rna-interference-aquatic-beetles-as-powerful-tool-for-manipulating

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

The Scripps Research Institute

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.

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

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.

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.

A Complex Lens for a Complex Eye.

Among animals with visual processing mechanisms, the leech  is a rare example in which all neurons can be identified. However, little is known about its visual system, which is composed of several pigmented head eyes and photosensitive non-pigmented sensilla that are distributed across its entire body. Although several interneurons are known to respond to visual stimuli, their response properties are poorly understood. Among these, the S-cell system is especially intriguing: it is multimodal, spans the entire body of the leech and is thought to be involved in sensory integration. To improve our understanding of the role of this system, we tested its spectral sensitivity, spatial integration and adaptation properties. The response of the S-cell system to visual stimuli was found to be strongly dependent on the size of the area stimulated, and adaptation was local. Furthermore, an adaptation experiment demonstrated that at least two color channels contributed to the response, and that their contribution was dependent on the adaptation to the background. The existence of at least two color channels was further supported by transcriptomic evidence, which indicated the existence of at least two distinct groups of putative opsins for leeches. Taken together, our results show that the S-cell system has response properties that could be involved in the processing of spatial and color information of visual stimuli. We propose the leech as a novel system to understand visual processing mechanisms with many practical advantages.

Electrophysiology and transcriptomics reveal two photoreceptor classes and complex visual integration in .

The tremendous diversity of Hymenoptera is commonly attributed to the evolution of parasitoidism in the last common ancestor of parasitoid sawflies (Orussidae) and wasp-waisted Hymenoptera (Apocrita). However, Apocrita and Orussidae differ dramatically in their species richness, indicating that the diversification of Apocrita was promoted by additional traits. These traits have remained elusive due to a paucity of sawfly genome sequences, in particular those of parasitoid sawflies. Here we present comparative analyses of draft genomes of the primarily phytophagous sawfly Athalia rosae and the parasitoid sawfly Orussus abietinus. Our analyses revealed that the ancestral hymenopteran genome exhibited traits that were previously considered unique to eusocial Apocrita (e.g., low transposable element content and activity) and a wider gene repertoire than previously thought (e.g., genes for CO2 detection). Moreover, we discovered that Apocrita evolved a significantly larger array of odorant receptors than sawflies, which could be relevant to the remarkable diversification of Apocrita by enabling efficient detection and reliable identification of hosts.

Aaron Stahl

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