Looking across the gap: Understanding the evolution of eyes and vision among insects.

The compound eyes of insects exhibit stunning variation in size, structure, and function, which has allowed these animals to use their vision to adapt to a huge range of different environments and lifestyles, and evolve complex behaviors. Much of our knowledge of eye development has been learned from Drosophila, while visual adaptations and behaviors are often more striking and better understood from studies of other insects. However, recent studies in Drosophila and other insects, including bees, beetles, and butterflies, have begun to address this gap by revealing the genetic and developmental bases of differences in eye morphology and key new aspects of compound eye structure and function. Furthermore, technical advances have facilitated the generation of high-resolution connectomic data from different insect species that enhances our understanding of visual information processing, and the impact of changes in these processes on the evolution of vision and behavior. Here, we review these recent breakthroughs and propose that future integrated research from the development to function of visual systems within and among insect species represents a great opportunity to understand the remarkable diversification of insect eyes and vision.

Evolution of compound eye morphology underlies differences in vision between closely related Drosophila species.

BACKGROUND: Insects have evolved complex visual systems and display an astonishing range of adaptations for diverse ecological niches. Species of Drosophila melanogaster subgroup exhibit extensive intra- and interspecific differences in compound eye size. These differences provide an excellent opportunity to better understand variation in insect eye structure and the impact on vision. Here we further explored the difference in eye size between D. mauritiana and its sibling species D. simulans. RESULTS: We confirmed that D. mauritiana have rapidly evolved larger eyes as a result of more and wider ommatidia than D. simulans since they recently diverged approximately 240,000 years ago. The functional impact of eye size, and specifically ommatidia size, is often only estimated based on the rigid surface morphology of the compound eye. Therefore, we used 3D synchrotron radiation tomography to measure optical parameters in 3D, predict optical capacity, and compare the modelled vision to in vivo optomotor responses. Our optical models predicted higher contrast sensitivity for D. mauritiana, which we verified by presenting sinusoidal gratings to tethered flies in a flight arena. Similarly, we confirmed the higher spatial acuity predicted for Drosophila simulans with smaller ommatidia and found evidence for higher temporal resolution. CONCLUSIONS: Our study demonstrates that even subtle differences in ommatidia size between closely related Drosophila species can impact the vision of these insects. Therefore, further comparative studies of intra- and interspecific variation in eye morphology and the consequences for vision among other Drosophila species, other dipterans and other insects are needed to better understand compound eye structure-function and how the diversification of eye size, shape, and function has helped insects to adapt to the vast range of ecological niches.

Sox21b underlies the rapid diversification of a novel male genital structure between Drosophila species.

The emergence and diversification of morphological novelties is a major feature of animal evolution.1,2,3,4,5,6,7,8,9 However, relatively little is known about the genetic basis of the evolution of novel structures and the mechanisms underlying their diversification. The epandrial posterior lobes of male genitalia are a novelty of particular Drosophila species.10,11,12,13 The lobes grasp the female ovipositor and insert between her abdominal tergites and, therefore, are important for copulation and species recognition.10,11,12,14,15,16,17 The posterior lobes likely evolved from co-option of a Hox-regulated gene network from the posterior spiracles10 and have since diversified in morphology in the D. simulans clade, in particular, over the last 240,000 years, driven by sexual selection.18,19,20,21 The genetic basis of this diversification is polygenic but, to the best of our knowledge, none of the causative genes have been identified.22,23,24,25,26,27,28,29,30 Identifying the genes underlying the diversification of these secondary sexual structures is essential to understanding the evolutionary impact on copulation and species recognition. Here, we show that Sox21b negatively regulates posterior lobe size. This is consistent with expanded Sox21b expression in D. mauritiana, which develops smaller posterior lobes than D. simulans. We tested this by generating reciprocal hemizygotes and confirmed that changes in Sox21b underlie posterior lobe evolution between these species. Furthermore, we found that posterior lobe size differences caused by the species-specific allele of Sox21b significantly affect copulation duration. Taken together, our study reveals the genetic basis for the sexual-selection-driven diversification of a novel morphological structure and its functional impact on copulatory behavior.

Evolution of the Spider Homeobox Gene Repertoire by Tandem and Whole Genome Duplication.

Gene duplication generates new genetic material that can contribute to the evolution of gene regulatory networks and phenotypes. Duplicated genes can undergo subfunctionalization to partition ancestral functions and/or neofunctionalization to assume a new function. We previously found there had been a whole genome duplication (WGD) in an ancestor of arachnopulmonates, the lineage including spiders and scorpions but excluding other arachnids like mites, ticks, and harvestmen. This WGD was evidenced by many duplicated homeobox genes, including two Hox clusters, in spiders. However, it was unclear which homeobox paralogues originated by WGD versus smaller-scale events such as tandem duplications. Understanding this is a key to determining the contribution of the WGD to arachnopulmonate genome evolution. Here we characterized the distribution of duplicated homeobox genes across eight chromosome-level spider genomes. We found that most duplicated homeobox genes in spiders are consistent with an origin by WGD. We also found two copies of conserved homeobox gene clusters, including the Hox, NK, HRO, Irx, and SINE clusters, in all eight species. Consistently, we observed one copy of each cluster was degenerated in terms of gene content and organization while the other remained more intact. Focussing on the NK cluster, we found evidence for regulatory subfunctionalization between the duplicated NK genes in the spider Parasteatoda tepidariorum compared to their single-copy orthologues in the harvestman Phalangium opilio. Our study provides new insights into the relative contributions of multiple modes of duplication to the homeobox gene repertoire during the evolution of spiders and the function of NK genes.

Characterisation of the role and regulation of Ultrabithorax in sculpting fine-scale leg morphology.

Hox genes are expressed during embryogenesis and determine the regional identity of animal bodies along the antero-posterior axis. However, they also function post-embryonically to sculpt fine-scale morphology. To better understand how Hox genes are integrated into post-embryonic gene regulatory networks, we further analysed the role and regulation of Ultrabithorax (Ubx) during leg development in Drosophila melanogaster. Ubx regulates several aspects of bristle and trichome patterning on the femurs of the second (T2) and third (T3) leg pairs. We found that repression of trichomes in the proximal posterior region of the T2 femur by Ubx is likely mediated by activation of the expression of microRNA-92a and microRNA-92b by this Hox protein. Furthermore, we identified a novel enhancer of Ubx that recapitulates the temporal and regional activity of this gene in T2 and T3 legs. We then used transcription factor (TF) binding motif analysis in regions of accessible chromatin in T2 leg cells to predict and functionally test TFs that may regulate the Ubx leg enhancer. We also tested the role of the Ubx co-factors Homothorax (Hth) and Extradenticle (Exd) in T2 and T3 femurs. We found several TFs that may act upstream or in concert with Ubx to modulate trichome patterning along the proximo-distal axis of developing femurs and that the repression of trichomes also requires Hth and Exd. Taken together our results provide insights into how Ubx is integrated into a post-embryonic gene regulatory network to determine fine-scale leg morphology.

A special issue of Essays in Biochemistry on evolutionary developmental biology.

Evolutionary developmental biology (or evo devo) is a broad field that aims to understand how developmental processes evolve and how this underpins phenotypic change and organismal diversification. This encompasses a need to understand theoretical concepts in evolutionary biology and how tissues, cells, genes, proteins and regulatory elements function and evolve. The articles in this special issue review key topics in the field of evo devo including advances in theory and methodology as well as our latest knowledge about molecular, cellular and organismal functionality and diversification.

Micromanagement of Drosophila Post-Embryonic Development by Hox Genes.

Hox genes function early in development to determine regional identity in animals. Consequently, the loss or gain of Hox gene expression can change this identity and cause homeotic transformations. Over 20 years ago, it was observed that the role of Hox genes in patterning animal body plans involves the fine-scale regulation of cell fate and identity during development, playing the role of 'micromanagers' as proposed by Michael Akam in key perspective papers. Therefore, as well as specifying where structures develop on animal bodies, Hox genes can help to precisely sculpt their morphology. Here, we review work that has provided important insights about the roles of Hox genes in influencing cell fate during post-embryonic development in Drosophila to regulate fine-scale patterning and morphology. We also explore how this is achieved through the regulation of Hox genes, specific co-factors and their complex regulation of hundreds of target genes. We argue that further investigating the regulation and roles of Hox genes in Drosophila post-embryonic development has great potential for understanding gene regulation, cell fate and phenotypic differentiation more generally.

Regulation of Eye Determination and Regionalization in the Spider Parasteatoda tepidariorum.

Animal visual systems are enormously diverse, but their development appears to be controlled by a set of conserved retinal determination genes (RDGs). Spiders are particular masters of visual system innovation, and offer an excellent opportunity to study the evolution of animal eyes. Several RDGs have been identified in spider eye primordia, but their interactions and regulation remain unclear. From our knowledge of RDG network regulation in Drosophila melanogaster, we hypothesize that orthologs of Pax6, eyegone, Wnt genes, hh, dpp, and atonal could play important roles in controlling eye development in spiders. We analyzed the expression of these genes in developing embryos of the spider Parasteatodatepidariorum, both independently and in relation to the eye primordia, marked using probes for the RDG sine oculis. Our results support conserved roles for Wnt genes in restricting the size and position of the eye field, as well as for atonal initiating photoreceptor differentiation. However, we found no strong evidence for an upstream role of Pax6 in eye development, despite its label as a master regulator of animal eye development; nor do eyg, hh or dpp compensate for the absence of Pax6. Conversely, our results indicate that hh may work with Wnt signaling to restrict eye growth, a role similar to that of Sonichedgehog (Shh) in vertebrates.

Widespread retention of ohnologs in key developmental gene families following whole-genome duplication in arachnopulmonates.

Whole-genome duplications (WGDs) have occurred multiple times during animal evolution, including in lineages leading to vertebrates, teleosts, horseshoe crabs, and arachnopulmonates. These dramatic events initially produce a wealth of new genetic material, generally followed by extensive gene loss. It appears, however, that developmental genes such as homeobox genes, signaling pathway components and microRNAs are frequently retained as duplicates (so-called ohnologs) following WGD. These not only provide the best evidence for WGD, but an opportunity to study its evolutionary consequences. Although these genes are well studied in the context of vertebrate WGD, similar comparisons across the extant arachnopulmonate orders are patchy. We sequenced embryonic transcriptomes from two spider species and two amblypygid species and surveyed three important gene families, Hox, Wnt, and frizzled, across these and 12 existing transcriptomic and genomic resources for chelicerates. We report extensive retention of putative ohnologs, further supporting the ancestral arachnopulmonate WGD. We also found evidence of consistent evolutionary trajectories in Hox and Wnt gene repertoires across three of the six arachnopulmonate orders, with interorder variation in the retention of specific paralogs. We identified variation between major clades in spiders and are better able to reconstruct the chronology of gene duplications and losses in spiders, amblypygids, and scorpions. These insights shed light on the evolution of the developmental toolkit in arachnopulmonates, highlight the importance of the comparative approach within lineages, and provide substantial new transcriptomic data for future study.

The Evolution of Sox Gene Repertoires and Regulation of Segmentation in Arachnids.

The Sox family of transcription factors regulates many processes during metazoan development, including stem cell maintenance and nervous system specification. Characterizing the repertoires and roles of these genes can therefore provide important insights into animal evolution and development. We further characterized the Sox repertoires of several arachnid species with and without an ancestral whole-genome duplication and compared their expression between the spider Parasteatoda tepidariorum and the harvestman Phalangium opilio. We found that most Sox families have been retained as ohnologs after whole-genome duplication and evidence for potential subfunctionalization and/or neofunctionalization events. Our results also suggest that Sox21b-1 likely regulated segmentation ancestrally in arachnids, playing a similar role to the closely related SoxB gene, Dichaete, in insects. We previously showed that Sox21b-1 is required for the simultaneous formation of prosomal segments and sequential addition of opisthosomal segments in P. tepidariorum. We studied the expression and function of Sox21b-1 further in this spider and found that although this gene regulates the generation of both prosomal and opisthosomal segments, it plays different roles in the formation of these tagmata reflecting their contrasting modes of segmentation and deployment of gene regulatory networks with different architectures.

A complex gene regulatory architecture underlies the development and evolution of cuticle morphology in Drosophila.

The cuticle of insects is decorated with non-sensory hairs called trichomes. A few Drosophila species independently lost most of the dorso-lateral trichomes on first instar larvae. Genetic experiments revealed that this naked cuticle phenotype was caused by the evolution of enhancer function at the ovo/shavenbaby (ovo/svb) locus. Here we explore how this discovery catalyzed major new insights into morphological evolution in different developmental contexts, enhancer pleiotropy in gene regulation and the functionality and evolution of the Svb gene regulatory network (GRN). Taken together this highlights the importance of understanding the architecture and evolution of gene regulatory networks in detail and the great potential for further study of the Svb GRN.

Unraveling the Genetic Basis for the Rapid Diversification of Male Genitalia between Drosophila Species.

In the last 240,000 years, males of the Drosophila simulans species clade have evolved striking differences in the morphology of their epandrial posterior lobes and claspers (surstyli). These appendages are used for grasping the female during mating and so their divergence is most likely driven by sexual selection. Mapping studies indicate a highly polygenic and generally additive genetic basis for these morphological differences. However, we have limited understanding of the gene regulatory networks that control the development of genital structures and how they evolved to result in this rapid phenotypic diversification. Here, we used new D. simulans/D. mauritiana introgression lines on chromosome arm 3L to generate higher resolution maps of posterior lobe and clasper differences between these species. We then carried out RNA-seq on the developing genitalia of both species to identify the expressed genes and those that are differentially expressed between the two species. This allowed us to test the function of expressed positional candidates during genital development in D. melanogaster. We identified several new genes involved in the development and possibly the evolution of these genital structures, including the transcription factors Hairy and Grunge. Furthermore, we discovered that during clasper development Hairy negatively regulates tartan (trn), a gene known to contribute to divergence in clasper morphology. Taken together, our results provide new insights into the regulation of genital development and how this has evolved between species.

The evolution and development of eye size in flies.

The compound eyes of flies exhibit striking variation in size, which has contributed to the adaptation of these animals to different habitats and their evolution of specialist behaviors. These differences in size are caused by differences in the number and/or size of ommatidia, which are specified during the development of the retinal field in the eye imaginal disc. While the genes and developmental mechanisms that regulate the formation of compound eyes are understood in great detail in the fruit fly Drosophila melanogaster, we know very little about the genetic changes and mechanistic alterations that lead to natural variation in ommatidia number and/or size, and thus overall eye size, within and between fly species. Understanding the genetic and developmental bases for this natural variation in eye size not only has great potential to help us understand adaptations in fly vision but also determine how eye size and organ size more generally are regulated. Here we explore the genetic and developmental mechanisms that could underlie natural differences in compound eye size within and among fly species based on our knowledge of eye development in D. melanogaster and the few cases where the causative genes and mechanisms have already been identified. We suggest that the fly eye provides an evolutionary and developmental framework to better understand the regulation and diversification of this crucial sensory organ globally at a systems level as well as the gene regulatory networks and mechanisms acting at the tissue, cellular and molecular levels. This article is categorized under: Establishment of Spatial and Temporal Patterns > Regulation of Size, Proportion, and Timing Invertebrate Organogenesis > Flies Comparative Development and Evolution > Regulation of Organ Diversity.

Wnt gene regulation and function during maxillary palp development in Drosophila melanogaster.

Wnt genes encode secreted ligands that play many important roles in the development of metazoans. There are thirteen known Wnt gene subfamilies and seven of these are represented in Drosophila melanogaster. While wingless (wg) is the best understood and most widely studied Wnt gene in Drosophila, the functions of many of the other Drosophila Wnt genes are less well understood. For example, relatively little is known about Wnt6, which is an ancient paralog of wg and they form a conserved Wnt cluster together with Wnt9 (Dwnt4) and Wnt10. Wg and Wnt6 encode similar proteins and exhibit overlapping expression in several tissues during development. Both wg and Wnt6 were previously shown to regulate the development of maxillary palps, important olfactory organs in flies, but it remained unclear how these two ligands may combine to carry out specific functions and how this is regulated. Here, we have further analysed Wnt6 function in the context of maxillary palp development. Surprisingly, we found that Wnt6 does not appear to be necessary for development of maxillary palps. While a deletion of the 5' region of Wnt6 results in very small maxillary palps, we show that this effect is more likely to be a consequence of removing cis-regulatory elements that may regulate wg expression in this tissue rather than through the loss of Wnt6 function. Although, we cannot completely exclude the possibility that Wnt6 may subtly regulate maxillary palp development in combination with wg, our analysis of Wnt6 loss of function mutants suggests this ligand plays a more general role in regulating growth during development. Taken together our results provide new insights into maxillary palp formation and Wnt6 functions in Drosophila, and further evidence for a complex cis-regulatory landscape in the Wnt9-wg-Wnt6-Wnt10 cluster, which may help explain its evolutionary conservation.

Gene content evolution in the arthropods.

BACKGROUND: Arthropods comprise the largest and most diverse phylum on Earth and play vital roles in nearly every ecosystem. Their diversity stems in part from variations on a conserved body plan, resulting from and recorded in adaptive changes in the genome. Dissection of the genomic record of sequence change enables broad questions regarding genome evolution to be addressed, even across hyper-diverse taxa within arthropods. RESULTS: Using 76 whole genome sequences representing 21 orders spanning more than 500 million years of arthropod evolution, we document changes in gene and protein domain content and provide temporal and phylogenetic context for interpreting these innovations. We identify many novel gene families that arose early in the evolution of arthropods and during the diversification of insects into modern orders. We reveal unexpected variation in patterns of DNA methylation across arthropods and examples of gene family and protein domain evolution coincident with the appearance of notable phenotypic and physiological adaptations such as flight, metamorphosis, sociality, and chemoperception. CONCLUSIONS: These analyses demonstrate how large-scale comparative genomics can provide broad new insights into the genotype to phenotype map and generate testable hypotheses about the evolution of animal diversity.

Characterization of the Genetic Architecture Underlying Eye Size Variation Within Drosophila melanogaster and Drosophila simulans.

The compound eyes of insects exhibit striking variation in size, reflecting adaptation to different lifestyles and habitats. However, the genetic and developmental bases of variation in insect eye size is poorly understood, which limits our understanding of how these important morphological differences evolve. To address this, we further explored natural variation in eye size within and between four species of the Drosophila melanogaster species subgroup. We found extensive variation in eye size among these species, and flies with larger eyes generally had a shorter inter-ocular distance and vice versa We then carried out quantitative trait loci (QTL) mapping of intra-specific variation in eye size and inter-ocular distance in both D. melanogaster and D. simulans This revealed that different genomic regions underlie variation in eye size and inter-ocular distance in both species, which we corroborated by introgression mapping in D. simulans This suggests that although there is a trade-off between eye size and inter-ocular distance, variation in these two traits is likely to be caused by different genes and so can be genetically decoupled. Finally, although we detected QTL for intra-specific variation in eye size at similar positions in D. melanogaster and D. simulans, we observed differences in eye fate commitment between strains of these two species. This indicates that different developmental mechanisms and therefore, most likely, different genes contribute to eye size variation in these species. Taken together with the results of previous studies, our findings suggest that the gene regulatory network that specifies eye size has evolved at multiple genetic nodes to give rise to natural variation in this trait within and among species.

tartan underlies the evolution of Drosophila male genital morphology.

Male genital structures are among the most rapidly evolving morphological traits and are often the only features that can distinguish closely related species. This process is thought to be driven by sexual selection and may reinforce species separation. However, while the genetic bases of many phenotypic differences have been identified, we still lack knowledge about the genes underlying evolutionary differences in male genital organs and organ size more generally. The claspers (surstyli) are periphallic structures that play an important role in copulation in insects. Here, we show that divergence in clasper size and bristle number between Drosophila mauritiana and Drosophila simulans is caused by evolutionary changes in tartan (trn), which encodes a transmembrane leucine-rich repeat domain protein that mediates cell-cell interactions and affinity. There are no fixed amino acid differences in trn between D. mauritiana and D. simulans, but differences in the expression of this gene in developing genitalia suggest that cis-regulatory changes in trn underlie the evolution of clasper morphology in these species. Finally, analyses of reciprocal hemizygotes that are genetically identical, except for the species from which the functional allele of trn originates, determined that the trn allele of D. mauritiana specifies larger claspers with more bristles than the allele of D. simulans Therefore, we have identified a gene underlying evolutionary change in the size of a male genital organ, which will help to better understand not only the rapid diversification of these structures, but also the regulation and evolution of organ size more broadly.

Sensory Organ Size Evolution: A View from Drosophila.

In this issue of Developmental Cell, Ramaekers et al. (2019) show that changes in eyeless/Pax6 expression cause differences in compound eye size within and between Drosophila species. These findings reveal how changes in the underlying gene regulatory network facilitate eye size evolution and provide insights into organ size regulation.

A standardized nomenclature and atlas of the male terminalia of Drosophila melanogaster.

Animal terminalia represent some of the most diverse and rapidly evolving structures in the animal kingdom, and for this reason have been a mainstay in the taxonomic description of species. The terminalia of Drosophila melanogaster, with its wide range of experimental tools, have recently become the focus of increased interest in the fields of development, evolution, and behavior. However, studies from different disciplines have often used discrepant terminologies for the same anatomical structures. Consequently, the terminology of genital parts has become a barrier to integrating results from different fields, rendering it difficult to determine what parts are being referenced. We formed a consortium of researchers studying the genitalia of D. melanogaster to help establish a set of naming conventions. Here, we present a detailed visual anatomy of male genital parts, including a list of synonymous terms, and suggest practices to avoid confusion when referring to anatomical parts in future studies. The goal of this effort is to facilitate interdisciplinary communication and help newcomers orient themselves within the exciting field of Drosophila genitalia.

Modulation and Evolution of Animal Development through microRNA Regulation of Gene Expression.

microRNAs regulate gene expression by blocking the translation of mRNAs and/or promoting their degradation. They, therefore, play important roles in gene regulatory networks (GRNs) by modulating the expression levels of specific genes and can tune GRN outputs more broadly as part of feedback loops. These roles for microRNAs provide developmental buffering on one hand but can facilitate evolution of development on the other. Here we review how microRNAs can modulate GRNs during animal development as part of feedback loops and through their individual or combinatorial targeting of multiple different genes in the same network. We then explore how changes in the expression of microRNAs and consequently targets can facilitate changes in GRNs that alter development and lead to phenotypic evolution. The reviewed studies exemplify the key roles played by microRNAs in the regulation and evolution of gene expression during developmental processes in animals.