between contexts such as pelagic zone, character state, and evolutionary history.

The mirror-based eyes of scallops demonstrate a light-evoked pupillary response.

Light levels in terrestrial and shallow-water environments can vary by ten orders of magnitude between clear days and overcast nights. Light-evoked pupillary responses help the eyes of animals perform optimally under these variable light conditions by balancing trade-offs between sensitivity and resolution [1]. Here, we document that the mirror-based eyes of the bay scallop Argopecten irradians and the sea scallop Placopecten magellanicus have pupils that constrict to ∼60% of their fully dilated areas within several minutes of light exposure. The eyes of scallops contain two separate retinas and our ray-tracing model indicates that, compared to eyes with fully constricted pupils, eyes from A. irradians with fully dilated pupils provide approximately three times the sensitivity and half the spatial resolution at the distal retina and five times the sensitivity and one third the spatial resolution at the proximal retina. We also identify radial and circular actin fibers associated with the corneas of A. irradians that may represent muscles whose contractions dilate and constrict the pupil, respectively.