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.

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.

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.

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.

Dynamics of oogenesis in ghost shrimp Callichirus major (Crustacea: Axiidea): a morphofunctional and histochemical study.

Callichirus major, popularly known as ghost shrimp, is a species of great importance in the fishing industry, because of its use as live bait. This study aimed to describe the different stages of the developing ovaries in C. major. Shrimps were collected along the Corujão beach, Piuma, Brazil (20°50'41.6"S 40°44'15.7"W), and the gonads were dissected for histological and histochemical analysis. The ovary consists of two elongated filaments covered by a connective tissue that divides the organ into lobules, where somatic and germ cells are found. It was possible to classify five types of germ cells: Oogonia (Oog), previtellogenic oocyte (Oc1), early vitellogenic oocyte (Oc2), late vitellogenic oocyte (Oc3) and mature oocyte (Oc4) based on their vitellogenic stage, cytoplasmic, nuclear and morphometric characteristics. The histochemical analysis demonstrated an intense reaction for proteins and polysaccharides in peripheral cytoplasm of Oc3 comparing to others cell types. According to size, volume, color intensity and distribution of oocyte types the gonads were classified into: immature, developing, developed and spent, being in females at this last stage, observed empty follicles and oocytes in reabsorption process. During oogenesis was observed a gradual increase in cytoplasmic acidophilia due to accumulation of yolk granules and the intense histochemical reaction in periphery of Oc3, which indicate the beginning of an extravitellogenic source of nutrients. Based on the microscopic analysis of the vitellogenesis, shrimp C. major showed the initial short phase of oocyte growth following with a fast vitellogenic cycle.

Neural organization of afferent pathways from the stomatopod compound eye.

Crustaceans and insects share many similarities of brain organization suggesting that their common ancestor possessed some components of those shared features. Stomatopods (mantis shrimps) are basal eumalacostracan crustaceans famous for their elaborate visual system, the most complex of which possesses 12 types of color photoreceptors and the ability to detect both linearly and circularly polarized light. Here, using a palette of histological methods we describe neurons and their neuropils most immediately associated with the stomatopod retina. We first provide a general overview of the major neuropil structures in the eyestalks lateral protocerebrum, with respect to the optical pathways originating from the six rows of specialized ommatidia in the stomatopod's eye, termed the midband. We then focus on the structure and neuronal types of the lamina, the first optic neuropil in the stomatopod visual system. Using Golgi impregnations to resolve single neurons we identify cells in different parts of the lamina corresponding to the three different regions of the stomatopod eye (midband and the upper and lower eye halves). While the optic cartridges relating to the spectral and polarization sensitive midband ommatidia show some specializations not found in the lamina serving the upper and lower eye halves, the general morphology of the midband lamina reflects cell types elsewhere in the lamina and cell types described for other species of Eumalacostraca.

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.

Sloppy morphological tuning in identified neurons of the crustacean stomatogastric ganglion.

Neuronal physiology depends on a neuron's ion channel composition and unique morphology. Variable ion channel compositions can produce similar neuronal physiologies across animals. Less is known regarding the morphological precision required to produce reliable neuronal physiology. Theoretical studies suggest that moraphology is tightly tuned to minimize wiring and conduction delay of synaptic events. We utilize high-resolution confocal microscopy and custom computational tools to characterize the morphologies of four neuron types in the stomatogastric ganglion (STG) of the crab Cancer borealis. Macroscopic branching patterns and fine cable properties are variable within and across neuron types. We compare these neuronal structures to synthetic minimal spanning neurite trees constrained by a wiring cost equation and find that STG neurons do not adhere to prevailing hypotheses regarding wiring optimization principles. In this highly modulated and oscillating circuit, neuronal structures appear to be governed by a space-filling mechanism that outweighs the cost of inefficient wiring.

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 involvement of MiR-1-clathrin pathway in the regulation of phagocytosis.

Phagocytosis, one of the most powerful immune responses, is a complicated process regulated by many factors. However the regulation of phagocytosis mediated by microRNAs has not been extensively investigated. To address this issue, the regulation of phagocytosis by miR-1 was characterized in this study. The results showed that miR-1 played an important role in the phagocytosis regulation in shrimp in vivo. The sequence analysis indicated that miR-1 was highly conserved from invertebrates to mammals, suggesting that miR-1 might share the similar or same functions in phagocytosis of shrimp hemocytes and mammalian macrophages. The data presented that miR-1 was significantly downregulated in cancerous macrophage RAW264.7 cells compared with those in the isolated murine macrophage and in the immortalized macrophage ANA-1. The findings showed that miR-1 had a great effect on the regulation of phagocytosis in cancerous macrophage by the inhibition of clathrin heavy chain 1 (CLTC1) gene. Therefore our study presented a novel miR-1-mediated regulation of phagocytosis both in invertebrate and in vertebrate.

The ovary structure and oogenesis in the basal crustaceans and hexapods. Possible phylogenetic significance.

Recent large-scale phylogenetic analyses of exclusively molecular or combined molecular and morphological characters support a close relationship between Crustacea and Hexapoda. The growing consensus on this phylogenetic link is reflected in uniting both taxa under the name Pancrustacea or Tetraconata. Several recent molecular phylogenies have also indicated that the monophyletic hexapods should be nested within paraphyletic crustaceans. However, it is still contentious exactly which crustacean taxon is the sister group to Hexapoda. Among the favored candidates are Branchiopoda, Malacostraca, Remipedia and Xenocarida (Remipedia + Cephalocarida). In this context, we review morphological and ultrastructural features of the ovary architecture and oogenesis in these crustacean groups in search of traits potentially suitable for phylogenetic considerations. We have identified a suite of morphological characters which may prove useful in further comparative studies.

The ventral nerve cord in Cephalocarida (Crustacea): new insights into the ground pattern of Tetraconata.

Cephalocarida are Crustacea with many anatomical features that have been interpreted as plesiomorphic with respect to crustaceans or Tetraconata. While the ventral nerve cord (VNC) has been investigated in many other arthropods to address phylogenetic and evolutionary questions, the few studies that exist on the cephalocarid VNC date back 20 years, and data pertaining to neuroactive substances in particular are too sparse for comparison. We reinvestigated the VNC of adult Hutchinsoniella macracantha in detail, combining immunolabeling (tubulin, serotonin, RFamide, histamine) and nuclear stains with confocal laser microscopy, complemented by 3D-reconstructions based on serial semithin sections. The subesophageal ganglion in Cephalocarida comprises three segmental neuromeres (Md, Mx1, Mx2), while a separate ganglion occurs in all thoracic segments and abdominal segments 1-8. Abdominal segments 9 and 10 and the telson are free of ganglia. The maxillar neuromere and the thoracic ganglia correspond closely in their limb innervation pattern, their pattern of mostly four segmental commissures and in displaying up to six individually identified serotonin-like immunoreactive neurons per body side, which exceeds the number found in most other tetraconates. Only two commissures and two serotonin-like immunoreactive neurons per side are present in abdominal ganglia. The stomatogastric nervous system in H. macracantha corresponds to that in other crustaceans and includes, among other structures, a pair of lateral neurite bundles. These innervate the gut as well as various trunk muscles and are, uniquely, linked to the unpaired median neurite bundle. We propose that most features of the cephalocarid ventral nerve cord (VNC) are plesiomorphic with respect to the tetraconate ground pattern. Further, we suggest that this ground pattern includes more serotonin-like neurons than hitherto assumed, and argue that a sister-group relationship between Cephalocarida and Remipedia, as favored by recent molecular analyses, finds no neuroanatomical support.

Larval development of Japanese "conchostracans": Part 3, larval development of Lynceus biformis (crustacea, branchiopoda, laevicaudata) based on scanning electron microscopy and fluorescence microscopy.

For comparison with the remarkable larvae of the laevicaudatan (clam shrimp) Lynceus brachyurus, a basic description of the larval sequence of another laevicaudatan branchiopod, the Japanese Lynceus biformis, is provided. Four larval stages have been identified, ranging in size from 258 to 560 µm in length. The first stage has no flattened dorsal shield, in contrast to the three following stages, in which such a shield is present. During development, the only significant changes to the naupliar appendages occur in the antenna at the molt from stage 1 to 2, with the addition of a fourth apical seta to the endopod and a change in the form of the naupliar process, used for food manipulation, from a long, unbranched, pointed spine to a bifid structure. In addition, buds of trunk limbs (five pairs) first appear externally in stage 4 but can be recognized through the cuticle in the previous stage. The larval sequence and larval morphology of L. biformis differ from those of L. brachyurus in at least two respects. L. brachyurus has a dorsal shield in the earliest known stages, but such a shield is lacking in the first stage of L. biformis. Another difference is that L. brachyurus has a huge, flattened, kidney-shaped labrum, whereas that of L. biformis is smaller and bears four robust, denticulate spines on the distal margin. Based on out-group comparison, the morphology of L. biformis, at least in these respects, is likely to represent the ancestral morphology. Despite the partly peculiar morphology of the larvae of Lynceus species, they share many similarities with other branchiopod larvae, at least two of which, the naupliar swimming/feeding apparatus and the mode of development of the trunk limbs, could be considered synapomorphies for the Branchiopoda.

Immunolocalization of estrogen receptor α in Neomysis japonica oocytes and follicle cells during ovarian development.

Estrogen induces oocytes development and vitellogenesis in crustacean by interacting with estrogen receptor (ER) subtypes. In the present study, we detect for the first time the ERα in oocytes and follicle cells and hepatopancreas cells of mysis by immunohistochemistry using a specific ERα antibody. ERα was mainly localized in the nuclei of oocytes and follicle cells, while mainly detected in nuclei of oogonia (OG), previtellogenic oocyte (PR) and endogenous vitellogenic oocyte (EN) at previtellogenic and early vitellogenic stage (I-early III). Follicle cells in all stages of ovary (all vitellogenic stages) showed strong ERα positive reaction, and they were able to gradually move to oocytes during the development of oocytes. In addition, ERα was also localized in the nuclei and cytoplasm of four hepatopancreas cells (including E-, R-, F- and B-cell) in all ovary stages. These findings suggest, for the first time to our knowledge, that there could be a close link between oogenesis, follicle cells, hepatopancreas cells and endocrine regulation, and estrogens might be involved in the regulation of oocytes at early ovarian stage in mysis.