Larval Eye Research Articles

Ultrastructure of cerebral eyes in Oweniidae and Chaetopteridae (Annelida) \u2013 implications for the evolution of eyes in Annelida

BackgroundRecent phylogenomic studies have revealed a robust, new hypothesis of annelid phylogeny. Most surprisingly, a few early branching lineages formed a basal grade, whereas the majority of taxa were categorized as monophyletic Pleistoannelida. Members of these basal groups show a comparatively simple organization lacking certain characters regarded to be annelid specific. Thus, the evolution of organ systems and the characteristics probably present in the last common annelid ancestor require reevaluation. With respect to light-sensitive organs, a pair of simple larval eyes is regarded as being present in their last common ancestor. However, the evolutionary origin and structure of adult eyes remain obscure. Typically, adult eyes are multicellular pigment cups or pinhole eyes with or without a lens comprising rhabdomeric photoreceptor cells (PRCs) and pigmented supportive cells (PSCs) in converse design. However, in the most basal lineages, eyes are only present in a few taxa, and thus far, their ultrastructure is unknown.ResultsUltrastructural investigations of members of Oweniidae and Chaetopteridae reveal a corresponding design of adult cerebral eyes and PRCs. The eyes in species of these groups are simple pigment spot eyes, either forming a flat patch or embedded in a tube-like invagination. They are part of the epidermis and comprise two cell types, PSCs and rhabdomeric PRCs. Both cell types bear microvilli and one more or less reduced cilium. However, the PRCs showed only a moderate increase in the apical membrane surface in the form of irregularly arranged microvilli intermingling with those of the PSCs; a densely arranged brush border of rhabdomeric microvilli was absent. Additionally, both cell types show certain characteristics elsewhere observable in typical epidermal supportive cells.ConclusionsThese findings shed new light on the evolutionary history of adult eyes in Annelida. Most likely, the adult eye of the annelid stem species was a pair of simple pigment spot eyes with only slightly specialized PSCs and PRCs being an integrative part of the epidermis. As is the case for the nuchal organs, typical pigment cup adult eyes presumably evolved later in the annelid phylogeny, namely, in the stem lineages of Amphinomida and Pleistoannelida.

Ultraviolet vision in larval Neogonodactylus oerstedii.

Stomatopod crustaceans have among the most complex eyes in the animal kingdom, with up to 12 different color detection channels. The capabilities of these unique eyes include photoreception of ultraviolet (UV) wavelengths (<400 nm). UV vision has been well characterized in adult stomatopods but has not been previously demonstrated in the comparatively simpler larval eye. Larval stomatopod eyes are developmentally distinct from their adult counterpart and have been described as lacking the visual pigment diversity and morphological specializations found in adult eyes. However, recent studies have provided evidence that larval stomatopod eyes are more complex than previously thought and warrant closer investigation. Using electroretinogram recordings in live animals we found physiological evidence of blue- and UV-sensitive photoreceptors in larvae of the Caribbean stomatopod species Neogonodactylus oerstedii. Transcriptomes of individual larvae were used to identify the expression of three distinct UV opsin mRNA transcripts, which may indicate the presence of multiple UV spectral channels. This is the first paper to document UV vision in any larval stomatopod, expanding our understanding of the importance of UV sensitivity in plankton. Larval stomatopod eyes are more complex and more similar to adult eyes than expected, showing previously uncharacterized molecular diversity and physiological functions.

A flow-through imaging system for automated measurement of ichthyoplankton

Microscopic imaging and morphometric measurement of fish embryos and larvae is essential in environmental monitoring of fish populations and to evaluate larvae development in aquaculture. Traditional microscopy methods require time-consuming, repetitive work by human experts.We present a method for fast imaging and analysis of millimetre-scale ichthyoplankton suspended in seawater. Our system can be easily built from common and off-the-shelf components and uses open-source software for image capture and analysis. Our system obtains images of similar quality to traditional microscopy, and biological measurements comparable to those by human experts, with minimal human interaction. This saves time and effort, while increasing the size of data sets obtained.We demonstrate our approach with cod eggs and larvae, and present results showing biologically relevant endpoints including egg diameter, larval standard length, yolk volume and eye diameter, with comparison to similar measurements reported in the literature.• High throughput, microscope-scale imaging of fish eggs and larvae• Automated measurement of biologically relevant endpoints• Easily built from off-the-shelf components and open-source software

EMC is required for biogenesis of Xport-A, anessential chaperone of Rhodopsin-1 and the TRPchannel.

The ER membrane protein complex (EMC) is required for the biogenesis of a subset of tail anchored (TA) and polytopic membrane proteins, including Rhodopsin-1 (Rh1) and the TRP channel. To understand the physiological implications of EMC-dependent membrane protein biogenesis, we perform a bioinformatic identification of Drosophila TA proteins. From 254 predicted TA proteins, screening in larval eye discs identified two proteins that require EMC for their biogenesis: fan and Xport-A. Fan is required for male fertility in Drosophila and we show that EMC is also required for this process. Xport-A is essential for the biogenesis of both Rh1 and TRP, raising the possibility that disruption of Rh1 and TRP biogenesis in EMC mutants is secondary to the Xport-A defect. We show that EMC is required for Xport-A TMD membrane insertion and that EMC-independent Xport-A mutants rescue Rh1 and TRP biogenesis in EMC mutants. Finally, our work also reveals a role for Xport-A in a glycosylation-dependent triage mechanism during Rh1 biogenesis in the endoplasmic reticulum.

Ultrastructure of the larval eyes of the hangingfly Terrobittacus implicatus (Mecoptera: Bittacidae)

The fine structure of the larval eyes of the hangingfly Terrobittacus implicatus (Huang & Hua) was investigated using scanning and transmission electron microscopy. The results show that the larval eyes of T. implicatus each consist of seven spaced ommatidia. Each ommatidium is composed of a corneal lens with about 45 lamellae, a tetrapartite eucone type of crystalline cone, eight retinula cells, two primary pigment cells, and an undetermined number of secondary pigment cells. The rhabdomeres of eight retinula cells effectively fuse into a centrally-fused, tiered funnel-shaped rhabdom extending from the base of the crystalline cone deeply into the ommatidium. In light of different positions in the ommatidium, the retinula cells can be divided into four distal and four proximal retinula cells. Pigment cells envelop the entire ommatidium. Electron-lucent vesicles are abundant throughout the cytoplasm of the eight retinula cells. The larval ommatidia of T. implicatus are similar to those of the Panorpidae, except for the distal retinula cells that also participate in the formation of the proximal rhabdom. In this case, the larval eyes of T. implicatus may lie in the transitional stage during the larval eye evolution of insects from ommatidia to stemmata.

Early life stage transient aristolochic acid exposure induces behavioral hyperactivity but not nephrotoxicity in larval zebrafish.

Aristolochic acids (AA) are nitrophenanthrene carboxylic acids found in plants of the Aristolochiaceae family. Humans are exposed to AA by deliberately taking herbal medicines or unintentionally as a result of environmental contamination. AA is notorious for its nephrotoxicity, however, fewer studies explore potential neurotoxicity associated with AA exposure. The developing nervous system is vulnerable to xenobiotics, and pregnant women exposed to AA may put their fetuses at risk. In the present study, we used the embryonic zebrafish model to evaluate the developmental neurotoxicity associated with AA exposure. At non-teratogenic concentrations (≤ 4µM), continuous AA exposure from 8 to 120 hours post fertilization (hpf) resulted in larval hyperactivity that was characterized by increased moving distance, elevated activity and faster swimming speeds in several behavioral assays. Further analysis revealed that 8-24 hpf is the most sensitive exposure window for AA-induced hyperactivity. AA exposures specifically increased motor neuron proliferation, increased apoptosis in the eye, and resulted in cellular oxidative stress. In addition, AA exposures increased larval eye size and perturbed the expression of vision genes. Our study, for the first time, demonstrates that AA is neurotoxic to the developmental zebrafish with a sensitive window distinct from its well-documented nephrotoxicity.

Distinct synaptic transfer functions in same-type photoreceptors.

Many sensory systems use ribbon-type synapses to transmit their signals to downstream circuits. The properties of this synaptic transfer fundamentally dictate which aspects in the original stimulus will be accentuated or suppressed, thereby partially defining the detection limits of the circuit. Accordingly, sensory neurons have evolved a wide variety of ribbon geometries and vesicle pool properties to best support their diverse functional requirements. However, the need for diverse synaptic functions does not only arise across neuron types, but also within. Here we show that UV-cones, a single type of photoreceptor of the larval zebrafish eye, exhibit striking differences in their synaptic ultrastructure and consequent calcium to glutamate transfer function depending on their location in the eye. We arrive at this conclusion by combining serial section electron microscopy and simultaneous 'dual-colour' two-photon imaging of calcium and glutamate signals from the same synapse in vivo. We further use the functional dataset to fit a cascade-like model of the ribbon synapse with different vesicle pool sizes, transfer rates, and other synaptic properties. Exploiting recent developments in simulation-based inference, we obtain full posterior estimates for the parameters and compare these across different retinal regions. The model enables us to extrapolate to new stimuli and to systematically investigate different response behaviours of various ribbon configurations. We also provide an interactive, easy-to-use version of this model as an online tool. Overall, we show that already on the synaptic level of single-neuron types there exist highly specialised mechanisms which are advantageous for the encoding of different visual features.

The Hippo pathway uses different machinery to control cell fate and organ size

SummaryThe Hippo pathway is a conserved signaling network that regulates organ growth and cell fate. One such cell fate decision is that of R8 photoreceptor cells in the Drosophila eye, where Hippo specifies whether cells sense blue or green light. We show that only a subset of proteins that control organ growth via the Hippo pathway also regulate R8 cell fate choice, including the STRIPAK complex, Tao, Pez, and 14-3-3 proteins. Furthermore, key Hippo pathway proteins were primarily cytoplasmic in R8 cells rather than localized to specific membrane domains, as in cells of growing epithelial organs. Additionally, Warts was the only Hippo pathway protein to be differentially expressed between R8 subtypes, while central Hippo pathway proteins were expressed at dramatically lower levels in adult and pupal eyes than in growing larval eyes. Therefore, we reveal several important differences in Hippo signaling in the contexts of organ growth and cell fate.

Hexagonal patterning of the Drosophila eye

A complex network of transcription factor interactions propagates across the larval eye disc to establish columns of evenly-spaced R8 precursor cells, the founding cells of Drosophila ommatidia. After the recruitment of additional photoreceptors to each ommatidium, the surrounding cells are organized into their stereotypical pattern during pupal development. These support cells – comprised of pigment and cone cells – are patterned to encapsulate the photoreceptors and separate ommatidia with an hexagonal honeycomb lattice. Since the proteins and processes essential for correct eye patterning are conserved, elucidating how these function and change during Drosophila eye patterning can substantially advance our understanding of transcription factor and signaling networks, cytoskeletal structures, adhesion complexes, and the biophysical properties of complex tissues during their morphogenesis. Our understanding of many of these aspects of Drosophila eye patterning is largely descriptive. Many important questions, especially relating to the regulation and integration of cellular events, remain.

New regulators of Drosophila eye development identified from temporal transcriptome changes.

In the last larval instar, uncommitted progenitor cells in the Drosophila eye primordium start to adopt individual retinal cell fates, arrest their growth and proliferation, and initiate terminal differentiation into photoreceptor neurons and other retinal cell types. To explore the regulation of these processes, we have performed mRNA-Seq studies of the larval eye and antennal primordial at multiple developmental stages. A total of 10,893 fly genes were expressed during these stages and could be adaptively clustered into gene groups, some of whose expression increases or decreases in parallel with the cessation of proliferation and onset of differentiation. Using in situ hybridization of a sample of 98 genes to verify spatial and temporal expression patterns, we estimate that 534 genes or more are transcriptionally upregulated during retinal differentiation, and 1367 or more downregulated as progenitor cells differentiate. Each group of co-expressed genes is enriched for regulatory motifs recognized by co-expressed transcription factors, suggesting that they represent coherent transcriptional regulatory programs. Using available mutant strains, we describe novel roles for the transcription factors SoxNeuro (SoxN), H6-like homeobox (Hmx), CG10253, without children (woc), Structure specific recognition protein (Ssrp), and multisex combs (mxc).

Early lineage segregation of the retinal basal glia in the Drosophila eye disc

The retinal basal glia (RBG) is a group of glia that migrates from the optic stalk into the third instar larval eye disc while the photoreceptor cells (PR) are differentiating. The RBGs are grouped into three major classes based on molecular and morphological characteristics: surface glia (SG), wrapping glia (WG) and carpet glia (CG). The SGs migrate and divide. The WGs are postmitotic and wraps PR axons. The CGs have giant nucleus and extensive membrane extension that each covers half of the eye disc. In this study, we used lineage tracing methods to determine the lineage relationships among these glia subtypes and the temporal profile of the lineage decisions for RBG development. We found that the CG lineage segregated from the other RBG very early in the embryonic stage. It has been proposed that the SGs migrate under the CG membrane, which prevented SGs from contacting with the PR axons lying above the CG membrane. Upon passing the front of the CG membrane, which is slightly behind the morphogenetic furrow that marks the front of PR differentiation, the migrating SG contact the nascent PR axon, which in turn release FGF to induce SGs’ differentiation into WG. Interestingly, we found that SGs are equally distributed apical and basal to the CG membrane, so that the apical SGs are not prevented from contacting PR axons by CG membrane. Clonal analysis reveals that the apical and basal RBG are derived from distinct lineages determined before they enter the eye disc. Moreover, the basal SG lack the competence to respond to FGFR signaling, preventing its differentiation into WG. Our findings suggest that this novel glia-to-glia differentiation is both dependent on early lineage decision and on a yet unidentified regulatory mechanism, which can provide spatiotemporal coordination of WG differentiation with the progressive differentiation of photoreceptor neurons.

Larval development of a new hybrid Malaysian mahseer (Barbonymus gonionotus ♀ × Tor tambroides ♂)

Hybrid Malaysian mahseer is a crossbreed between the male Malaysian mahseer, Tor tambroides and female silver barb, Barbonymus gonionotus through induced breeding. The study was carried out to observe the morpho-histological development of the F1 hybrid Malaysian mahseer larvae for the entire duration of its larval stage. Newly hatched larvae were reared in three 120 L aquaria at a stocking density of 5 larvae per liter. Larval development was observed daily from 1 to 9 DAH (day after hatching) and at 2-day intervals until 25 DAH by means of light and scanning electron microscopy (SEM), and histology. Newly hatched larvae had tiny body with closed mouth and simple gut formation. The hybrid larvae commenced exogenous feeding by 3 DAH using the taste buds and neuromasts to detect the presence of foods. Larval eyes were underdeveloped in early stage. They became fully functional as early as 9 DAH with larval TL and BW of 8.36 ± 0.24 mm and 4.3 ± 0.14 mg, respectively. By 7 DAH the stomach and intestinal tract were completely developed while complete tissue layers (mucosa, submucosa, muscularis and serosa) were visible in the larval gut by 18–21 DAH indicating the end of the larval stage. The post larval stage of the hybrid Malaysian mahseer was short and completed by 21–25 DAH when the scales fully covered its body. The rapid completion of larval stages and morphological features took after the maternal brood-fish, Barbonymus gonionotus but with larger scales. These positive attributes in larval development could be of interest to the aquaculture industry.

Abnormal Cone and Rod Photoreceptor Morphogenesis in gdf6a Mutant Zebrafish.

PurposeAnalysis of photoreceptor morphology and gene expression in mispatterned eyes of zebrafish growth differentiation factor 6a (gdf6a) mutants.MethodsRod and cone photoreceptors were compared between gdf6a mutant and control zebrafish from larval to late adult stages using transgenic labels, immunofluorescence, and confocal microscopy, as well as by transmission electron microscopy. To compare transcriptomes between larval gdf6a mutant and control zebrafish, RNA-Seq was performed on isolated eyes.ResultsAlthough rod and cone photoreceptors differentiate in gdf6a mutant zebrafish, the cells display aberrant growth and morphology. The cone outer segments, the light-detecting sensory endings, are reduced in size in the mutant larvae and fail to recover to control size at subsequent stages. In contrast, rods form temporarily expanded outer segments. The inner segments, which generate the required energy and proteins for the outer segments, are shortened in both rods and cones at all stages. RNA-Seq analysis provides a set of misregulated genes associated with the observed abnormal photoreceptor morphogenesis.Conclusions GDF6 mutations were previously identified in patients with Leber congenital amaurosis. Here, we reveal a unique photoreceptor phenotype in the gdf6a mutant zebrafish whereby rods and cones undergo abnormal maturation distinct for each cell type. Further, subsequent development shows partial recovery of cell morphology and maintenance of the photoreceptor layer. By conducting a transcriptomic analysis of the gdf6a larval eyes, we identified a collection of genes that are candidate regulators of photoreceptor size and morphology.

Deficiency in classical nonhomologous end-joining–mediated repair of transcribed genes is linked to SCA3 pathogenesis

Spinocerebellar ataxia type 3 (SCA3) is a dominantly inherited neurodegenerative disease caused by CAG (encoding glutamine) repeat expansion in the Ataxin-3 (ATXN3) gene. We have shown previously that ATXN3-depleted or pathogenic ATXN3-expressing cells abrogate polynucleotide kinase 3'-phosphatase (PNKP) activity. Here, we report that ATXN3 associates with RNA polymerase II (RNAP II) and the classical nonhomologous end-joining (C-NHEJ) proteins, including PNKP, along with nascent RNAs under physiological conditions. Notably, ATXN3 depletion significantly decreased global transcription, repair of transcribed genes, and error-free double-strand break repair of a 3'-phosphate-containing terminally gapped, linearized reporter plasmid. The missing sequence at the terminal break site was restored in the recircularized plasmid in control cells by using the endogenous homologous transcript as a template, indicating ATXN3's role in PNKP-mediated error-free C-NHEJ. Furthermore, brain extracts from SCA3 patients and mice show significantly lower PNKP activity, elevated p53BP1 level, more abundant strand-breaks in the transcribed genes, and degradation of RNAP II relative to controls. A similar RNAP II degradation is also evident in mutant ATXN3-expressing Drosophila larval brains and eyes. Importantly, SCA3 phenotype in Drosophila was completely amenable to PNKP complementation. Hence, salvaging PNKP's activity can be a promising therapeutic strategy for SCA3.

Development of the visual system of anchovy larvae, Engraulis anchoita: A microanatomical description

During the early ontogeny of fish larvae, the accurate development of the visual system plays a key role, because it is involved in locating food, orientation, selection of favorable habitat, and evasion of predators. The structure of the eye of the fish is typical of vertebrates, with some modifications related to the aquatic environment. In the present work, we describe the development of the larval eye of Engraulis anchoita for the first time. Larvae were collected at the Permanent Station of Environmental Studies (EPEA) in coastal waters of the Southwestern Atlantic Ocean during research cruises in 2015 and 2016. We describe the histology of the retina layers, determine the beginning of the functionality of the eye, and discuss a possible synchronization with the development of the digestive tract. This study provides information about the biology of E. anchoita, the most abundant fish species in the southwestern Atlantic Ocean. Also, recent studies have shown responses of the retina and other tissues to the increase in environmental acidity. Therefore, results of this study are also discussed with respect to the possible effect of acidification on the larvae of this species. The continuity of the time series developed at the EPEA will allow monitoring the effect of long-term environmental and biological variables on the early ontogeny of anchovy in the context of climate change. The high commercial fishing potential of E. anchoita due to its high abundance, as well as its essential role in the trophic web of other commercially valuable fishing resources of Argentina, reinforce the need to continue deepening knowledge about this species. Research highlights: Eyes of Engraulis anchoita larvae are functional from early larval stages. At hatching, the retina is formed by only few layers from which the other layers differentiates during ontogeny. Focal distance increases with larval growth.

Influence of salinity, temperature and photoperiod on eye asymmetry of Amphirion ocellaris larvae

Fluctuating asymmetry (FA) is the deviation from perfect bilateral symmetry caused by environmental stresses, developmental instability, and genetic problems during ontogeny. We tested FA levels in larvae of Amphirion ocellaris will change with different salinities (25, 30 and 35), temperatures (20, 25 and 30 °C), and photoperiods (6, 8 and 10 h). A total of 45 larvae of A. ocellaris were analyzed. The survival of larvae did not differ significantly under different salinities (PERMANOVA; F = 0.5, p = 1), temperatures (PERMANOVA; F = 0.8; p = .68) and photoperiods (PERMANOVA; F = 0.13; p = .99). The absence of a significant relationship between FA and survival may have occurred because larvae still have a yolk sac, which justifies the low mortality rate. Eye asymmetry changed significantly among salinities (PERMANOVA; F = 6.02; p = .006), suggesting that the higher salinity (35) was more stressful for A. ocellaris larvaes. Eye asymmetry not changed significantly among temperature (PERMANOVA; F = 1.65; p = .19) and photoperiod (PERMANOVA; F = 1.05; p = .35). For A. ocellaris larvaes it is important to maintain the salinity of 25, due to the better zootechnical performance due to the lower energy expenditure of the species, for osmoregulation. The present work is a pioneer in the detection of AF in the larval eye diameter of A. ocellaris. Fluctuating asymmetry in fish farming has a simple and effective way to detect anomalies that can lead to economic losses.

Zika virus non-structural protein NS4A restricts eye growth in Drosophila through regulation of JAK/STAT signaling.

ABSTRACTTo gain a comprehensive view of the changes in host gene expression underlying Zika virus (ZIKV) pathogenesis, we performed whole-genome RNA sequencing (RNA-seq) of ZIKV-infected Drosophila adult flies. RNA-seq analysis revealed that ZIKV infection alters several and diverse biological processes, including stress, locomotion, lipid metabolism, imaginal disc morphogenesis and regulation of JAK/STAT signaling. To explore the interaction between ZIKV infection and JAK/STAT signaling regulation, we generated genetic constructs overexpressing ZIKV-specific non-structural proteins NS2A, NS2B, NS4A and NS4B. We found that ectopic expression of non-structural proteins in the developing Drosophila eye significantly restricts growth of the larval and adult eye and correlates with considerable repression of the in vivo JAK/STAT reporter, 10XStat92E-GFP. At the cellular level, eye growth defects are associated with reduced rate of proliferation without affecting the overall rate of apoptosis. In addition, ZIKV NS4A genetically interacts with the JAK/STAT signaling components; co-expression of NS4A along with the dominant-negative form of domeless or StatRNAi results in aggravated reduction in eye size, while co-expression of NS4A in HopTuml (also known as hopTum) mutant background partially rescues the hop-induced eye overgrowth phenotype. The function of ZIKV NS4A in regulating growth is maintained in the wing, where ZIKV NS4A overexpression in the pouch domain results in reduced growth linked with diminished expression of Notch targets, Wingless (Wg) and Cut, and the Notch reporter, NRE-GFP. Thus, our study provides evidence that ZIKV infection in Drosophila results in restricted growth of the developing eye and wing, wherein eye phenotype is induced through regulation of JAK/STAT signaling, whereas restricted wing growth is induced through regulation of Notch signaling. The interaction of ZIKV non-structural proteins with the conserved host signaling pathways further advance our understanding of ZIKV-induced pathogenesis.This article has an associated First Person interview with the first author of the paper.

Ambient Illumination Influence on Photuris Firefly Larval Surface Movements is not Mediated by the Stemmata

Movements of fireflies visible on the surface of soil were measured under controlled laboratory conditions consisting of high and low ambient illumination. High illumination approximating the intensity of light prior to sunset constituted our light condition. Low illumination was consistent with ambient light levels after civil sunset, which we referred to as our dark condition. Surface movements were significantly more frequent during dark conditions compared to light conditions. Stemmata are the larval form of the insect eye and were the only identified visual organs present in Photuris larvae. We investigated whether stemmata provided larvae with the sensory information facilitating the light dependency of surface movements. We disrupted transmission of visual information from the larval eyes to the brain by severing the optic nerves. The amount of surface movement was compared between larvae with intact and severed optic nerves, under light and dark conditions. Light dependency of surface movements was preserved in larvae with cut optic nerves. The presence of the light dependency after cutting the optic nerves indicated that an alternative, extrastemmatal sensory pathway must be providing light intensity information to the animal. Light dependency was abolished upon removal of the head. Thus, these results suggested that the extraocular system providing light intensity information for regulating the frequency of surface movement was located in the head. The precise location of the suggested extraocular receptor and the nature of the associated sensory system remains unknown.

Remnants of ancestral larval eyes in an eyeless mollusk? Molecular characterization of photoreceptors in the scaphopod Antalis entalis

BackgroundEyes have evolved and been lost multiple times during animal evolution, however, the process of eye loss has only been reconstructed in a few cases. Mollusks exhibit eyes as varied as the octopod camera eye or the gastropod cup eye and are ideal systems for studying the evolution of eyes, photoreceptors, and opsins.ResultsHere, we identify genes related to photoreceptor formation and function in an eyeless conchiferan mollusk, the scaphopod Antalis entalis, and investigate their spatial and temporal expression patterns during development. Our study reveals that the scaphopod early mid-stage trochophore larva has putative photoreceptors in a similar location and with a similar gene expression profile as the trochophore of polyplacophoran mollusks. The apical and post-trochal putative photoreceptors appear to co-express go-opsin, six1/2, myoV, and eya, while expression domains in the posterior foot and pavilion (posterior mantle opening) show co-expression of several other candidate genes but not go-opsin. Sequence analysis reveals that the scaphopod Go-opsin amino acid sequence lacks the functionally important lysine (K296; Schiff base) in the retinal-binding domain, but has not accumulated nonsense mutations and still exhibits the canonical G-protein activation domain.ConclusionsThe scaphopod Go-opsin sequence reported here is the only known example of a bilaterian opsin that lacks lysine K296 in the retinal-binding domain. Although this may render the Go-opsin unable to detect light, the protein may still perform sensory functions. The location, innervation, development, and gene expression profiles of the scaphopod and polyplacophoran apical and post-trochal photoreceptors suggest that they are homologous, even though the scaphopod post-trochal photoreceptors have degenerated. This indicates that post-trochal eyes are not a polyplacophoran apomorphy but likely a molluscan synapomorphy lost in other mollusks. Scaphopod eye degeneration is probably a result of the transition to an infaunal life history and is reflected in the likely functional degeneration of Go-opsin, the loss of photoreceptor shielding pigments, and the scarce expression of genes involved in phototransduction and eye development. Our results emphasize the importance of studying a phylogenetically broad range of taxa to infer the mechanisms and direction of body plan evolution.

Ultrastructure of pigmented eyes in Onuphidae and Eunicidae (Annelida: Errantia: Eunicida) and its importance in understanding the evolution of eyes in Annelida

Annelida displays enormous eye and photoreceptor cell (PRC) diversity. In polychaetes, larval and adult eyes can be readily distinguished as the former are small, inverse and comprised of only two or three cells, and the latter are usually everse and multicellular. However, there are some species in which adult eyes are small, fewer in number or even absent. Recent studies show that two pairs of multicellular adult eyes belong to the ground pattern of a clade comprising Amphinomida/Sipuncula and Pleistoannelida with Errantia and Sedentaria. As ultrastructural data in one higher taxon of Errantia, Eunicida, are scarce or completely lacking for certain subgroups, we investigated the structure of pigmented eyes in three species of eunicidan bristle worms: Aponuphis bilineata (Onuphidae), Paucibranchia bellii (Eunicidae) and Leodice cf. torquata (Eunicidae). All had two pairs of pigmented eyes possessing typical adult structures: rhabdomeric PRCs (rPRC), pigmented supportive cells (PSCs) and additional cell types (in some species). The PSCs formed a shading pigment cup housing the sensory processes of the PRCs in everse orientation. Both Eunicidae species examined possess a lens-like structure formed by extensions of the PSCs as typical for members of Errantia, suggesting that lens-like structures formed by PSC processes were acquired in the stem lineage of Errantia and represent an autapomorphy of this clade. Our observations provide further evidence for the presence of two pairs of adult eyes in the ground pattern of Amphinomida/Sipuncula and Pleistoannelida.