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.

Exploring the molecular makeup of support cells in insect camera eyes.

Animals typically have either compound eyes, or camera-type eyes, both of which have evolved repeatedly in the animal kingdom. Both eye types include two important kinds of cells: photoreceptor cells, which can be excited by light, and non-neuronal support cells (SupCs), which provide essential support to photoreceptors. At the molecular level deeply conserved genes that relate to the differentiation of photoreceptor cells have fueled a discussion on whether or not a shared evolutionary origin might be considered for this cell type. In contrast, only a handful of studies, primarily on the compound eyes of Drosophila melanogaster, have demonstrated molecular similarities in SupCs. D. melanogaster SupCs (Semper cells and primary pigment cells) are specialized eye glia that share several molecular similarities with certain vertebrate eye glia, including Müller glia. This led us to question if there could be conserved molecular signatures of SupCs, even in functionally different eyes such as the image-forming larval camera eyes of the sunburst diving beetle Thermonectus marmoratus. To investigate this possibility, we used an in-depth comparative whole-tissue transcriptomics approach. Specifically, we dissected the larval principal camera eyes into SupC- and retina-containing regions and generated the respective transcriptomes. Our analysis revealed several common features of SupCs including enrichment of genes that are important for glial function (e.g. gap junction proteins such as innexin 3), glycogen production (glycogenin), and energy metabolism (glutamine synthetase 1 and 2). To evaluate similarities, we compared our transcriptomes with those of fly (Semper cells) and vertebrate (Müller glia) eye glia as well as respective retinas. T. marmoratus SupCs were found to have distinct genetic overlap with both fly and vertebrate eye glia. These results suggest that T. marmoratus SupCs are a form of glia, and like photoreceptors, may be deeply conserved.

Exploring the molecular makeup of support cells in insect camera eyes.

Animals generally have either compound eyes, which have evolved repeatedly in different invertebrates, or camera eyes, which have evolved many times across the animal kingdom. Both eye types include two important kinds of cells: photoreceptor cells, which can be excited by light, and non-neuronal support cells (SupCs), which provide essential support to photoreceptors. Despite many examples of convergence in eye evolution, similarities in the gross developmental plan and molecular signatures have been discovered, even between phylogenetically distant and functionally different eye types. For this reason, a shared evolutionary origin has been considered for photoreceptors. In contrast, only a handful of studies, primarily on the compound eyes of Drosophila melanogaster , have demonstrated molecular similarities in SupCs. D. melanogaster SupCs (Semper cells and primary pigment cells) are specialized eye glia that share several molecular similarities with certain vertebrate eye glia, including Müller glia. This led us to speculate whether there are conserved molecular signatures of SupCs, even in functionally different eyes such as the image-forming larval camera eyes of the sunburst diving beetle Thermonectus marmoratus . To investigate this possibility, we used an in-depth comparative whole-tissue transcriptomics approach. Specifically, we dissected the larval principal camera eyes into SupC- and retina-containing regions and generated the respective transcriptomes. Our analysis revealed several conserved features of SupCs including enrichment of genes that are important for glial function (e.g. gap junction proteins such as innexin 3), glycogen production (glycogenin), and energy metabolism (glutamine synthetase 1 and 2). To evaluate the extent of conservation, we compared our transcriptomes with those of fly (Semper cells) and vertebrate (Müller glia) eye glia as well as respective retinas. T. marmoratus SupCs were found to have distinct genetic overlap with both fly and vertebrate eye glia. These results provide molecular evidence for the deep conservation of SupCs in addition to photoreceptor cells, raising essential questions about the evolutionary origin of eye-specific glia in animals.

Probing the conserved roles of cut in the development and function of optically different insect compound eyes.

Astonishing functional diversity exists among arthropod eyes, yet eye development relies on deeply conserved genes. This phenomenon is best understood for early events, whereas fewer investigations have focused on the influence of later transcriptional regulators on diverse eye organizations and the contribution of critical support cells, such as Semper cells (SCs). As SCs in Drosophila melanogaster secrete the lens and function as glia, they are critical components of ommatidia. Here, we perform RNAi-based knockdowns of the transcription factor cut (CUX in vertebrates), a marker of SCs, the function of which has remained untested in these cell types. To probe for the conserved roles of cut, we investigate two optically different compound eyes: the apposition optics of D. melanogaster and the superposition optics of the diving beetle Thermonectus marmoratus. In both cases, we find that multiple aspects of ocular formation are disrupted, including lens facet organization and optics as well as photoreceptor morphogenesis. Together, our findings support the possibility of a generalized role for SCs in arthropod ommatidial form and function and introduces Cut as a central player in mediating this role.

Nutrition-induced macular-degeneration-like photoreceptor damage in jumping spider eyes.

Age-related macular degeneration (AMD) is a leading cause of vision loss in humans. Despite its prevalence and medical significance, many aspects of AMD remain elusive and treatment options are limited. Here, we present data that suggest jumping spiders offer a unique opportunity for understanding the fundamentals underlying retinal degeneration, thereby shedding light on a process that impacts millions of people globally. Using a micro-ophthalmoscope and histological evidence, we demonstrate that significant photoreceptor damage can occur during development in the image-forming anterior lateral eyes of the jumping spider Phidippus audax. Furthermore, we find that this photoreceptor degeneration is exacerbated by inadequate nutrition and is most prevalent in the high-density region of the retina, like AMD in humans. This suggests that similar to those in vertebrates, the retinas in P. audax are challenged to meet high-energy cellular demands.

EyeVolve, a modular PYTHON based model for simulating developmental eye type diversification.

Vision is among the oldest and arguably most important sensory modalities for animals to interact with their external environment. Although many different eye types exist within the animal kingdom, mounting evidence indicates that the genetic networks required for visual system formation and function are relatively well conserved between species. This raises the question as to how common developmental programs are modified in functionally different eye types. Here, we approached this issue through EyeVolve, an open-source PYTHON-based model that recapitulates eye development based on developmental principles originally identified in Drosophila melanogaster. Proof-of-principle experiments showed that this program's animated timeline successfully simulates early eye tissue expansion, neurogenesis, and pigment cell formation, sequentially transitioning from a disorganized pool of progenitor cells to a highly organized lattice of photoreceptor clusters wrapped with support cells. Further, tweaking just five parameters (precursor pool size, founder cell distance and placement from edge, photoreceptor subtype number, and cell death decisions) predicted a multitude of visual system layouts, reminiscent of the varied eye types found in larval and adult arthropods. This suggests that there are universal underlying mechanisms that can explain much of the existing arthropod eye diversity. Thus, EyeVolve sheds light on common principles of eye development and provides a new computational system for generating specific testable predictions about how development gives rise to diverse visual systems from a commonly specified neuroepithelial ground plan.

Evolution of visual system specialization in predatory arthropods.

Under strong selective pressure for survival, image-forming vision set off an ongoing predatory arms race 500 million years ago. Since then, and particularly so in the arthropods, predatory behavior has driven a myriad of eye adaptations that increase visual performance. In this review, we provide examples of how different arthropod predators have achieved improvements in key visual features such as spatial and temporal resolution of their retina. We then describe morphological, neural and behavioral strategies used by animals in this group to gather crucial information about the prey, such as its distance, velocity and size. We also highlight the importance of head and body tracking movements to aid in categorizing the potential prey, and briefly mention the ongoing work on the sensorimotor transformations necessary for target interception.

Functionalized carbon nanotube microfibers for chronic neural implants.

BACKGROUND: Much progress has been made at the interface between neural tissue and electrodes for neurophysiology. However, there continues to be a need for novel materials that integrate well with the nervous system and facilitate neural recordings with longer-term sustainability and stability. Such materials have the potential to improve clinical approaches and provide important tools for basic neuroscience research. NEW METHOD: In this paper, we explore the use of dry-spun untreated or functionalized carbon nanotube fibers as implantable electrodes for neural recordings from insects over extended time periods. RESULTS: Measurements of fly eyes responding to light flashes illustrate the suitability of these materials for recording both the low- and high-frequency components of neural signals. Repeated recordings show good sustainability, especially with functionalized carbon nanotube fibers. In particular, recordings from the optic lobes of Madagascar hissing cockroaches last for at least 8 weeks. COMPARISON WITH EXISTING METHOD(S): Electrophysiological research continues to rely heavily on metal electrodes that are good for short-lived preparations but less suitable for longer-term recordings, as scar tissue formation and cytotoxicity tend to cause a gradual reduction in signals. CONCLUSIONS: Functionalized carbon nanotubes are a promising novel material that can be used to obtain long-term or repeated stable recordings, which are necessary for longitudinal studies, or to maintain other neural tissue interfaces such as those in insect-machine hybrid robots. The introduced insect preparation can also be used for the relatively rapid and cost-efficient testing of novel electrode materials.

Stark trade-offs and elegant solutions in arthropod visual systems.

Vision is one of the most important senses for humans and animals alike. Diverse elegant specializations have evolved among insects and other arthropods in response to specific visual challenges and ecological needs. These specializations are the subject of this Review, and they are best understood in light of the physical limitations of vision. For example, to achieve high spatial resolution, fine sampling in different directions is necessary, as demonstrated by the well-studied large eyes of dragonflies. However, it has recently been shown that a comparatively tiny robber fly (Holcocephala) has similarly high visual resolution in the frontal visual field, despite their eyes being a fraction of the size of those of dragonflies. Other visual specializations in arthropods include the ability to discern colors, which relies on parallel inputs that are tuned to spectral content. Color vision is important for detection of objects such as mates, flowers and oviposition sites, and is particularly well developed in butterflies, stomatopods and jumping spiders. Analogous to color vision, the visual systems of many arthropods are specialized for the detection of polarized light, which in addition to communication with conspecifics, can be used for orientation and navigation. For vision in low light, optical superposition compound eyes perform particularly well. Other modifications to maximize photon capture involve large lenses, stout photoreceptors and, as has been suggested for nocturnal bees, the neural pooling of information. Extreme adaptations even allow insects to see colors at very low light levels or to navigate using the Milky Way.

Establishment of correctly focused eyes may not require visual input in arthropods.

For proper function, vertebrate and invertebrate visual systems must be able to achieve and maintain emmetropia, a state where distant objects are in focus on the retina. In vertebrates, this is accomplished through a combination of genetic control during early development and homeostatic visual input that fine-tunes the optics of the eye. While emmetropization has long been researched in vertebrates, it is largely unknown how emmetropia is established in arthropods. We used a micro-ophthalmoscope to directly measure how the lens projects images onto the retina in the eyes of small, live arthropods, allowing us to compare the refractive states of light-reared and dark-reared arthropods. First, we measured the image-forming larval eyes of diving beetles (Thermonectus marmoratus), which are known to grow rapidly and dramatically between larval instars. Then, we measured the image-forming principal anterior-median eyes of jumping spiders (Phidippus audax) after emergence from their egg cases. Finally, we measured individual ommatidia in the compound eyes of flesh flies (Sarcophaga bullata) that had developed and emerged under either light or dark conditions. Surprisingly, and in sharp contrast to vertebrates, our data for this diverse set of arthropods suggest that visual input is inconsequential in regard to achieving well-focused eyes. Although it remains unclear whether visual input that is received after the initial development further improves focusing, these results suggest that at least the initial coordination between the lens refractive power and eye size in arthropods may be more strongly predetermined by developmental factors than is typically the case in vertebrates.

Growing tiny eyes: How juvenile jumping spiders retain high visual performance in the face of size limitations and developmental constraints.

Adult jumping spiders are known for their extraordinary eyesight and complex, visually guided behaviors, including elaborate communicatory displays, navigational abilities, and prey-specific predatory strategies. Juvenile spiders also exhibit many of these behaviors, yet their visual systems are many times smaller. How do juveniles retain high visually guided performance despite severe size constraints on their visual systems? We investigated developmental changes in eye morphology and visual function in the jumping spider Phidippus audax using morphology, histology, ophthalmoscopy, and optical measurements. We find that juvenile spiders have proportionally larger lenses in relation to their body size than adults. This should alleviate some of the costs of small body size on visual function. However, photoreceptor number in the anterior lateral eyes (ALE) remains constant from early development onward, consistent with a developmental constraint on photoreceptor differentiation. To accommodate these photoreceptors within the diminutive volume of the spiderling cephalothorax, ALE rhabdoms in early life stages are more tightly packed and significantly smaller in diameter and length, properties that expand across development. Lens focal lengths increase as eyes and retinas grow, resulting in a remarkable maintenance of ALE spatial acuity and field-of-view across life stages. However, this maintenance of acuity comes at a sensitivity cost given the small rhabdomal volumes required by space constraints early in life. Taken together, our results indicate that young jumping spiders have eyes already equipped for high acuity vision, but these young spiders may struggle to perform visually demanding behaviors in low-light environments, a notion that warrants further testing.

Xenos peckii vision inspires an ultrathin digital camera.

Increased demand for compact devices leads to rapid development of miniaturized digital cameras. However, conventional camera modules contain multiple lenses along the optical axis to compensate for optical aberrations that introduce technical challenges in reducing the total thickness of the camera module. Here, we report an ultrathin digital camera inspired by the vision principle of Xenos peckii, an endoparasite of paper wasps. The male Xenos peckii has an unusual visual system that exhibits distinct benefits for high resolution and high sensitivity, unlike the compound eyes found in most insects and some crustaceans. The biologically inspired camera features a sandwiched configuration of concave microprisms, microlenses, and pinhole arrays on a flat image sensor. The camera shows a field-of-view (FOV) of 68 degrees with a diameter of 3.4 mm and a total track length of 1.4 mm. The biologically inspired camera offers a new opportunity for developing ultrathin cameras in medical, industrial, and military fields.

Molecular Evolution of Spider Vision: New Opportunities, Familiar Players.

Spiders are among the world's most species-rich animal lineages, and their visual systems are likewise highly diverse. These modular visual systems, composed of four pairs of image-forming "camera" eyes, have taken on a huge variety of forms, exhibiting variation in eye size, eye placement, image resolution, and field of view, as well as sensitivity to color, polarization, light levels, and motion cues. However, despite this conspicuous diversity, our understanding of the genetic underpinnings of these visual systems remains shallow. Here, we review the current literature, analyze publicly available transcriptomic data, and discuss hypotheses about the origins and development of spider eyes. Our efforts highlight that there are many new things to discover from spider eyes, and yet these opportunities are set against a backdrop of deep homology with other arthropod lineages. For example, many (but not all) of the genes that appear important for early eye development in spiders are familiar players known from the developmental networks of other model systems (e.g., Drosophila). Similarly, our analyses of opsins and related phototransduction genes suggest that spider photoreceptors employ many of the same genes and molecular mechanisms known from other arthropods, with a hypothesized ancestral spider set of four visual and four nonvisual opsins. This deep homology provides a number of useful footholds into new work on spider vision and the molecular basis of its extant variety. We therefore discuss what some of these first steps might be in the hopes of convincing others to join us in studying the vision of these fascinating creatures.

Giving invertebrates an eye exam: an ophthalmoscope that utilizes the autofluorescence of photoreceptors.

One of the most important functional features of eyes is focusing light, as both nearsightedness and farsightedness have major functional implications. Accordingly, refractive errors are frequently assessed in vertebrates, but not in the very small invertebrate eyes. We describe a micro-ophthalmoscope that takes advantage of autofluorescent properties of invertebrate photoreceptors and test the device on the relatively well-understood eyes of jumping spiders and flies. In each case, our measurements confirmed previous findings with a greater degree of accuracy. For example, we could precisely resolve the layering of the anterior median eyes and could map out the extensive retina of the anterior lateral eyes of the spider. Measurements also confirmed that fly ommatidia are focused into infinity, but showed that their focal plane is situated slightly below the receptor surface. In contrast to other approaches, this device does not rely on reflective tapeta and allows for precise optical assessment of diverse invertebrate eyes.

A Complex Lens for a Complex Eye.

A key innovation for high resolution eyes is a sophisticated lens that precisely focuses light onto photoreceptors. The eyes of holometabolous larvae range from very simple eyes that merely detect light to eyes that are capable of high spatial resolution. Particularly interesting are the bifocal lenses of Thermonectus marmoratus larvae, which differentially focus light on spectrally-distinct retinas. While functional aspects of insect lenses have been relatively well studied, little work has explored their molecular makeup, especially in regard to more complex eye types. To investigate this question, we took a transcriptomic and proteomic approach to identify the major proteins contributing to the principal bifocal lenses of T. marmoratus larvae. Mass spectrometry revealed 10 major lens proteins. Six of these share sequence homology with cuticular proteins, a large class of proteins that are also major components of corneal lenses from adult compound eyes of Drosophila melanogaster and Anopheles gambiae. Two proteins were identified as house-keeping genes and the final two lack any sequence homologies to known genes. Overall the composition seems to follow a pattern of co-opting transparent and optically dense proteins, similar to what has been described for other animal lenses. To identify cells responsible for the secretion of specific lens proteins, we performed in situ hybridization studies and found some expression differences between distal and proximal corneagenous cells. Since the distal cells likely give rise to the periphery and the proximal cells to the center of the lens, our findings highlight a possible mechanism for establishing structural differences that are in line with the bifocal nature of these lenses. A better understanding of lens composition provides insights into the evolution of proper focusing, which is an important step in the transition between low-resolution and high-resolution eyes.

Multifunctional glial support by Semper cells in the Drosophila retina.

Glial cells play structural and functional roles central to the formation, activity and integrity of neurons throughout the nervous system. In the retina of vertebrates, the high energetic demand of photoreceptors is sustained in part by Müller glia, an intrinsic, atypical radial glia with features common to many glial subtypes. Accessory and support glial cells also exist in invertebrates, but which cells play this function in the insect retina is largely undefined. Using cell-restricted transcriptome analysis, here we show that the ommatidial cone cells (aka Semper cells) in the Drosophila compound eye are enriched for glial regulators and effectors, including signature characteristics of the vertebrate visual system. In addition, cone cell-targeted gene knockdowns demonstrate that such glia-associated factors are required to support the structural and functional integrity of neighboring photoreceptors. Specifically, we show that distinct support functions (neuronal activity, structural integrity and sustained neurotransmission) can be genetically separated in cone cells by down-regulating transcription factors associated with vertebrate gliogenesis (pros/Prox1, Pax2/5/8, and Oli/Olig1,2, respectively). Further, we find that specific factors critical for glial function in other species are also critical in cone cells to support Drosophila photoreceptor activity. These include ion-transport proteins (Na/K+-ATPase, Eaat1, and Kir4.1-related channels) and metabolic homeostatic factors (dLDH and Glut1). These data define genetically distinct glial signatures in cone/Semper cells that regulate their structural, functional and homeostatic interactions with photoreceptor neurons in the compound eye of Drosophila. In addition to providing a new high-throughput model to study neuron-glia interactions, the fly eye will further help elucidate glial conserved "support networks" between invertebrates and vertebrates.

The cuticular nature of corneal lenses in Drosophila melanogaster.

The dioptric visual system relies on precisely focusing lenses that project light onto a neural retina. While the proteins that constitute the lenses of many vertebrates are relatively well characterized, less is known about the proteins that constitute invertebrate lenses, especially the lens facets in insect compound eyes. To address this question, we used mass spectrophotometry to define the major proteins that comprise the corneal lenses from the adult Drosophila melanogaster compound eye. This led to the identification of four cuticular proteins: two previously identified lens proteins, drosocrystallin and retinin, and two newly identified proteins, Cpr66D and Cpr72Ec. To determine which ommatidial cells contribute each of these proteins to the lens, we conducted in situ hybridization at 50% pupal development, a key age for lens secretion. Our results confirm previous reports that drosocrystallin and retinin are expressed in the two primary corneagenous cells-cone cells and primary pigment cells. Cpr72Ec and Cpr66D, on the other hand, are more highly expressed in higher order interommatidial pigment cells. These data suggest that the complementary expression of cuticular proteins give rise to the center vs periphery of the corneal lens facet, possibly facilitating a refractive gradient that is known to reduce spherical aberration. Moreover, these studies provide a framework for future studies aimed at understanding the cuticular basis of corneal lens function in holometabolous insect eyes.

The unusual eyes of Xenos peckii (Strepsiptera: Xenidae) have green- and UV--sensitive photoreceptors.

The highly specialized evolution of Strepsiptera has produced one of the most unusual eyes among mature insects, perhaps in line with their extremely complex and challenging life cycle. This relatively rare insect order is one of the few for which it has been unclear what spectral classes of photoreceptors any of its members may possess, an even more apt question given the nocturnal evolution of the group. To address this question, we performed electroretinograms on adult male Xenos peckii: we measured spectral responses to equi-quantal monochromatic light flashes of different wavelengths, and established VlogI relationships to calculate spectral sensitivities. Based on opsin template fits, we found maximal spectral sensitivity (λmax) in the green domain at 539 nm. Application of a green light to 'bleach' green receptors revealed that a UV peak was contributed to by an independent UV opsin with a λmax of 346 nm. Transcriptomics and a phylogenetic analysis including 50 other opsin sequences further confirmed the presence of these two opsin classes. While these findings do not necessarily indicate that these unorthodox insects have color vision, they raise the possibility that UV vision plays an important role in the ability of X. peckii males to find the very cryptic strepsipteran females that are situated within their wasp hosts.

Embryonic development of the larval eyes of the Sunburst Diving Beetle, Thermonectus marmoratus (Insecta: Dytiscidae): a morphological study.

Stemmata, the larval eyes of holometabolous insects are extremely diverse, ranging from full compound eyes, to a few ommatidial units as are typical in compound eyes, to sophisticated and functionally specialized image-forming camera-type eyes. Stemmata evolved from a compound eye ommatidial ancestor, an eye type that is morphologically well conserved in regards to cellular composition, and well studied in regards to development. However, despite this evolutionary origin it remains largely unknown how stemmata develop. In addition, it is completely unclear how development is altered to give rise to some of the functionally most complex stemmata, such as those of the sunburst diving beetle, Thermonectus marmoratus. In this study, we used histological methods to investigate the embryonic development of the functionally complex principal stemmata Eye 1 and Eye 2 of the larval visual system of T. marmoratus. To gain insights into how cellular components of their sophisticated camera-type eyes might have evolved from the cellular components of ommatidial ancestors, we contrast our findings against known features of ommatidia development, which are particularly well understood in Drosophila. We find many similarities, such as the early presence of a pseudostratified epithelium, and the order in which specific cell types are recruited. However, in Thermonectus each cell type is represented by a large number of cells from early on and major tissue re-orientation occurs as eye development progresses. This study provides insights into the timing of morphological features and represents the basis for future molecular studies.

Rapid and step-wise eye growth in molting diving beetle larvae.

However complex a visual system is, the size (and growth rate) of all its components-lens, retina and nervous system-must be precisely tuned to each other for the system to be functional. As organisms grow, their eyes must be able to achieve and maintain emmetropia, a state in which photoreceptors receive sharp images of objects that are at infinity. While there has been ample research into how vertebrates coordinate eyes growth, this has never been addressed in arthropods with camera eyes, which tend to grow dramatically and typically in a step-wise manner with each molt (ecdysis). Here, we used histological and optical methods to measure how the larval eyes of Sunburst Diving Beetles (Thermonectus marmoratus, Coleoptera, Dytiscidae) grow, and how well optical and morphological parameters match, during the dramatic growth that occurs between two consecutive larval stages. We find that the eye tubes of the principal eyes of T. marmoratus grow substantially around molt, with the vitreous-like crystalline cone contributing the most to the overall growth. Lenses also reform relatively quickly, undergoing a period of dysfunction and then regaining the ability to project sharp images onto the retina around 8 h post-molt.