RNA Interference in Aquatic Beetles as a Powerful Tool for Manipulating Gene Expression at Specific Developmental Time Points

RNA interference (RNAi) remains a powerful technique that allows for the targeted reduction of gene expression through mRNA degradation. This technique is ...

University of Cincinnati

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.

The development of a long, coiled, optic nerve in the stalk-eyed fly Cyrtodiopsis whitei.

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.

Scanning behavior by larvae of the predacious diving beetle, Thermonectus marmoratus (Coleoptera: Dytiscidae) enlarges visual field prior to prey capture.

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.

The visual system of male scale insects.

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.

Multitasking in an eye: the unusual organization of the Thermonectus marmoratus principal larval eyes allows for far and near vision and might aid in depth perception.

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. 

Escaping compound eye ancestry: the evolution of single-chamber eyes in holometabolous 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.

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

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) 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 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.

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

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 cuticular nature of corneal lenses in Drosophila melanogaster.

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.

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

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 (), which are known to grow rapidly and dramatically between larval instars. Then, we measured the image-forming principal anterior-median eyes of jumping spiders () after emergence from their egg cases. Finally, we measured individual ommatidia in the compound eyes of flesh flies () 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.

Elke K. Bushbeck

Department of Biological Sciences

University of Cincinnati

RNA Interference in Aquatic Beetles as a Powerful Tool for Manipulating Gene Expression at Specific Developmental Time Points

Elke K. Bushbeck

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Department of Biological Sciences

University of Cincinnati

RNA Interference in Aquatic Beetles as a Powerful Tool for Manipulating Gene Expression at Specific Developmental Time Points

Department of Biological Sciences