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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., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 Elsevier Inc. All rights reserved.)

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., (© 2023. The Author(s).)

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., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023. Published by Elsevier Ltd.)

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., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Rathore, Meece, Charlton-Perkins, Cook and Buschbeck.)

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., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Lavin, Rathore, Bauer, Disalvo, Mosley, Shearer, Elia, Cook and Buschbeck.)

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., Competing Interests: Declaration of Competing Interest Nothing declared., (Copyright © 2022 Elsevier Inc. All rights reserved.)

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., (Copyright © 2021 Elsevier B.V. All rights reserved.)

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., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2021. Published by The Company of Biologists Ltd.)

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., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2020. Published by The Company of Biologists Ltd.)

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., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

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., Competing Interests: The authors declare that they have no conflict of interest.

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., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2017. Published by The Company of Biologists Ltd.)

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., (© The Author 2017. Published by Oxford University Press on behalf of the Society for Integrative and Comparative Biology. All rights reserved. For permissions please email: journals.permissions@oup.com.)

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.

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.

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 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 (λ max ) 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 λ max 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., (© 2016. Published by The Company of Biologists Ltd.)

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., (© 2016 Wiley Periodicals, Inc.)

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.

A particularly unusual visual system exists in the visually guided aquatic predator, the Sunburst Diving Beetle, Thermonectus marmoratus (Coleoptera: Dytiscidae). The question arises: how does this peculiar visual system function? A series of experiments suggests that their principal eyes (E1 and E2) are highly specialized for hunting. These eyes are tubular and have relatively long focal lengths leading to high image magnification. Their retinae are linear, and are divided into distinct green-sensitive distal and UV and polarization-sensitive proximal portions. Each distal retina, moreover, has many tiers of photoreceptors with rhabdomeres the long axis of which are peculiarly oriented perpendicular to the light path. Based on detailed optical investigations, the lenses of these eyes are bifocal and project focused images onto specific retinal tiers. Behavioral experiments suggest that these larvae approach prey within their eyes' near-fields, and that they can correctly gauge prey distances even when conventional distance-vision mechanisms are unavailable. In the near-field of these eyes object distance determines which of the many retinal layers receive the best-focused images. This retinal organization could facilitate an unusual distance-vision mechanism. We here summarize past findings and discuss how these eyes allow Thermonectus larvae to be such successful predators.

Stemmata, the eyes of holometabolous insect larvae, have gained little attention, even though they exhibit remarkably different optical solutions, ranging from compound eyes with upright images, to sophisticated single-chamber eyes with inverted images. Such optical differences raise the question of how major transitions may have occurred. Stemmata evolved from compound eye ancestry, and optical differences are apparent even in some of the simplest systems that share strong cellular homology with adult ommatidia. The transition to sophisticated single-chamber eyes occurred many times independently, and in at least two different ways: through the fusion of many ommatidia [as in the sawfly (Hymenoptera)], and through the expansion of single ommatidia [as in tiger beetles (Coleoptera), antlions (Neuroptera) and dobsonflies (Megaloptera)]. Although ommatidia-like units frequently have multiple photoreceptor layers (tiers), sophisticated image-forming stemmata tend to only have one photoreceptor tier, presumably a consequence of the lens only being able to efficiently focus light on to one photoreceptor layer. An interesting exception is found in some diving beetles [Dytiscidae (Coleoptera)], in which two retinas receive sharp images from a bifocal lens. Taken together, stemmata represent a great model system to study an impressive set of optical solutions that evolved from a relatively simple ancestral organization., (© 2014. Published by The Company of Biologists Ltd.)

Very few visual systems diverge fundamentally from the basic plans of well-studied animal eyes. However, investigating those that do can provide novel insights into visual system function. A particularly unusual system exists in the principal larval eyes of a visually guided aquatic predator, the sunburst diving beetle, Thermonectus marmoratus (Coleoptera: Dystiscidae). These eyes are characterized by complex layered distal and proximal retinas. We previously reported that their principal eye E2 has a bifocal lens, and previous behavioral experiments suggested that these larvae have a unilateral range-finding mechanism that may involve their bizarre eye organization. In the present study, we expanded our optical measurements and found that: (1) E1 also has a bifocal lens, (2) E1 is best suited for far vision while E2 is best suited for near vision and (3) throughout their typical hunting range, the positions of focused images shift across specific retinal layers. This anatomical and optical organization in principle could support unilateral range finding. Taken together, our findings outline an unusual visual mechanism that is likely to be essential for the extraordinary hunting ability of these larvae., (© 2014. Published by The Company of Biologists Ltd.)

One of the biggest challenges that predators, such as the larvae of the diving beetle Thermonectus marmoratus (Coleoptera: Dytiscidae), are faced with is to accurately assess the distance of their prey. Most animals derive distance information from disparities of images that are viewed from different angles, from information that is obtained from well-controlled translational movements (motion parallax) or from the image size of known objects. Using a behavioral assay we demonstrated that T. marmoratus larvae continue to accurately strike at artificial prey, even if none of these typical distance estimation cues are available to them. Specifically, we excluded bilateral binocular stereopsis by occlusion, confounded possible motion parallax cues with an artificially moving prey, and excluded the possibility that beetle larvae simply approached their targets based on known prey size by presenting different prey sizes. Despite these constraints, larvae consistently struck our artificial targets from a distance of ~4.5 mm. Based on these findings we conclude that T. marmoratus likely employ an unusual mechanism to accurately determine prey distances, possibly mediated by the object-distance-dependent activation of specific subsets of their many-tiered and peculiarly positioned photoreceptors.

Sensorineural hearing loss is genetically heterogeneous. Here, we report that mutations in CIB2, which encodes a calcium- and integrin-binding protein, are associated with nonsyndromic deafness (DFNB48) and Usher syndrome type 1J (USH1J). One mutation in CIB2 is a prevalent cause of deafness DFNB48 in Pakistan; other CIB2 mutations contribute to deafness elsewhere in the world. In mice, CIB2 is localized to the mechanosensory stereocilia of inner ear hair cells and to retinal photoreceptor and pigmented epithelium cells. Consistent with molecular modeling predictions of calcium binding, CIB2 significantly decreased the ATP-induced calcium responses in heterologous cells, whereas mutations in deafness DFNB48 altered CIB2 effects on calcium responses. Furthermore, in zebrafish and Drosophila melanogaster, CIB2 is essential for the function and proper development of hair cells and retinal photoreceptor cells. We also show that CIB2 is a new member of the vertebrate Usher interactome.

Polarization sensitivity has most often been studied in mature insects, yet it is likely that larvae also make use of this visual modality. The aquatic larvae of the predacious diving beetle Thermonectus marmoratus are highly successful visually guided predators, with a UV-sensitive proximal retina that, according to its ultrastructure, has three distinct cell types with anatomical attributes that are consistent with polarization sensitivity. In the present study we used electrophysiological methods and single-cell staining to confirm polarization sensitivity in the proximal retinas of both principal eyes of these larvae. As expected from their microvillar orientation, cells of type T1 are most sensitive to vertically polarized light, while cells of type T2 are most sensitive to horizontally polarized light. In addition, T3 cells probably constitute a second population of cells that are most sensitive to light with vertical e-vector orientation, characterized by shallower polarization modulations, and smaller polarization sensitivity (PS) values than are typical for T1 cells. The level of PS values found in this study suggests that polarization sensitivity probably plays an important role in the visual system of these larvae. Based on their natural history and behavior, possible functions are: (1) finding water after hatching, (2) finding the shore before pupation, and (3) making prey more visible, by filtering out horizontally polarized haze, and/or using polarization features for prey detection.

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

Almost all animal eyes follow a few, relatively well-understood functional plans. Only rarely do researchers discover an eye that diverges fundamentally from known types. The principal eye E2 of sunburst diving beetle (Thermonectus marmoratus) larvae clearly falls into the rarer category. On the basis of two different tests, we here report that it has truly bifocal lenses, something that has been previously suggested only for certain trilobites. Our evidence comes from (1) the relative contrast in images of a square wave grating and (2) the refraction of a narrow laser beam projected through the lens. T. marmoratus larvae have two retinas at different depths behind the lens, and these are situated so that each can receive its own focused image. This is consistent with a novel eye organization that possibly comprises "two eyes in one." Moreover, we find that in contrast to most commercial bifocal lenses, the lens of E2 exhibits asymmetry, which results in separation of the images both dorsoventrally and rostrocaudally within the layered retina. Visual contrast might thus be improved over conventional bifocal lenses because the unfocused version of one image is shifted away from the focused version of the other, an organization which could potentially be exploited in optical engineering., (Copyright 2010 Elsevier Ltd. All rights reserved.)

Larvae of the sunburst diving beetle, Thermonectus marmoratus, have a cluster of six stemmata (E1-6) and one eye patch on each side of the head. Each eye has two retinas: a distal retina that is closer to the lens, and a proximal retina that lies directly underneath. The distal retinas of E1 and E2 are made of a dorsal and a ventral stack of at least twelve photoreceptor layers. Could this arrangement be used to compensate for lens chromatic aberration, with shorter wavelengths detected by the distal layers and longer wavelengths by the proximal layers? To answer this question we molecularly identified opsins and their expression patterns in these eyes. We found three opsin-encoding genes. The distal retinas of all six eyes express long-wavelength opsin (TmLW) mRNA, whereas the proximal retinas express ultraviolet opsin (TmUV I) mRNA. In the proximal retinas of E1 and E2, the TmUV I mRNA is expressed only in the dorsal stack. A second ultraviolet opsin mRNA (TmUV II), is expressed in the proximal retinas of E1 and E2 (both stacks). The finding that longer-wavelength opsins are expressed distally to shorter-wavelength opsins makes it unlikely that this retinal arrangement is used to compensate for lens chromatic aberration. In addition, we also described opsin expression patterns in the medial retina of E1 and in the non-tiered retina of the lensless eye patch. To our knowledge, this is also the first report of multiple UV opsins being expressed in the same stemma.

Animal eyes generally fall into two categories: (1) their photoreceptive array is convex, as is typical for camera eyes, including the human eye, or (2) their photoreceptive array is concave, as is typical for the compound eye of insects. There are a few rare examples of the latter eye type having secondarily evolved into the former one. When viewed in a phylogenetic framework, the head morphology of a variety of male scale insects suggests that this group could be one such example. In the Margarodidae (Hemiptera, Coccoidea), males have been described as having compound eyes, while males of some more derived groups only have two single-chamber eyes on each side of the head. Those eyes are situated in the place occupied by the compound eye of other insects. Since male scale insects tend to be rare, little is known about how their visual systems are organized, and what anatomical traits are associated with this evolutionary transition. In adult male Margarodidae, one single-chamber eye (stemmateran ocellus) is present in addition to a compound eye-like region. Our histological investigation reveals that the stemmateran ocellus has an extended retina which is formed by concrete clusters of receptor cells that connect to its own first-order neuropil. In addition, we find that the ommatidia of the compound eyes also share several anatomical characteristics with simple camera eyes. These include shallow units with extended retinas, each of which is connected by its own small nerve to the lamina. These anatomical changes suggest that the margarodid compound eye represents a transitional form to the giant unicornal eyes that have been described in more derived species.

Nearly nothing is known about the transition that visual brain regions undergo during metamorphosis, except for Drosophila in which larval eyes and the underlying neural structure are strongly reduced. We have studied the larvae of the sunburst diving beetle, Thermonectus marmoratus (Coleoptera: Dytiscidae), which are sophisticated visually oriented predators characterized by six elaborate stemmata on each side of the head and an associated large optic lobe. We used general neurohistological staining and 3D reconstruction to determine how the eyes and optic lobe of T. marmoratus change morphologically during metamorphosis. We find that in third (last) instar larvae, the adult neuropils are already forming de novo dorsally and slightly anteriorly to the larval neuropils, while the latter rapidly degenerate. Larval eyes are eventually reduced to distinct areas with dark pigmentation. This complete reorganization, which may be an evolutionarily conserved trait in holometabolous insects, occurs despite the considerable costs that must apply to such a visually complex animal. Our findings are consistent with the concept that stemmata are homologous to the most posterior ommatidia of hemimetabolous insects, an idea also recently supported by molecular data.

Larvae of the predaceous diving beetle Thermonectus marmoratus bear six stemmata on each side of their head, two of which form relatively long tubes with linear retinas at their proximal ends. The physical organization of these eyes results in extremely narrow visual fields that extend only laterally in the horizontal body plane. There are other examples of animals possessing eyes with predominantly linear retinas, or with linear arrangements of specific receptor types. In these animals, the eyes, or parts of the eyes, are movable and perform scanning movements to increase the visual field. Based on anatomical data and observations of relatively transparent, immobilized young larvae, we report here that T. marmoratus larvae are incapable of moving their eyes or any part of their eyes within the head capsule. However, they do perform a series of bodily dorso-ventral pivots prior to prey capture, behaviorally extending the vertical visual field from 2 degrees to up to 50 degrees. Frame-by-frame analysis shows that such behavior is performed within a characteristic distance to the prey. These data provide first insights into the function of the very peculiar anatomical eye organization of T. marmoratus larvae.

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

The larvae of the sunburst diving beetle, Thermonectus marmoratus (Coleoptera: Dytiscidae), are highly efficient visually guided predators. Their visual system consists of a cluster of six stemmata and one eye patch on each side of the head capsule. Histological investigations show that the organization of individual stemmata differs strongly from any eye that has previously been described. Based on general morphology, ultrastructural data, and the presence of actin-rich areas that are typical for rhabdoms, we find that each eye is characterized by several retinas. The most dorsal eye on each side is relatively long and tubular, and we have identified three spatially distinct areas with retinula cells: 1) a band of two rows of rhabdoms along the medial side of the eye tube; 2) a flattened cone-shaped region towards the bottom of the tube that is formed by the rhabdoms of retinula cells that are oriented perpendicular to the light path; and 3) two horizontal rows of long rhabdoms parallel to the light path at the base of the tube. A second large eye is organized similarly but lacks the medial band. The remaining four eyes are nearly spherical and each has two distinct retinas. The 12 eyes hence account for a total of 26 retinas, and two further retinas are present in eye patches lacking lenses. Our anatomical findings suggest that this is an example of a visual system in which specific visual tasks are distributed among the eyes, and which relies on a variety of highly specialized retinas., (Copyright 2006 Wiley-Liss, Inc.)

In the stalk-eyed fly Cyrtodiopsis whitei (Diopsidae; Diptera), the relatively long optic nerve develops within the tight lumen of a very short eyestalk. Axonal growth is generally considered in terms of path finding, selective fasciculation, and towing. Physical forces that are necessary for axon lengthening are generated either by the growth cone or by the growth of surrounding tissues. Therefore, it is surprising to encounter a loosely coiled nerve apparently lacking any attachments that could allow for pull, or towing, of the nerve. In this study, we used histological sections and whole-mount preparations to confirm that the optic nerve of the stalk-eyed fly indeed elongates without the external application of tension to the nerve. Secondly, we examined the distribution of cytoskeletal elements and selected proteins that may be involved in axon extension. Staining against the vesicle fusion proteins SNAP-24 and SNAP-25 consistently results in stronger staining in the rapidly extending optic nerve than in a control nerve, suggesting a possible role of these proteins in the extension process. On a gross morphological level, SNAP-24/25 as well as the cytoskeletal elements actin and tubulin are uniformly distributed throughout the lengths of the growing nerve, suggesting that nerve elongation is distributed rather than localized. Finally, we identified glia as a possible source for tension within the nerve bundle. Glia proliferate rapidly in the optic nerve but not in the control nerve. Much work continues to focus on the growth of axons in culture, but this study is one of the few that considers the dynamics of nerve bundle extension as a whole.

Adult males of the insect order Strepsiptera are characterized by an unusual visual system that may use design principles from compound as well as simple eyes. The lenses of this eye are unusually large and focus images onto extended retinae. The light-gathering ability of the lens is sufficient to resolve multiple points of an image in each optical unit. We regard each unit as an independent image-forming eye that contributes an inverted partial image. Each partial image is re-inverted by optic chiasmata between the retinae and the lamina, where the complete image could be assembled from the neighboring units. The lamina, medulla and lobula are present, but their organization into cartridges is not clearly discernable. Fluorescent fills, whole-tissue stains, and synaptotagmin immunohistochemistry show that the optic neuropils nevertheless are densely packed, and that several parallel channels within the medulla underlie each of the lenses. The size and shape of the rhabdoms, as well as a relatively slow flicker-fusion frequency could suggest that these eyes evolved through a nocturnal life stage.

After emergence from the puparium, stalk-eyed flies of the family Diopsidae rapidly expand their head capsule so that the eyes and optic lobes are displaced at the ends of stalks that extend from the central head. Because the expansion takes place in only 15 minutes, we are especially interested in ontogenetic modifications that may facilitate such a rapid and dramatic change. To examine the pupal development of the brain, we used Bodian staining in the stalk-eyed fly, Cyrtodiopsis whitei and compared it with development in the fruit fly, Drosophila melanogaster, which serves as a "typical" dipteran example without eye stalks. Early in pupal development, the neuropil organization of the two species is fairly similar. In both species, columns are present in the outer medulla and giant fibers are discernible in the lobula plate. In contrast to D. melanogaster, C. whitei shows a small, neck-like constriction between the optic lobes and the rest of the brain. By 20% of pupal development, the divergence is more apparent, and by 30%, the future eye stalk and optic nerve of C. whitei has started to form. During the remaining 70% of development, the initially thick optic nerve narrows, and becomes gradually elongated, eventually coiling and folding throughout the short eye stalk. Similarly, the cuticle of the surrounding region becomes constricted, slightly elongated, and gradually appears more and more densely corrugated, like an accordion bellows. However, except for the formation of the optic nerve, the dense aggregation of cuticle around it, and a shift in orientation of the neuropils, the developmental programs of the two species are remarkably similar. This suggests that only a few aspects of development have been modified during the course of evolution to generate the stalk-eyed phenotype. At eclosion, the imago of C. whitei goes through a pumping process to inflate the eye stalks to their full length. Measurements of the diameter of the optic nerve before and after the expansion reveal only a small decrease. We propose that the cuticular folding of the eye stalk as well as the coiling of the optic nerve prepare the pupa well for the rapid and dramatic eye-stalk inflation after eclosion., (Copyright 2001 Wiley-Liss, Inc.)

Much uncertainty still exists regarding higher level phylogenetic relationships in the insect order Diptera, and the need for independent analyses is apparent. In this paper, I present a parsimony analysis that is based on details of the nervous system of flies. Because neural characters have received little attention in modern phylogenetic analyses and the stability of neural traits has been debated, special emphasis is given to testing the robustness of the analysis itself and to evaluating how neurobiological constraints (such as levels of neural processing) influence the phylogenetic information content. The phylogenetic study is based on 14 species in three nematoceran and nine brachyceran families. All characters used in the analysis are based on anatomical details of the neural organization of the fly visual system. For the most part they relate to uniquely identifiable neurons, which are cells or cell types that can be confidently recognized as homologues among different species and thus compared. Parsimony analysis results in a phylogenetic hypothesis that favors specific previously suggested phylogenetic relationships and suggests alternatives regarding other placements. For example, several heterodactylan families (Bombyliidae, Asilidae, and Dolichopodidae) are supported in their placement as suggested by Sinclair et al. (1993), but Tipulidae and Syrphidae are placed differently. Tipulidae are placed at a derived rather than ancestral position within the Nematocera, and Syrphidae are placed within the Schizophora. The analysis suggests that neural characters generally maintain phylogenetic information well. However, by "forcing" neural characters onto conventional phylogenetic analyses it becomes apparent that not all neural centers maintain such information equally well. For example, neurons of the second-order visual neuropil, the medulla, contain stronger phylogenetic "signal" than do characters of the deeper visual center, the lobula plate. These differences may relate to different functional constraints in the two neuropils.

Diopsid flies have eye stalks up to a centimeter in length, displacing the retina laterally from the rest of the head. This bizarre condition, called hypercephaly, is rare, but has evolved independently among several insect orders and is most common in flies (Diptera). Earlier studies of geometrical optics and behavior have led to various hypotheses about possible adaptive advantages of eye stalks, such as enhanced stereoscopic vision while other hypothesis suggest that eye stalks are an outcome of sexual selection. Here, we focus on how these curious distortions of head/eye morphology are accompanied by changes in the neural organization of the visual system of Cyrtodiopsis quinqueguttata. Histological examinations reveal that the optic lobes, lamina (La), medulla (Me), lobula (Lo), and lobula plate (LP) are contained entirely within the fly's eye bulbs, which are located at the distal ends of the eye stalks. We report that the organization of the peripheral visual system (La and Me) is similar to that of other Diptera (e.g., Musca and Drosophila), but deeper visual areas (Lo and LP) have been more strongly modified. For example, in both the lobula and lobula plate, fewer but larger giant collector neurons are found. The most pronounced difference is the reduction in the number of wide-field vertical cells of the lobula plate, where there are only four relatively large fibers, as opposed to 11 in Musca. The "fewer but larger" neural organization may enhance the conduction velocities of these cells, but may result in a loss of spatial resolution. At the base of the eye bulb, axon bundles collect and form a long optic nerve that extends the length of the eye stalk. We suggest that this organization of the diopsid visual system provides evidence for the costs of possessing long eye stalks.

In cyclorrhaphan flies, giant tangential neurons in the lobula plate are supplied by isomorphic arrays of evolutionarily conserved achromatic elementary motion detecting circuits originating in the retina. The arrangements among giant tangential neurons is characteristic of a taxon and can differ between taxa having different visual performances. Observations of 12 brachyceran and 4 nematoceran species have identified different behaviors associated with visually stabilized flight. Neuroanatomical comparisons between closely related species having different behaviors and phylogenetically distant species that have similar behaviors suggest that such differences relate to differences of giant tangential cell architecture in the lobula plate. These functionally related differences contrast to anatomical features that reflect phylogenetic affinities. For example, the lobula plates of robber flies, typified by ballistic flight behavior, all differ from other taxa in lacking cyclorrhaphan-type vertical motion-sensitive neurons; instead, they possess an extra complement of horizontal cells in their place. The results suggest that, although circuits that compute elementary motion are conserved across the Diptera, selective pressure has resulted in modifications of their target neurons, thus contributing to the wide variety of visual behaviors observed within this group of insects.

The Hassenstein-Reichardt autocorrelation model for motion computation was derived originally from studies of optomotor turning reactions of beetles and further refined from studies of houseflies. Its applicaton for explaining a variety of optokinetic behaviors in other insects assumes that neural correlates to the model are principally similar across taxa. This account examines whether this assumption is warranted. The results demonstrate that an evolutionarily conserved subset of neurons corresponds to small retinotopic neurons implicated in motion-detecting circuits that link the retina to motion-sensitive neuropils of the lobula plate. The occurrence of these neurons in basal groups suggests that they must have evolved at least 240 million years before the present time. Functional contiguity among the neurons is suggested by their having layer relationships that are independent of taxon-specific neurons, or the absence of orientation-specific motion-sensitive levels in the lobula plate.