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.

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.

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.

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.

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.

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.

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.

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.

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.

Core-shell nanospheres behind the blue eyes of the bay scallop Argopecten irradians.

The bay scallop Argopecten irradians (Mollusca: Bivalvia) has dozens of iridescent blue eyes that focus light using mirror-based optics. Here, we test the hypothesis that these eyes appear blue because of photonic nanostructures that preferentially scatter short-wavelength light. Using transmission electron microscopy, we found that the epithelial cells covering the eyes of A. irradians have three distinct layers: an outer layer of microvilli, a middle layer of random close-packed nanospheres and an inner layer of pigment granules. The nanospheres are approximately 180 nm in diameter and consist of electron-dense cores approximately 140 nm in diameter surrounded by less electron-dense shells 20 nm thick. They are packed at a volume density of approximately 60% and energy-dispersive X-ray spectroscopy indicates that they are not mineralized. Optical modelling revealed that the nanospheres are an ideal size for producing angle-weighted scattering that is bright and blue. A comparative perspective supports our hypothesis: epithelial cells from the black eyes of the sea scallop Placopecten magellanicus have an outer layer of microvilli and an inner layer of pigment granules but lack a layer of nanospheres between them. We speculate that light-scattering nanospheres help to prevent UV wavelengths from damaging the internal structures of the eyes of A. irradians and other blue-eyed scallops.

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.

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.

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.

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.

Colorful seashells: Identification of haem pathway genes associated with the synthesis of porphyrin shell color in marine snails.

Very little is known about the evolution of molluskan shell pigments, although Mollusca is a highly diverse, species rich, and ecologically important group of animals comprised of many brightly colored taxa. The marine snail genus Clanculus was chosen as an exceptional model for studying the evolution of shell color, first, because in Clanculus margaritarius and Clanculus pharaonius both shell and foot share similar colors and patterns; and second, because recent studies have identified the pigments, trochopuniceus (pink-red), and trochoxouthos (yellow-brown), both comprised of uroporphyrin I and uroporphyrin III, in both shell and colored foot tissue of these species. These unusual characteristics provide a rare opportunity to identify the genes involved in color production because, as the same pigments occur in the shell and colored foot tissue, the same color-related genes may be simultaneously expressed in both mantle (which produces the shell) and foot tissue. In this study, the transcriptomes of these two Clanculus species along with a third species, Calliostoma zizyphinum, were sequenced to identify genes associated with the synthesis of porphyrins. Calliostoma zizyphinum was selected as a negative control as trochopuniceus and trochoxouthos were not found to occur in this species. As expected, genes necessary for the production of uroporphyrin I and III were found in all three species, but gene expression levels were consistent with synthesis of uroporphyrins in mantle and colored foot tissue only in Clanculus. These results are relevant not only to understanding the evolution of shell pigmentation in Clanculus but also to understanding the evolution of color in other species with uroporphyrin pigmentation, including (mainly marine) mollusks soft tissues and shells, annelid and platyhelminth worms, and some bird feathers.

Expression of G Proteins in the Eyes and Parietovisceral Ganglion of the Bay Scallop Argopecten irradians.

A multitude of image-forming eyes are spread across the bodies of certain invertebrates. Recent efforts have characterized how these eyes function, but less progress has been made toward describing the neural structures associated with them. Scallops, for example, have a distributed visual system that includes dozens of eyes whose optic nerves project to the lateral lobes of the parietovisceral ganglion (PVG). To identify sensory receptors and chemical synapses associated with the scallop visual system, we studied the expression of four G protein α subunits (Gαi, Gαo, Gαq, and Gαs) in the eyes and PVG of the bay scallop Argopecten irradians (Lamarck, 1819). In the eyes of A. irradians, we noted expression of Gαo by the ciliary photoreceptors of the distal retina, expression of Gαq by the rhabdomeric photoreceptors of the proximal retina, and the expression of Gαo and Gαq by the cells of the cornea; we did not, however, detect expression of Gαi or Gαs in the eyes. In the PVG of A. irradians, we noted widespread expression of Gαi, Gαo, and Gαq. The expression of Gαs was limited to fine neurites in the lateral and ventral central lobes, as well as large unipolar neurons in the dorsal central lobes. Our findings suggest that light detection by the eyes of A. irradians is conferred primarily by photoreceptors that express Gαo or Gαq, that the corneal cells of scallops may contain sensory receptors and/or receive neural input, and that G protein labeling is useful for visualizing substructures and identifying specific populations of cells within the nervous systems of invertebrates.

Examining the Effects of Chromatic Aberration, Object Distance, and Eye Shape on Image-Formation in the Mirror-Based Eyes of the Bay Scallop Argopecten irradians.

The eyes of scallops form images using a concave spherical mirror and contain two separate retinas, one layered on top of the other. Behavioral and electrophysiological studies indicate that the images formed by these eyes have angular resolutions of about 2°. Based on previous ray-tracing models, it has been thought that the more distal of the two retinas lies near the focal point of the mirror and that the proximal retina, positioned closer to the mirror at the back of the eye, receives light that is out-of-focus. Here, we propose three mechanisms through which both retinas may receive focused light: (1) chromatic aberration produced by the lens may cause the focal points for longer and shorter wavelengths to fall near the distal and proximal retinas, respectively; (2) focused light from near and far objects may fall on the distal and proximal retinas, respectively; and (3) the eyes of scallops may be dynamic structures that change shape to determine which retina receives focused light. To test our hypotheses, we used optical coherence tomography (OCT), a method of near-infrared optical depth-ranging, to acquire virtual cross-sections of live, intact eyes from the bay scallop Argopecten irradians Next, we used a custom-built ray-tracing model to estimate the qualities of the images that fall on an eye's distal and proximal retinas as functions of the wavelengths of light entering the eye (400-700 nm), object distances (0.01-1 m), and the overall shape of the eye. When we assume 550 nm wavelength light and object distances greater than 0.01 m, our model predicts that the angular resolutions of the distal and proximal retinas are 2° and 7°, respectively. Our model also predicts that neither chromatic aberration nor differences in object distance lead to focused light falling on the distal and proximal retinas simultaneously. However, if scallops can manipulate the shapes of their eyes, perhaps through muscle contractions, we speculate that they may be able to influence the qualities of the images that fall on their proximal retinas and-to a lesser extent-those that fall on their distal retinas as well.