Exosomes drive ferroptosis by stimulating iron accumulation to inhibit bacterial infection in crustaceans.

Ferroptosis, characterized by iron-dependent cell death, has recently emerged as a critical defense mechanism against microbial infections. The present study aims to investigate the involvement of exosomes in the induction of ferroptosis and the inhibition of bacterial infection in crustaceans. Our findings provide compelling evidence for the pivotal role of exosomes in the immune response of crustaceans, wherein they facilitate intracellular iron accumulation and activate the ferroptotic pathways. Using RNA-seq and bioinformatic analysis, we demonstrate that cytochrome P450 (CYP) can effectively trigger ferroptosis. Moreover, by conducting an analysis of exosome cargo proteins, we have identified the participation of six-transmembrane epithelial antigen of prostate 4 in the regulation of hemocyte ferroptotic sensitivity. Subsequent functional investigations unveil that six-transmembrane epithelial antigen of prostate 4 enhances cellular Fe2+ levels, thereby triggering Fenton reactions and accelerating CYP-mediated lipid peroxidation, ultimately culminating in ferroptotic cell death. Additionally, the Fe2+-dependent CYP catalyzes the conversion of arachidonic acid into 20-hydroxyeicosatetraenoic acid, which activates the peroxisome proliferator-activated receptor. Consequently, the downstream target of peroxisome proliferator-activated receptor, cluster of differentiation 36, promotes intracellular fatty acid accumulation, lipid peroxidation, and ferroptosis. These significant findings shed light on the immune defense mechanisms employed by crustaceans and provide potential strategies for combating bacterial infections in this species.

CeLaVi: an interactive cell lineage visualization tool.

Recent innovations in genetics and imaging are providing the means to reconstruct cell lineages, either by tracking cell divisions using live microscopy, or by deducing the history of cells using molecular recorders. A cell lineage on its own, however, is simply a description of cell divisions as branching events. A major goal of current research is to integrate this description of cell relationships with information about the spatial distribution and identities of the cells those divisions produce. Visualizing, interpreting and exploring these complex data in an intuitive manner requires the development of new tools. Here we present CeLaVi, a web-based visualization tool that allows users to navigate and interact with a representation of cell lineages, whilst simultaneously visualizing the spatial distribution, identities and properties of cells. CeLaVi's principal functions include the ability to explore and manipulate the cell lineage tree; to visualise the spatial distribution of cell clones at different depths of the tree; to colour cells in the 3D viewer based on lineage relationships; to visualise various cell qualities on the 3D viewer (e.g. gene expression, cell type) and to annotate selected cells/clones. All these capabilities are demonstrated with four different example data sets. CeLaVi is available at http://www.celavi.pro.

Optic lobe organization in stomatopod crustacean species possessing different degrees of retinal complexity.

Stomatopod crustaceans possess tripartite compound eyes; upper and lower hemispheres are separated by an equatorial midband of several ommatidial rows. The organization of stomatopod retinas is well established, but their optic lobes have been studied less. We used histological staining, immunolabeling, and fluorescent tracer injections to compare optic lobes in two 6-row midband species, Neogonodactylus oerstedii and Pseudosquilla ciliata, to those in two 2-row midband species, Squilla empusa and Alima pacifica. Compared to the 6-row species, we found structural differences in all optic neuropils in both 2-row species. Photoreceptor axons from 2-row midband ommatidia supply two sets of lamina cartridges; however, conspicuous spaces lacking lamina cartridges are observed in locations corresponding to where the cartridges of the upper four ommatidial rows of 6-row species would exist. The tripartite arrangement and enlarged projections containing fibers associated with the two rows of midband ommatidia can be traced throughout the entire optic lobe. However, 2-row species lack some features of medullar and lobular neuropils in 6-row species. Our results support the hypothesis that 2-row midband species are derived from a 6-row ancestor, and suggest specializations in the medulla and lobula found solely in 6-row species are important for color and polarization analysis.

Neural architecture: from cells to circuits.

Circuit operations are determined jointly by the properties of the circuit elements and the properties of the connections among these elements. In the nervous system, neurons exhibit diverse morphologies and branching patterns, allowing rich compartmentalization within individual cells and complex synaptic interactions among groups of cells. In this review, we summarize work detailing how neuronal morphology impacts neural circuit function. In particular, we consider example neurons in the retina, cerebral cortex, and the stomatogastric ganglion of crustaceans. We also explore molecular coregulators of morphology and circuit function to begin bridging the gap between molecular and systems approaches. By identifying motifs in different systems, we move closer to understanding the structure-function relationships that are present in neural circuits.

Comparative genomic analysis of innate immunity reveals novel and conserved components in crustacean food crop species.

BACKGROUND: Growing global demands for crustacean food crop species have driven large investments in aquaculture research worldwide. However, large-scale production is susceptible to pathogen-mediated destruction particularly in developing economies. Thus, a thorough understanding of the immune system components of food crop species is imperative for research to combat pathogens. RESULTS: Through a comparative genomics approach utilising extant data from 55 species, we describe the innate immune system of the class Malacostraca, which includes all food crop species. We identify 7407 malacostracan genes from 39 gene families implicated in different aspects of host defence and demonstrate dynamic evolution of innate immunity components within this group. Malacostracans have achieved flexibility in recognising infectious agents through divergent evolution and expansion of pathogen recognition receptors genes. Antiviral RNAi, Toll and JAK-STAT signal transduction pathways have remained conserved within Malacostraca, although the Imd pathway appears to lack several key components. Immune effectors such as the antimicrobial peptides (AMPs) have unique evolutionary profiles, with many malacostracan AMPs not found in other arthropods. Lastly, we describe four putative novel immune gene families, potentially representing important evolutionary novelties of the malacostracan immune system. CONCLUSION: Our analyses across the broader Malacostraca have allowed us to not only draw analogies with other arthropods but also to identify evolutionary novelties in immune modulation components and form strong hypotheses as to when key pathways have evolved or diverged. This will serve as a key resource for future immunology research in crustacean food crops.

Removal of extracellular coat from giant sperm in female receptacle induces sperm motility in Mytilocypris mytiloides (Cyprididae, Ostracoda, Crustacea).

Previous studies of cypridoidean ostracods have noted that (1) their giant spermatozoa are immotile inside the male, (2) these spermatozoa are motile in the female seminal receptacle and (3) these receptacles are often filled with empty sperm coats. Such findings have led previous authors to hypothesize that sperm must shed their coats in the female receptacle to become motile. We present light and electron microscopy results and video recordings of mating experiments with virgin specimens of Mytilocypris mytiloides. We show that the empty sperm coats frequently found in the female receptacles are not the result of sperm molting but are the resistant inner coats of exhausted sperm not used for egg fertilization. In contrast, we show that an outer granular coating material is successively removed from the sperm while resident inside the female receptacles before first oviposition occurs. During this period, previously immotile sperm gain motility, showing strong movement shortly before first oviposition takes place. By correlation of these phenomena, we suggest that dissolution of the outer coat material is required for motility to develop.

Olfactory pathway in Xibalbanus tulumensis: remipedian hemiellipsoid body as homologue of hexapod mushroom body.

The Remipedia have been proposed to be the crustacean sister group of the Hexapoda. These blind cave animals heavily rely on their chemical sense and are thus rewarding subjects for the analysis of olfactory pathways. The evolution of these pathways as a character for arthropod phylogeny has recently received increasing attention. Here, we investigate the situation in Xibalbanus tulumensis by focal dye injections and immunolabelling of the catalytic subunit of the cAMP-dependent protein kinase (DC0), an enzyme particularly enriched in insect mushroom bodies. DC0 labelling of the hemiellipsoid body suggests its subdivision into a cap-like and a core neuropil. Immunofluorescence of the enzyme glutamic acid decarboxylase (GAD), which synthesizes γ-aminobutyric acid (GABA), has revealed a cluster of GABAergic interneurons in the hemiellipsoid body, reminiscent of the characteristic feedback neurons of the mushroom body. Thus, the hemiellipsoid body of Xibalbanus shares many of the characteristics of insect mushroom bodies. Nevertheless, the general neuroanatomy of the olfactory pathway in the Remipedia strongly corresponds to the malacostracan ground pattern. Given that the Remipedia are probably the sister group of the Hexapoda, the phylogenetic appearance of the typical neuropilar compartments in the insect mushroom body has to be assigned to the origins of the Hexapoda.

Genome size estimates for crustaceans using Feulgen image analysis densitometry of ethanol-preserved tissues.

Crustaceans are enormously diverse both phylogenetically and ecologically, but they remain substantially underrepresented in the existing genome size database. An expansion of this dataset could be facilitated if it were possible to obtain genome size estimates from ethanol-preserved specimens. In this study, two tests were performed in order to assess the reliability of genome size data generated using preserved material. First, the results of estimates based on flash-frozen versus ethanol-preserved material were compared across 37 species of crustaceans that differ widely in genome size. Second, a comparison was made of specimens from a single species that had been stored in ethanol for 1-14 years. In both cases, the use of gill tissue in Feulgen image analysis densitometry proved to be a very viable approach. This finding is of direct relevance to both new studies of field-collected crustaceans as well as potential studies based on existing collections.

The functional relationship between ectodermal and mesodermal segmentation in the crustacean, Parhyale hawaiensis.

In arthropods, annelids and chordates, segmentation of the body axis encompasses both ectodermal and mesodermal derivatives. In vertebrates, trunk mesoderm segments autonomously and induces segmental arrangement of the ectoderm-derived nervous system. In contrast, in the arthropod Drosophila melanogaster, the ectoderm segments autonomously and mesoderm segmentation is at least partially dependent on the ectoderm. While segmentation has been proposed to be a feature of the common ancestor of vertebrates and arthropods, considering vertebrates and Drosophila alone, it is impossible to conclude whether the ancestral primary segmented tissue was the ectoderm or the mesoderm. Furthermore, much of Drosophila segmentation occurs before gastrulation and thus may not accurately represent the mechanisms of segmentation in all arthropods. To better understand the relationship between segmented germ layers in arthropods, we asked whether segmentation is an intrinsic property of the ectoderm and/or the mesoderm in the crustacean Parhyale hawaiensis by ablating either the ectoderm or the mesoderm and then assaying for segmentation in the remaining tissue layer. We found that the ectoderm segments autonomously. However, mesoderm segmentation requires at least a permissive signal from the ectoderm. Although mesodermal stem cells undergo normal rounds of division in the absence of ectoderm, they do not migrate properly in respect to migration direction and distance. In addition, their progeny neither divide nor express the mesoderm segmentation markers Ph-twist and Ph-Even-skipped. As segmentation is ectoderm-dependent in both Parhyale and holometabola insects, we hypothesize that segmentation is primarily a property of the ectoderm in pancrustacea.

Silicon: The key element in early stages of biocalcification.

Biocalcification is a widespread process of forming hard tissues like bone and teeth in vertebrates. It is also a topic connecting life sciences and earth sciences: calcified skeletons and shells deposited as sediments represent the earth's fossil record and are of paramount interest for biogeochemists trying to get an insight into the past of our planet. This study reports on the role of silicon in the early biocalcification steps, where silicon and calcium were detected on the surface of cyanobacteria (initial stage of lacustrine calcite precipitation) and in crustacean cuticles. By using innovative methodological approaches of correlative microscopy (AFM in combination with analytical TEM: EFTEM, EELS) the chemical form of silicon in biocalcifying matrices and organic-inorganic particles is determined. Previously, silicon was reported to be localized in active growth areas in the young bone of vertebrates. We have found evidence that biocalcification in evolutionarily distant organisms involves very similar initial phases with silicon as a key element at the organic-inorganic interface.

Mesoderm and ectoderm lineages in the crustacean Parhyale hawaiensis display intra-germ layer compensation.

In Parhyale hawaiensis, the first three divisions are holoblastic and asymmetric, resulting in an embryo comprised of eight cells-four macromeres and four micromeres. Lineage studies performed at this stage demonstrate that the progeny of each cell contribute to specific portions of different germ layers. However, it is not known if this lineage pattern means a given blastomere is committed to its specific fate, indicative of mosaic development, or if regulation can occur between blastomere progeny so that the loss of a blastomere could be compensated for during development. Furthermore, if compensation occurs, what would be the source of such replacement? To investigate these possibilities, we performed ablation experiments at the eight-cell stage. We find that loss of blastomeres results in compensation. To determine the compensation pattern, we combined ablation and cell lineage tracing to reveal that progeny of mesoderm and ectoderm producing blastomeres display intra-germ layer compensation. Furthermore, by ablating lineages later in development, we identify a key interval between gastrulation and germband elongation after which compensation no longer occurs. Our results suggest that Parhyale possesses a mechanism to assess the status of mesoderm and ectoderm formation and alter development to replace the missing portions of these lineages.

Mechanisms of apoptosis in Crustacea: What conditions induce versus suppress cell death?

Arthropoda is the largest of all animal phyla and includes about 90% of extant species. Our knowledge about regulation of apoptosis in this phylum is largely based on findings for the fruit fly Drosophila melanogaster. Recent work with crustaceans shows that apoptotic proteins, and presumably mechanisms of cell death regulation, are more diverse in arthropods than appreciated based solely on the excellent work with fruit flies. Crustacean homologs exist for many major proteins in the apoptotic networks of mammals and D. melanogaster, but integration of these proteins into the physiology and pathophysiology of crustaceans is far from complete. Whether apoptosis in crustaceans is mainly transcriptionally regulated as in D. melanogaster (e.g., RHG 'killer' proteins), or rather is controlled by pro- and anti-apoptotic Bcl-2 family proteins as in vertebrates needs to be clarified. Some phenomena like the calcium-induced opening of the mitochondrial permeability transition pore (MPTP) are apparently lacking in crustaceans and may represent a vertebrate invention. We speculate that differences in regulation of the intrinsic pathway of crustacean apoptosis might represent a prerequisite for some species to survive harsh environmental insults. Pro-apoptotic stimuli described for crustaceans include UV radiation, environmental toxins, and a diatom-produced chemical that promotes apoptosis in offspring of a copepod. Mechanisms that serve to depress apoptosis include the inhibition of caspase activity by high potassium in energetically healthy cells, alterations in nucleotide abundance during energy-limited states like diapause and anoxia, resistance to opening of the calcium-induced MPTP, and viral accommodation during persistent viral infection. Characterization of the players, pathways, and their significance in the core machinery of crustacean apoptosis is revealing new insights for the field of cell death.

Cleavage and gastrulation in the Kuruma shrimp Penaeus (Marsupenaeus) japonicus (Bate): a revised cell lineage and identification of a presumptive germ cell marker.

A previous study suggested that mesendoderm (ME) cell arrest occurred at the 64-cell stage and a ring of eight presumptive naupliar mesoderm cells or crown cells surrounded the blastopore in the Kuruma shrimp Penaeus (Marsupenaeus) japonicus. Since this varied from the pattern observed in other penaeoidean shrimp, cleavage and gastrulation was re-examined in P. japonicus using the nucleic acid stain Sytox Green and confocal microscopy. In contrast to the earlier study, cleavage and gastrulation followed the pattern observed in other penaeoidean shrimp. The ME cells arrested at the 32-cell stage, ingressed into the blastocoel, and resumed division after a three cell cycle delay. Nine naupliar mesoderm or crown cells surrounded the blastopore and their descendants invaginated during gastrulation. An intracellular body (ICB) was detected by Sytox Green and SYTO RNASelect staining to be segregated to one ME cell in P. japonicus, as described previously in Penaeus monodon. Staining of the ICB was eliminated by pre-treatment with RNase but not DNase. The ICB was also found in two other penaeoidean shrimp, Penaeus vannamei (Family Penaeidae) and Sicyonia ingentis (Family Sicyoniidae). The results support the hypothesis that the ICB is a germ granule found in the Dendrobranchiata.

Geometry and dynamics of activity-dependent homeostatic regulation in neurons.

To maintain activity in a functional range, neurons constantly adjust membrane excitability to changing intra- and extracellular conditions. Such activity-dependent homeostatic regulation (ADHR) is critical for normal processing of the nervous system and avoiding pathological conditions. Here, we posed a homeostatic regulation problem for the classical Morris-Lecar (ML) model. The problem was motivated by the phenomenon of the functional recovery of stomatogastric neurons in crustaceans in the absence of neuromodulation. In our study, the regulation of the ionic conductances in the ML model depended on the calcium current or the intracellular calcium concentration. We found an asymptotic solution to the problem under the assumption of slow regulation. The solution provides a full account of the regulation in the case of correlated or anticorrelated changes of the maximal conductances of the calcium and potassium currents. In particular, the solution shows how the target and parameters of the regulation determine which perturbations of the conductances can be compensated by the ADHR. In some cases, the sets of compensated initial perturbations are not convex. On the basis of our analysis we formulated specific questions for subsequent experimental and theoretical studies of ADHR.

Hemocyte-Mediated Phagocytosis in Crustaceans.

Phagocytosis is an ancient, highly conserved process in all multicellular organisms, through which the host can protect itself against invading microorganisms and environmental particles, as well as remove self-apoptotic cells/cell debris to maintain tissue homeostasis. In crustacean, phagocytosis by hemocyte has also been well-recognized as a crucial defense mechanism for the host against infectious agents such as bacteria and viruses. In this review, we summarized the current knowledge of hemocyte-mediated phagocytosis, in particular focusing on the related receptors for recognition and internalization of pathogens as well as the downstream signal pathways and intracellular regulators involved in the process of hemocyte phagocytosis. We attempted to gain a deeper understanding of the phagocytic mechanism of different hemocytes and their contribution to the host defense immunity in crustaceans.

Establishment of long-term ostracod epidermal culture.

Primary crustacean cell culture was introduced in the 1960s, but to date limited cell lines have been established. Skogsbergia lerneri is a myodocopid ostracod, which has a body enclosed within a thin, durable, transparent bivalved carapace, through which the eye can see. The epidermal layer lines the inner surface of the carapace and is responsible for carapace synthesis. The purpose of the present study was to develop an in vitro epidermal tissue and cell culture method for S. lerneri. First, an optimal environment for the viability of this epidermal tissue was ascertained, while maintaining its cell proliferative capacity. Next, a microdissection technique to remove the epidermal layer for explant culture was established and finally, a cell dissociation method for epidermal cell culture was determined. Maintenance of sterility, cell viability and proliferation were key throughout these processes. This novel approach for viable S. lerneri epidermal tissue and cell culture augments our understanding of crustacean cell biology and the complex biosynthesis of the ostracod carapace. In addition, these techniques have great potential in the fields of biomaterial manufacture, the military and fisheries, for example, in vitro toxicity testing.

Cardiac muscle of the horseshoe crab, Limulus polyphemus. I. Ultrastructure.

The fine structure of the cardiac muscle of the horseshoe crab, Limulus polyphemus, has been studied with respect to the organization of its contractile material, and the structure of its organelles and the cell junctions. Longitudinal sections show long sarcomeres (5.37 micro at L(max)), wide A bands (2.7 micro), irregular Z lines, no M line, and no apparent H zone. Transverse sections through the S zone of the A band show that each thick filament is ca. 180 A in diameter, is circular in profile with a center of low density, and is surrounded by an orbit of 9-12 thin filaments, each 60 A in diameter. Thick filaments are confined to the A band: thin filaments originate at the Z band, extend through the I band, and pass into the A band between the thick filaments. The sarcolemmal surface area is increased significantly by intercellular clefts. Extending into the fiber from these clefts and from the sarcolemma, T tubules pass into the fiber at the A-I level. Each fibril is enveloped by a profuse membranous covering of sarcoplasmic reticulum (SR). Sacculations of the SR occur at the A-I boundary where they make diadic contact with longitudinal branches of the T system. These branches also extend toward the Z, enlarge at the Z line, and pass into the next sarcomere. Infrequently noted were intercalated discs possessing terminal insertion and desmosome modifications, but lacking close junctions (fasciae occludentes). These structural details are compared with those of mammalian cardiac and invertebrate muscles.

Cell junctions in ommatidia of Limulus.

The intercellular relationships in the ommatidia of the lateral eye of Limulus have been investigated. The distal process of the eccentric cell gives origin to microvilli which interdigitate with the microvilli of the retinular cells. Therefore, both types of visual cells contribute to form the rhabdom and may have an analogous photoreceptor function. Quintuple-layered junctions are found within the rhabdom at the lines of demarcation between adjoining microvilli, whether the microvilli originate from a single retinular cell, from two adjacent retinular cells, or from a retinular cell and the eccentric cell. Furthermore, quintuple-layered junctions between the eccentric cell and the tips of the microvilli of the retinular cells occur at the boundary between the distal process and the rhabdom. These findings are interpreted to indicate that the rhabdom provides an extensive electrotonic junction relating retinular cells to one another and to the eccentric cell. Quintuple-layered junctions between glial and visual cells, as well as other structural features of the ommatidial cells, are also described.

Ultrastructure of the prawn nerve sheaths. Role of fixative and osmotic pressure in vesiculation of thin cytoplasmic laminae.

The sheaths from freshly teased nerve fibers of the prawn exhibit a positive radial birefringence, consistent with their EM appearance as highly organized laminated structures composed of numerous thin cytoplasmic sheets or laminae bordered by unit membranes and arranged concentrically around the axon. The closely apposed membranes in these sheaths are fragile and often break down into rows of vesicles during fixation. Desmosome-like attachment zones occur in many regions of the sheath. The membranes within these zones resist vesiculation and thereby provide a "control" region for relating the type of vesicles formed in the fragile portions of the sheaths to the specific fixation conditions. It is proposed that during fixation the production of artifactual vesicles is governed by an interplay of three factors: (a) direct chemical action of the fixative on the polar strata of adjacent unit membranes, (b) osmotic forces applied to membranes during fixation, and (c) the pre-existing natural relations between adjacent membranes. It is found that permanganate best preserves the continuity of the membranes but will still produce vesicles if the fixative exerts severe osmotic forces. These results support other reports (19) of the importance of comparing tissues fixed by complementary procedures so that systematic artifacts will not be described as characteristic of the natural state.