Impact of Initial Feeding and Molting on Tachypleus tridentatus Gut Microbiota.

Tri-spine horseshoe crabs (HSCs) Tachypleus tridentatus have been facing the threat of population depletion for decades, and the physiology and microbiology of their early life stages are lacking. To explore what directs the change of juvenile T. tridentatus gut microbiota and how gut microbiota change, by using 16S rRNA sequencing of gut samples we detected the intestinal microbiome of juvenile HSCs and compared the impact of initial molting and initial feeding, as well as the effect of environment. Results showed that the predominant phyla in the gut microbial community of juvenile HSCs are Proteobacteria and Bacteroidetes. The richness and diversity of intestinal microbes greatly decreased after initial molting. Microbial-mediated functions predicted by PICRUSt showed that "Signal Transduction", "Cellular Processes and Signaling", "Infective Diseases" and "Digestive System" pathways significantly increased in 2nd instars. As for the effect of environment, the connection between living environment and the intestinal microbiome started to manifest after initial molting. Unexpectedly, initial feeding treatment slightly affected the intestinal microbiome of T. tridentatus in the early life stage, whereas the effect of initial molting was significant. The present study provided the first insight into the gut microbiota of T. tridentatus, and the findings led a new sight to explain what guide the change of gut microbiota.

Immune Responses to Gram-Negative Bacteria in Hemolymph of the Chinese Horseshoe Crab, Tachypleus tridentatus.

Chinese horseshoe crab, Tachypleus tridentatus, is an ancient marine arthropod with a long evolutionary history. As a kind of living fossil species, the pathogen defenses of horseshoe crabs entirely depend on the innate immune system. Although, there are abundant immune molecules found in the horseshoe crab hemolymph, the biological mechanisms underlying their abilities of distinguishing and defending against invading microbes are still unclear. In this study, we used high-throughput sequencing at mRNA and protein levels and bioinformatics analysis methods to systematically analyze the innate immune response to Gram-negative bacteria in hemolymph of Chinese horseshoe crab. These results showed that many genes in the complement and coagulation cascades, Toll, NF-κB, C-type lectin receptor, JAK-STAT, and MAPK signaling pathways, and antimicrobial substances were activated at 12 and 24 h post-infection, suggesting that Gram-negative bacteria could activate the hemolymph coagulation cascade and antibacterial substances release via the above pathways. In addition, we conjectured that Toll and NF-κB signaling pathway were most likely to participate in the immune response to Gram-negative bacteria in hemolymph of horseshoe crab through an integral signal cascade. These findings will provide a useful reference for exploring the ancient original innate immune mechanism.

Fusarium solani species complex associated with carapace lesions and branchitis in captive American horseshoe crabs Limulus polyphemus.

Captive American horseshoe crabs Limulus polyphemus housed at the National Aquarium presented with a variety of shell and gill lesions over a 3 yr period. Carapace lesions were located on both the dorsal and ventral prosoma and opisthosoma and included multifocal circular areas of tan discoloration, ulcerations, and/or pitting lesions, extending from superficial to full thickness. Gill lesions involved both the book gill cover (operculum) and individual book gill leaflets and included multifocal circular areas of tan discoloration, tan to off-white opaque proliferative lesions, and/or areas of black discoloration. Histopathology revealed fungal hyphae, with variable morphology throughout the thickened and irregular cuticle of the carapace and occasionally penetrating into subcuticular tissues, with associated amebocytic inflammation. Book gill leaflets were infiltrated by fungal hyphae and contained necrotic debris and amebocytes. Thirty-eight of 39 animals (97%) evaluated via histopathological examination had intralesional fungal hyphae. Fungal cultures of carapace and gill lesions were attempted in 26 tissue samples from 15 individuals and were positive in 13 samples (50%), with 10 cultures (77%) yielding identification to genus. Fusarium sp. was identified in 8 of the 10 cultures (80%) via culture morphology. The Fusarium solani species complex was confirmed in 6 of these 8 (75%) via polymerase chain reaction amplification of 2 different ribosomal-specific sequences of isolated fungal DNA. Ante-mortem systemic and topical treatments were performed on some affected individuals, but no appreciable change in lesions was observed. Mycotic dermatitis and branchitis are serious health issues for captive American horseshoe crabs.

A putative link between phagocytosis-induced apoptosis and hemocyanin-derived phenoloxidase activation.

Apoptosis and phagocytosis are crucial processes required for developmental morphogenesis, pathogen deterrence and immunomodulation in metazoans. We present data showing that amebocytes of the chelicerate, Limulus polyphemus, undergo phagocytosis-induced cell death after ingesting spores of the fungus, Beauveria bassiana, in vitro. The observed biochemical and morphological modifications associated with dying amebocytes are congruent with the hallmarks of apoptosis, including: extracellularisation of phosphatidylserine, intranucleosomal DNA fragmentation and an increase in caspase 3/7-like activities. Previous studies have demonstrated that phosphatidylserine is a putative endogenous activator of hemocyanin-derived phenoloxidase, inducing conformational changes that permit phenolic substrate access to the active site. Here, we observed extracellular hemocyanin-derived phenoloxidase activity levels increase in the presence of apoptotic amebocytes. Enzyme activity induced by phosphatidylserine or apoptotic amebocytes was reduced completely upon incubation with the phosphatidylserine binding protein, annexin V. We propose that phosphatidylserine redistributed to the outer plasma membrane of amebocytes undergoing phagocytosis-induced apoptosis could interact with hemocyanin, thus facilitating its conversion into a phenoloxidase-like enzyme, during immune challenge.

Microbe-specific C3b deposition in the horseshoe crab complement system in a C2/factor B-dependent or -independent manner.

Complement C3 plays an essential role in the opsonization of pathogens in the mammalian complement system, whereas the molecular mechanism underlying C3 activation in invertebrates remains unknown. To understand the molecular mechanism of C3b deposition on microbes, we characterized two types of C2/factor B homologs (designated TtC2/Bf-1 and TtC2/Bf-2) identified from the horseshoe crab Tachypleus tridentatus. Although the domain architectures of TtC2/Bf-1 and TtC2/Bf-2 were identical to those of mammalian homologs, they contained five-repeated and seven-repeated complement control protein domains at their N-terminal regions, respectively. TtC2/Bf-1 and TtC2/Bf-2 were synthesized and glycosylated in hemocytes and secreted to hemolymph plasma, which existed in a complex with C3 (TtC3), and their activation by microbes was absolutely Mg(2+)-dependent. Flow cytometric analysis revealed that TtC3b deposition was Mg(2+)-dependent on Gram-positive bacteria or fungi, but not on Gram-negative bacteria. Moreover, this analysis demonstrated that Ca(2+)-dependent lectins (C-reactive protein-1 and tachylectin-5A) were required for TtC3b deposition on Gram-positive bacteria, and that a Ca(2+)-independent lectin (Tachypleus plasma lectin-1) was definitely indispensable for TtC3b deposition on fungi. In contrast, a horseshoe crab lipopolysaccharide-sensitive protease factor C was necessary and sufficient to deposit TtC3b on Gram-negative bacteria. We conclude that plasma lectins and factor C play key roles in microbe-specific TtC3b deposition in a C2/factor B-dependent or -independent manner.

A portable test system for determination of bacterial endotoxins in 18F-FDG, 99mTc, and lyophilized reagents for labeling with 99mTc.

UNLABELLED: A rapid quantitative kinetic chromogenic test in an automated portable test system has been developed for in-process and end-product determination of bacterial endotoxins in water using the Limulus amebocyte lysate. The aim of this work was to validate the method for (18)F-FDG, (99m)Tc, and the lyophilized reagents methylene diphosphonic acid (MDP) and pyrophosphate for labeling with (99m)Tc radiopharmaceuticals with no interfering factors. METHODS: Experiments were performed on 3 consecutive batches of (18)F-FDG, (99m)Tc, MDP, and pyrophosphate produced at the Nuclear Energy and Research Institute of São Paulo, Brazil, using a portable test system. The maximum valid dilution (=500) was calculated to establish the extent of dilution to avoid interfering test conditions. RESULTS: Better results were obtained above a 1:5 dilution factor for (18)F-FDG and (99m)Tc, 1:20 for MDP, and 1:100 for pyrophosphate. The requirements of the test were satisfied (R ≤ 0.980, recovery of product positive control between 50% and 200%, and coefficient variation of samples < 25%), and the endotoxin concentration was lower than the lowest concentration of the standard curve (0.05 endotoxin unit mL(-1)) and therefore less than the established limit in pharmacopoeias. CONCLUSION: The portable test system is a rapid, simple, and accurate technique using the quantitative kinetic chromogenic method for bacterial endotoxin determination. For this reason, the test is practical for radiopharmaceutical uses and tends to be the method of choice for the pyrogen test. For (18)F-FDG, (99m)Tc, MDP, and pyrophosphate, the validation was successfully performed.

A novel serine protease inhibitor acts as an immunomodulatory switch while maintaining homeostasis.

Serine protease cascades boost immune responses while maintaining homeostasis. These crucial actions are intricately regulated by cognate serine protease inhibitors. However, the mechanism underlying such a dynamic immunomodulation during acute phase infection remains obscure, particularly where the pathogen's serine protease adds a new challenge to the host. Here, we found that infection of horseshoe crab, Carcinoscorpius rotundicauda, induced reciprocal profiles of CrSPI (serine protease inhibitor) and CrFurin (serine protease) with respect to their transcription and protein activities. Using recombinant rCrSPI, we explored its inhibitory activity against various microbial proteases and found it most efficacious against a model serine protease, subtilisin A. rCrSPI inhibited subtilisin at Ki 10(-9)M with a molar ratio of 1 rCrSPI:2 subtilisin. The rCrSPI also inhibited plasma CrFurin, suppressed subtilisin-mediated activation of prophenoloxidase (PPO) and interacted with complement C3. Taken together, CrSPI acts as a key immunomodulatory 'on-off' switch in a 2-way regulation of serine protease microbial subtilisin and host serine proteases (CrFurin and CrC3), thereby controlling immune responses involving the complements and the PPO-mediated antimicrobial activities, while maintaining homeostasis.

SARM: a novel Toll-like receptor adaptor, is functionally conserved from arthropod to human.

Sterile-alpha and Armadillo motif containing protein (SARM) was recently identified as the fifth member of the Toll-like receptor (TLR) adaptor family. Whilst the Caenorhabditis elegans SARM homologue, TIR-1, is crucial for efficient immune responses against bacterial infections, human SARM was demonstrated to function as a specific inhibitor of TRIF-dependent TLR signaling. The opposing role of SARM in C. elegans and human is intriguing, prompting us to seek clarification on the enigmatic function of SARM in an ancient species which relies solely on innate immunity for survival. Here, we report the discovery of a primitive but functional SARM (CrSARM) in the immune defense of a "living fossil", the horseshoe crab, Carcinoscorpius rotundicauda. CrSARM shares numerous signature motifs and displays significant homology with vertebrate and invertebrate SARM homologues. CrSARM downregulates TRIF-dependent TLR signaling suggesting the conservation of SARM function from horseshoe crab to human. During infection by Pseudomonas aeruginosa, CrSARM is rapidly upregulated within 3h and strongly repressed at 6h, coinciding with the timing of bacterial clearance, thus demonstrating its dynamic role in innate immunity. Furthermore, yeast-two-hybrid screening revealed several potential interaction partners of CrSARM implying the role of SARM in downregulating TLR signaling events. Altogether, our study shows that, although C. elegans SARM upregulates immune signaling, its disparate role as a suppressor of TLR signaling, specifically via TRIF and not MyD88, is well-conserved from horseshoe crab to human.

Elucidating the function of an ancient NF-kappaB p100 homologue, CrRelish, in antibacterial defense.

The family of NF-kappaB transcription factors essentially regulates immune-related gene expression. Recently, we isolated and characterized the classical NF-kappaB/inhibitor kappaB (IkappaB) homologues from a "living fossil," the horseshoe crab, Carcinoscorpius rotundicauda. Interestingly, this ancient species also harbors another class I NF-kappaB p100 homologue, C. rotundicauda Relish (CrRelish). Similar to Drosophila Relish and the mammalian p100, CrRelish contains both the Rel-homology domains (RHD) and the IkappaB-like domain. In this study, we found that the RHD of CrRelish can recognize horseshoe crab and human kappaB response elements and activate the downstream reporter in vitro, thereby suggesting the evolutionary conservation of this molecule. Pseudomonas aeruginosa infection transcriptionally upregulates CrRelish, which exhibits a dynamic protein profile over the time course of infection. Surprisingly, secondary infection reinduced an upsurge in CrRelish protein expression to a level which overrode the protein degradation at 12 h postinfection. These observations strongly suggest the involvement of CrRelish in antibacterial defense. Secondary infection causes (i) the maintenance of a favorable expression-competent sequence context of the CrRelish gene and/or (ii) the derepression or stabilization of the CrRelish transcript resulting from the primary infection to enable the more rapid expression and accumulation of the CrRelish protein, reflecting apparent signal/immune priming in a repeated infection.

Recognition of pathogens and activation of immune responses in Drosophila and horseshoe crab innate immunity.

In innate immunity, pattern recognition receptors discriminate between self- and infectious non-self-matter. Mammalian homologs of the Drosophila Toll protein, which are collectively referred to as Toll-like receptors (TLRs), recognize pathogen-associated molecular patterns (PAMPs), including lipopolysaccharides (LPS) and lipoproteins, whereas the Drosophila Toll protein does not act as a PAMP receptor, but rather binds to Spätzle, an endogenous peptide. In Drosophila, innate immune surveillance is mediated by members of the peptidoglycan recognition protein (PGRP) family, which recognize diverse bacteria-derived peptidoglycans and initiate appropriate immune reactions including the release of antimicrobial peptides and the activation of the prophenoloxidase cascade, the latter effecting localized wound healing, melanization, and microbial phagocytosis. In the horseshoe crab, LPS induces hemocyte exocytotic degranulation, resulting in the secretion of various defense molecules, such as coagulation factors, antimicrobial peptides, and lectins. Recent studies have demonstrated that the zymogen form of the serine protease factor C, a major granular component of hemocyte, also exists on the hemocyte surface and functions as a biosensor for LPS. The proteolytic activity of activated factor C initiates hemocyte exocytosis via a G protein mediated signal transduction pathway. Furthermore, it has become clear that an endogenous mechanism for the feedback amplification of the innate immune response exists and is dependent upon a granular component of the horseshoe crab hemocyte.

Proteases and protease inhibitors: a balance of activities in host-pathogen interaction.

The immune system is the collection of effector molecules and cells of the host that act against invading parasites and their products. Secreted proteases serve important roles in parasitic metabolism and virulence and the several families of protein protease inhibitors of the plasma and blood cells play an important role in immunity by inactivating and clearing the protease virulence factors of parasites. The protease inhibitors are of two classes, the active-site inhibitors and the alpha2-macroglobulins. Inhibitors for the first class bind and inactivate the active site of the target protease. Proteins of the second class bind proteases by a unique molecular trap mechanism and deliver the bound protease to a receptor-mediated endocytic system for degradation in secondary lysosomes. Proteins of the alpha2-macroglobulin family are present in a variety of animal phyla, including the nematodes, arthropods, mollusks, echinoderms, urochordates, and vertebrates. A shared suite of unique functional characteristics have been documented for the alpha2-macroglobulins of vertebrates, arthropods, and mollusks. The alpha2-macroglobulins of nematodes, arthropods, mollusks, and vertebrates show significant sequence identity in key functional domains. Thus, the alpha2-macroglobulins comprise an evolutionarily conserved arm of the innate immune system with similar structure and function in animal phyla separated by 0.6 billion years of evolution.

Evidence for the ancient origin of the NF-kappaB/IkappaB cascade: its archaic role in pathogen infection and immunity.

The evolutionary conservation of the NF-kappaB transcription factors, from Drosophila to humans, underscores its pivotal role in immune response. Unexpectedly, the canonical NF-kappaB signaling pathway is not functional in the immune system of Caenorhabditis elegans. Therefore, the ancient origin of the NF-kappaB signaling pathway is still unknown. Here, we report the discovery and characterization of a primitive and functional NF-kappaB/IkappaB pathway in the immune defense of a "living fossil," the horseshoe crab, Carcinoscorpius rotundicauda. The ancient NF-kappaB/IkappaB homologues, CrNFkappaB, CrRelish, and CrIkappaB, share numerous signature motifs with their vertebrate orthologues. CrNFkappaB recognizes both horseshoe crab and mammalian kappaB response elements. CrIkappaB interacts with CrNFkappaB and inhibits its nuclear translocation and DNA-binding activity. The activation of the CrNFkappaB is autoregulated by a feedback mechanism mediated by CrIkappaB, the natural inhibitor of CrNFkappaB. We further show that Gram-negative bacteria infection causes rapid degradation of CrIkappaB and nuclear translocation of CrNFkappaB. Infection also leads to an increase in the kappaB-binding activity and up-regulation of immune-related gene expression, like inducible nitric oxide synthase and Factor C, an LPS-activated serine protease. Altogether, our study shows that, although absent in C. elegans, the NF-kappaB/IkappaB signaling cascade remains well conserved from horseshoe crab to humans, playing an archaic but fundamental role in regulating the expression of critical immune defense molecules.

Spatial and temporal coordination of expression of immune response genes during Pseudomonas infection of horseshoe crab, Carcinoscorpius rotundicauda.

Knowledge on how genes are turned on/off during infection and immunity is lacking. Here, we report the co-regulation of diverse clusters of functionally related immune response genes in a horseshoe crab, Carcinoscorpius rotundicauda. Expressed sequence tag (EST) clusters for frontline immune defense, cell signalling, apoptosis and stress response genes were expressed or repressed spatio-temporally during the acute phase of Pseudomonas infection. An infection time course monitored by virtual Northern evaluation indicates upregulation of genes in blood cells (amebocytes) at 3-h postinfection, whereas most of the hepatopancreas genes remained down regulated over 72 h of infection. Thus, the two tissues orchestrate a coordinated and timely response to infection. The hepatopancreas probably immuno-modulates the expression of other genes and serves as a reservoir for later response, if/when chronic infection ensues. On the other hand, being the first to encounter pathogens, we reasoned that amebocytes would respond acutely to infection. Besides acute transactivation of the immune genes, the amebocytes maintained morphological integrity, indicating their ability to synthesise and store/secrete the immune proteins and effectors to sustain the frontline innate immune defense, while simultaneously elicit complement-mediated phagocytosis of the invading pathogen. Our results show that the immune response against Pseudomonas infection is spatially and temporally coordinated.

Mineralization of soft-part anatomy and invading microbes in the horseshoe crab Mesolimulus from the Upper Jurassic Lagerstätte of Nusplingen, Germany.

A remarkable specimen of Mesolimulus from the Upper Jurassic (Kimmeridgian) of Nusplingen, Germany, preserves the musculature of the prosoma and associated microbes in three dimensions in calcium phosphate (apatite). The musculature of Mesolimulus conforms closely to that of modern horseshoe crabs. Associated with the muscles are patches of mineralized biofilm with spiral and coccoid forms. This discovery emphasizes the potential of soft-bodied fossils as a source for increasing our knowledge of the diversity of fossil microbes in particular settings.

The horseshoe crab: a model for gram-negative sepsis in marine organisms and humans.

The roles of the amebocyte in providing hemostasis and controlling infection, and its reaction to endotoxin, suggest that the response of platelets and the blood coagulation system in various mammals to gram-negative infection or endotoxin is an evolutionary remnant of this ancient mechanism. In humans, this mechanism occasionally subverts its presumed protective function by overresponding in a manner that results in pathophysiologic thrombosis or hemorrhage. (In this regard, it is interesting that human platelets are much more resistant to the effects of bacterial endotoxins than are other species.) Similarly, the rudimentary ability of mammalian platelets to phagocytose particles and kill bacteria may be another remnant of functions that are more important in amebocytes (or the thrombocytes of other invertebrates). Thus, these two cells, one from an ancient invertebrate and the other from mammals, have remarkably similar characteristics, although the relative importance of their various functions has changed as evolution has taken place. Nevertheless, after at least 400,000,000 years of evolution, coagulation and anti-bacterial mechanisms remain at least partially linked.

Presence of microorganisms in hemolymph of the horseshoe crab Limulus polyphemus.

Hemolymph samples obtained from Limulus polyphemus at the time of collection and after a 1-week holding period exhibited a significant increase in bacterial levels. No differences were observed in the ability of amoebocyte lysate, prepared from these same samples, to gel in the presence of lipopolysaccharide.