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 the ribbon-like organization of the Golgi apparatus in animal cells.

The "ribbon," a structural arrangement in which Golgi stacks connect to each other, is considered to be restricted to vertebrate cells. Although ribbon disruption is linked to various human pathologies, its functional role in cellular processes remains unclear. In this study, we investigate the evolutionary origin of the Golgi ribbon. We observe a ribbon-like architecture in the cells of several metazoan taxa suggesting its early emergence in animal evolution predating the appearance of vertebrates. Supported by AlphaFold2 modeling, we propose that the evolution of Golgi reassembly and stacking protein (GRASP) binding by golgin tethers may have driven the joining of Golgi stacks resulting in the ribbon-like configuration. Additionally, we find that Golgi ribbon assembly is a shared developmental feature of deuterostomes, implying a role in embryogenesis. Overall, our study points to the functional significance of the Golgi ribbon beyond vertebrates and underscores the need for further investigations to unravel its elusive biological roles.

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.

Electron Microscopy Techniques for 3D Plant ER Imaging.

The endoplasmic reticulum (ER) forms an extensive network in plant cells. In leaf cells and vacuolated root cells it is mainly restricted to the cortex, whereas in the root meristem the cortical and cytoplasmic ER takes up a large volume throughout the entire cell. Only 3D electron microscopy provides sufficient resolution to understand the spatial organization of the ER in the root. Here we present two protocols for 3D EM imaging of the ER across a range of scales. For large-scale ER structure analysis, we describe selective ER staining with ZIO that allows for automated or semi-automated ER segmentation. For smaller regions of ER, we describe high-pressure freezing, which enables almost instantaneous fixation of plant tissues but without organelle specific staining. These fixation and staining techniques are suitable for a range of imaging modalities, including serial sections, array tomography, serial block face-scanning electron microscopy (SBF-SEM), or focused ion beam (FIB) SEM.

Syncytial nerve net in a ctenophore adds insights on the evolution of nervous systems.

A fundamental breakthrough in neurobiology has been the formulation of the neuron doctrine by Santiago Ramón y Cajal, which stated that the nervous system is composed of discrete cells. Electron microscopy later confirmed the doctrine and allowed the identification of synaptic connections. In this work, we used volume electron microscopy and three-dimensional reconstructions to characterize the nerve net of a ctenophore, a marine invertebrate that belongs to one of the earliest-branching animal lineages. We found that neurons in the subepithelial nerve net have a continuous plasma membrane that forms a syncytium. Our findings suggest fundamental differences of nerve net architectures between ctenophores and cnidarians or bilaterians and offer an alternative perspective on neural network organization and neurotransmission.

Sexual morph specialisation in a trioecious nematode balances opposing selective forces.

The coexistence of different mating strategies, whereby a species can reproduce both by selfing and outcrossing, is an evolutionary enigma. Theory predicts two predominant stable mating states: outcrossing with strong inbreeding depression or selfing with weak inbreeding depression. As these two mating strategies are subject to opposing selective forces, mixed breeding systems are thought to be a rare transitory state yet can persist even after multiple speciation events. We hypothesise that if each mating strategy plays a distinctive role during some part of the species life history, opposing selective pressures could be balanced, permitting the stable co-existence of selfing and outcrossing sexual morphs. In this scenario, we would expect each morph to be specialised in their respective roles. Here we show, using behavioural, physiological and gene expression studies, that the selfing (hermaphrodite) and outcrossing (female) sexual morphs of the trioecious nematode Auanema freiburgensis have distinct adaptations optimised for their different roles during the life cycle. A. freiburgensis hermaphrodites are known to be produced under stressful conditions and are specialised for dispersal to new habitat patches. Here we show that they exhibit metabolic and intestinal changes enabling them to meet the cost of dispersal and reproduction. In contrast, A. freiburgensis females are produced in favourable conditions and facilitate rapid population growth. We found that females compensate for the lack of reproductive assurance by reallocating resources from intestinal development to mate-finding behaviour. The specialisation of each mating system for its role in the life cycle could balance opposing selective forces allowing the stable maintenance of both mating systems in A. freiburgensis.

Neuropeptide repertoire and 3D anatomy of the ctenophore nervous system.

Ctenophores are gelatinous marine animals famous for locomotion by ciliary combs. Due to the uncertainties of the phylogenetic placement of ctenophores and the absence of some key bilaterian neuronal genes, it has been hypothesized that their neurons evolved independently. Additionally, recent whole-body, single-cell RNA sequencing (scRNA-seq) analysis failed to identify ctenophore neurons using any of the known neuronal molecular markers. To reveal the molecular machinery of ctenophore neurons, we have characterized the neuropeptide repertoire of the ctenophore Mnemiopsis leidyi. Using the machine learning NeuroPID tool, we predicted 129 new putative neuropeptide precursors. Sixteen of them were localized to the subepithelial nerve net (SNN), sensory aboral organ (AO), and epithelial sensory cells (ESCs), providing evidence that they are neuropeptide precursors. Four of these putative neuropeptides had a behavioral effect and increased the animals' swimming speed. Intriguingly, these putative neuropeptides finally allowed us to identify neuronal cell types in single-cell transcriptomic data and reveal the molecular identity of ctenophore neurons. High-resolution electron microscopy and 3D reconstructions of the nerve net underlying the comb plates confirmed a more than 100-year-old hypothesis of anastomoses between neurites of the same cell in ctenophores and revealed that they occur through a continuous membrane. Our work demonstrates the unique ultrastructure of the peptidergic nerve net and a rich neuropeptide repertoire of ctenophores, supporting the hypothesis that the first nervous system(s) evolved as nets of peptidergic cells.

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.

Quantitative analysis of plant ER architecture and dynamics.

The endoplasmic reticulum (ER) is a highly dynamic polygonal membrane network composed of interconnected tubules and sheets (cisternae) that forms the first compartment in the secretory pathway involved in protein translocation, folding, glycosylation, quality control, lipid synthesis, calcium signalling, and metabolon formation. Despite its central role in this plethora of biosynthetic, metabolic and physiological processes, there is little quantitative information on ER structure, morphology or dynamics. Here we describe a software package (AnalyzER) to automatically extract ER tubules and cisternae from multi-dimensional fluorescence images of plant ER. The structure, topology, protein-localisation patterns, and dynamics are automatically quantified using spatial, intensity and graph-theoretic metrics. We validate the method against manually-traced ground-truth networks, and calibrate the sub-resolution width estimates against ER profiles identified in serial block-face SEM images. We apply the approach to quantify the effects on ER morphology of drug treatments, abiotic stress and over-expression of ER tubule-shaping and cisternal-modifying proteins.

Protein Storage Vacuoles Originate from Remodeled Preexisting Vacuoles in Arabidopsis thaliana.

Protein storage vacuoles (PSV) are the main repository of protein in dicotyledonous seeds, but little is known about the origins of these transient organelles. PSV are hypothesized to either arise de novo or originate from the preexisting embryonic vacuole (EV) during seed maturation. Here, we tested these hypotheses by studying PSV formation in Arabidopsis (Arabidopsis thaliana) embryos at different stages of seed maturation and recapitulated this process in Arabidopsis leaves reprogrammed to an embryogenic fate by inducing expression of the LEAFY COTYLEDON2 transcription factor. Confocal and immunoelectron microscopy indicated that both storage proteins and tonoplast proteins typical of PSV were delivered to the preexisting EV in embryos or to the lytic vacuole in reprogrammed leaf cells. In addition, sectioning through embryos at several developmental stages using serial block face scanning electron microscopy revealed the 3D architecture of forming PSV. Our results indicate that the preexisting EV is reprogrammed to become a PSV in Arabidopsis.

The odd one out: Arabidopsis reticulon 20 does not bend ER membranes but has a role in lipid regulation.

Reticulons are integral ER membrane proteins characterised by a reticulon homology domain comprising four transmembrane domains which results in the proteins sitting in the membrane in a W-topology. Here we report on a novel subgroup of reticulons with an extended N-terminal domain and in particular on arabidopsis reticulon 20. Using high resolution confocal microscopy we show that reticulon 20 is located in a unique punctate pattern on the ER membrane. Its closest homologue reticulon 19 labels the whole ER. Other than demonstrated for the other members of the reticulon protein family RTN20 and 19 do not display ER constriction phenotypes on over expression. We show that mutants in RTN20 or RTN19, respectively, display a significant change in sterol composition in roots indicating a role in lipid regulation. A third homologue in this family -3BETAHSD/D1- is unexpectedly localised to ER exit sites resulting in an intriguing location difference for the three proteins.

3D Electron Microscopy of the ER.

The endoplasmic reticulum (ER) forms an extensive network in plant cells. In leaf cells and vacuolated root cells it is mainly restricted to the cortex whereas in the root meristem the cortical and cytoplasmic ER takes up a large volume throughout the entire cell. Only 3D electron microscopy provides sufficient resolution to understand the spatial organization of the ER in the root. However, high contrast staining and optimally ER specific staining is essential. Here we describe a protocol for selective ER staining that allows automated or semiautomated segmentation of the organelle in 3D datasets obtained from serial sections, Array Tomography, Serial Block Face Scanning Electron Microscopy (SBFSEM), or Focused Ion Beam (FIB) SEM.

Actin-dependent vacuolar occupancy of the cell determines auxin-induced growth repression.

The cytoskeleton is an early attribute of cellular life, and its main components are composed of conserved proteins. The actin cytoskeleton has a direct impact on the control of cell size in animal cells, but its mechanistic contribution to cellular growth in plants remains largely elusive. Here, we reveal a role of actin in regulating cell size in plants. The actin cytoskeleton shows proximity to vacuoles, and the phytohormone auxin not only controls the organization of actin filaments but also impacts vacuolar morphogenesis in an actin-dependent manner. Pharmacological and genetic interference with the actin-myosin system abolishes the effect of auxin on vacuoles and thus disrupts its negative influence on cellular growth. SEM-based 3D nanometer-resolution imaging of the vacuoles revealed that auxin controls the constriction and luminal size of the vacuole. We show that this actin-dependent mechanism controls the relative vacuolar occupancy of the cell, thus suggesting an unanticipated mechanism for cytosol homeostasis during cellular growth.

Diverse Roles of Axonemal Dyneins in Drosophila Auditory Neuron Function and Mechanical Amplification in Hearing.

Much like vertebrate hair cells, the chordotonal sensory neurons that mediate hearing in Drosophila are motile and amplify the mechanical input of the ear. Because the neurons bear mechanosensory primary cilia whose microtubule axonemes display dynein arms, we hypothesized that their motility is powered by dyneins. Here, we describe two axonemal dynein proteins that are required for Drosophila auditory neuron function, localize to their primary cilia, and differently contribute to mechanical amplification in hearing. Promoter fusions revealed that the two axonemal dynein genes Dmdnah3 (=CG17150) and Dmdnai2 (=CG6053) are expressed in chordotonal neurons, including the auditory ones in the fly's ear. Null alleles of both dyneins equally abolished electrical auditory neuron responses, yet whereas mutations in Dmdnah3 facilitated mechanical amplification, amplification was abolished by mutations in Dmdnai2. Epistasis analysis revealed that Dmdnah3 acts downstream of Nan-Iav channels in controlling the amplificatory gain. Dmdnai2, in addition to being required for amplification, was essential for outer dynein arms in auditory neuron cilia. This establishes diverse roles of axonemal dyneins in Drosophila auditory neuron function and links auditory neuron motility to primary cilia and axonemal dyneins. Mutant defects in sperm competition suggest that both dyneins also function in sperm motility.

Ankyrin Repeats Convey Force to Gate the NOMPC Mechanotransduction Channel.

How metazoan mechanotransduction channels sense mechanical stimuli is not well understood. The NOMPC channel in the transient receptor potential (TRP) family, a mechanotransduction channel for Drosophila touch sensation and hearing, contains 29 Ankyrin repeats (ARs) that associate with microtubules. These ARs have been postulated to act as a tether that conveys force to the channel. Here, we report that these N-terminal ARs form a cytoplasmic domain essential for NOMPC mechanogating in vitro, mechanosensitivity of touch receptor neurons in vivo, and touch-induced behaviors of Drosophila larvae. Duplicating the ARs elongates the filaments that tether NOMPC to microtubules in mechanosensory neurons. Moreover, microtubule association is required for NOMPC mechanogating. Importantly, transferring the NOMPC ARs to mechanoinsensitive voltage-gated potassium channels confers mechanosensitivity to the chimeric channels. These experiments strongly support a tether mechanism of mechanogating for the NOMPC channel, providing insights into the basis of mechanosensitivity of mechanotransduction channels.

Novel cell types, neurosecretory cells, and body plan of the early-diverging metazoan Trichoplax adhaerens.

BACKGROUND: Trichoplax adhaerens is the best-known member of the phylum Placozoa, one of the earliest-diverging metazoan phyla. It is a small disk-shaped animal that glides on surfaces in warm oceans to feed on algae. Prior anatomical studies of Trichoplax revealed that it has a simple three-layered organization with four somatic cell types. RESULTS: We reinvestigate the cellular organization of Trichoplax using advanced freezing and microscopy techniques to identify localize and count cells. Six somatic cell types are deployed in stereotyped positions. A thick ventral plate, comprising the majority of the cells, includes ciliated epithelial cells, newly identified lipophil cells packed with large lipid granules, and gland cells. Lipophils project deep into the interior, where they alternate with regularly spaced fiber cells whose branches contact all other cell types, including cells of the dorsal and ventral epithelium. Crystal cells, each containing a birefringent crystal, are arrayed around the rim. Gland cells express several proteins typical of neurosecretory cells, and a subset of them, around the rim, also expresses an FMRFamide-like neuropeptide. CONCLUSIONS: Structural analysis of Trichoplax with significantly improved techniques provides an advance in understanding its cell types and their distributions. We find two previously undetected cell types, lipohil and crystal cells, and an organized body plan in which different cell types are arranged in distinct patterns. The composition of gland cells suggests that they are neurosecretory cells and could control locomotor and feeding behavior.

A novel CaM kinase II pathway controls the location of neuropeptide release from Caenorhabditis elegans motor neurons.

Neurons release neuropeptides via the regulated exocytosis of dense core vesicles (DCVs) to evoke or modulate behaviors. We found that Caenorhabditis elegans motor neurons send most of their DCVs to axons, leaving very few in the cell somas. How neurons maintain this skewed distribution and the extent to which it can be altered to control DCV numbers in axons or to drive release from somas for different behavioral impacts is unknown. Using a forward genetic screen, we identified loss-of-function mutations in UNC-43 (CaM kinase II) that reduce axonal DCV levels by ∼90% and cell soma/dendrite DCV levels by ∼80%, leaving small synaptic vesicles largely unaffected. Blocking regulated secretion in unc-43 mutants restored near wild-type axonal levels of DCVs. Time-lapse video microscopy showed no role for CaM kinase II in the transport of DCVs from cell somas to axons. In vivo secretion assays revealed that much of the missing neuropeptide in unc-43 mutants is secreted via a regulated secretory pathway requiring UNC-31 (CAPS) and UNC-18 (nSec1). DCV cargo levels in unc-43 mutants are similarly low in cell somas and the axon initial segment, indicating that the secretion occurs prior to axonal transport. Genetic pathway analysis suggests that abnormal neuropeptide function contributes to the sluggish basal locomotion rate of unc-43 mutants. These results reveal a novel pathway controlling the location of DCV exocytosis and describe a major new function for CaM kinase II.

Liprin-α/SYD-2 determines the size of dense projections in presynaptic active zones in C. elegans.

Synaptic vesicle (SV) release is spatially and temporally regulated by a network of proteins that form the presynaptic active zone (AZ). The hallmark of most AZs is an electron-dense projection (DP) surrounded by SVs. Despite their importance for our understanding of triggered SV release, high-resolution analyses of DP structures are limited. Using electron microscopy, we show that DPs at Caenorhabditis elegans neuromuscular junctions (NMJs) were highly structured, composed of building units forming bays in which SVs are docked to the AZ membrane. Furthermore, larger ribbonlike DPs that were multimers of the NMJ building unit are found at synapses between inter- and motoneurons. We also demonstrate that DP size is determined by the activity of the AZ protein SYD-2/Liprin-α. Whereas loss of syd-2 function led to smaller DPs, syd-2 gain-of-function mutants displayed larger ribbonlike DPs through increased recruitment of ELKS-1/ELKS. Therefore, our data suggest that a main role of SYD-2/Liprin-α in synaptogenesis is to regulate the polymerization of DPs.