Comparative transcriptome profiling of horseshoe crab Tachypleus gigas hemocytes in response to lipopolysaccharides.

Horseshoe crabs (HSCs) are living fossil species of marine arthropods with a long evolutionary history spanning approximately 500 million years. Their survival is helped by their innate immune system that comprises cellular and humoral immune components to protect them against invading pathogens. To help understand the genetic mechanisms involved, the present study utilised the Illumina HiSeq platform to perform transcriptomic analysis of hemocytes from the HSC, Tachypleus gigas, that were challenged with lipopolysaccharides (LPS). The high-throughput sequencing resulted in 352,077,208 and 386,749,136 raw reads corresponding to 282,490,910 and 305,709,830 high-quality mappable reads for the control and LPS-treated hemocyte samples, respectively. Based on the log-fold change of > 0.3 or < -0.3, 1338 genes were significantly upregulated and 215 genes were significantly downregulated following LPS stimulation. The differentially expressed genes (DEGs) were further identified to be associated with multiple pathways such as those related to immune defence, stress response, cytoskeleton function and signal transduction. This study provides insights into the underlying molecular and regulatory mechanisms in hemocytes exposed to LPS, which has relevance for the study of the immune response of HSCs to infection.

Enhanced immunity and hemocytes proliferation by three immunostimulants in tri-spine horseshoe crab Tachypleus tridentatus.

Tachypleus amebocyte lysate (TAL) is crucial in medical testing, but its industry in China has been restricted due to the decline of horseshoe crab population in recent years. Exploring methods of enhancing immunity and rapid hemocytes proliferation is urgent for the industrial horseshoe crab culture. In this study, ß-glucan (G), peptidoglycan (P), and squalene (S) were injected to horseshoe crabs at two concentrations (5 and 10 mg/kg), in order to compare their effects on total hemocyte count (THC), reactive oxygen species (ROS), and non-specific immune enzyme activities. Results showed that the THC, superoxide dismutase (SOD), catalase (CAT), and total antioxidant capacity (T-AOC) were significantly increased by three immunostimulants at different points of time; ROS was significantly increased except at two squalene groups; lysozyme (LZM) and alkaline phosphatase (AKP) activity were increased except at low dose (5 mg/kg) squalene group; malondialdehyde (MDA) activity was decreased in all treatments; and hemocyanin concentration (HC) changed little during the experiment. At the 48th hour, THC, ROS, SOD, CAT, T-AOC, LZM, and AKP activities were significantly higher in the two peptidoglycan groups than those in the control group; the low dose ß-glucan and squalene groups showed significantly higher SOD and CAT, but their THC and AKP were not significantly different from those of the control group. In general, all three immunostimulants stimulated the hemolymph parameters of horseshoe crabs, notably, peptidoglycan could significantly increase the THC and enzyme activities, suggesting that peptidoglycan can be developed as an efficient immunostimulant for horseshoe crabs.

2-PHENOXYETHANOL (2-PE) AND TRICAINE METHANESULFONATE (MS-222) IMMERSION ANESTHESIA OF AMERICAN HORSESHOE CRABS (LIMULUS POLYPHEMUS).

Despite extensive literature examining American horseshoe crab physiology, there are comparatively few publications addressing their medical care. Establishing anesthesia protocols for horseshoe crabs is integral to limiting the potential stress and pain associated with invasive procedures and for advancing euthanasia techniques. The objective of this study was to compare the effects of two immersion anesthetics, tricaine methanesulfonate (MS-222) at 1 g/L (buffered with sodium carbonate) and 2-phenoxyethanol (2-PE) at 2 mL/L, on horseshoe crabs. Twenty horseshoe crabs were assigned to one of two anesthetic treatment groups and individually anesthetized in natural seawater. Water quality, cardiac contractility, and hemolymph gas analytes were measured prior to anesthesia and at 30 min Animals were monitored via heart rate, gilling rate, and sedation score every 5 min until recovered. Transcarapacial ultrasonography was used to obtain heart rate, gilling rate, and percent fractional shortening. Light or surgical anesthesia was produced in 10/10 animals in the 2-PE group and 8/10 animals in the MS-222 group. There was no significant difference in sedation scores, induction time (median 15 min), or recovery time (median 20.5 min). Gilling rate and cardiac contractility decreased during anesthesia, whereas heart rate did not. Hemolymph pH and pO2 were not different among treatment groups or time points. Baseline pCO2 was higher than pCO2 at 30 min for both groups but significantly elevated only in the MS-222 group. This is attributed to increased activity during the handling of awake animals. Invasive blood pressure obtained via cardiac catheterization in two animals was markedly decreased during surgical anesthesia. In conclusion, 2-PE and MS-222 provided effective anesthesia with clinically useful induction and recovery times. 2-PE provided a subjectively more reliable and smoother anesthesia compared to MS-222.

Nigritoxin is a bacterial toxin for crustaceans and insects.

The Tetraconata (Pancrustacea) concept proposes that insects are more closely related to aquatic crustaceans than to terrestrial centipedes or millipedes. The question therefore arises whether insects have kept crustacean-specific genetic traits that could be targeted by specific toxins. Here we show that a toxin (nigritoxin), originally identified in a bacterial pathogen of shrimp, is lethal for organisms within the Tetraconata and non-toxic to other animals. X-ray crystallography reveals that nigritoxin possesses a new protein fold of the α/ß type. The nigritoxin N-terminal domain is essential for cellular translocation and likely encodes specificity for Tetraconata. Once internalized by eukaryotic cells, nigritoxin induces apoptotic cell death through structural features that are localized in the C-terminal domain of the protein. We propose that nigritoxin will be an effective means to identify a Tetraconata evolutionarily conserved pathway and speculate that nigritoxin holds promise as an insecticidal protein.

Low Oxygen Levels Slow Embryonic Development of Limulus polyphemus.

The American horseshoe crab Limulus polyphemus typically spawns in the upper intertidal zone, where the developing embryos are exposed to large variations in abiotic factors such as temperature, humidity, salinity, and oxygen, which affect the rate of development. It has been shown that embryonic development is slowed at both high and low salinities and temperatures, and that late embryos close to hatching tolerate periodic hypoxia. In this study we investigated the influence of hypoxia on both early and late embryonic development in L. polyphemus under controlled laboratory conditions. Embryos were exposed to four different oxygen levels and their developmental stage was scored every second day. Embryos developed more slowly at both 5% O2 and 10% O2 than at the 21% O2 treatment; late development was arrested when oxygen was reduced to 2%. Our study confims that L. polyphemus not only tolerates pronounced hypoxia in later embryonic developmental stages, but also in earlier, previously unexplored, developmental stages.

Environmental exposure of Atlantic horseshoe crab (Limulus polyphemus) early life stages to essential trace elements.

This study investigated the accumulation Co, Cu, Fe, Mn, Ni, Se, and Zn in Atlantic horseshoe crab (Limulus polyphemus) early life stages (egg, embryo and larvae) and compared the concentrations to the concentration of each element in sediment, pore water and overlying water for 5 sites across Long Island, NY. For the majority of the sites, all essential trace elements accumulated in the embryos and larvae. However, many of the embryos and larvae at specific sites presented different concentration patterns which had no apparent relationship with the local habitat sediment and water values. Generally, Cu, Fe, and Se sequentially increased from egg stage through larval stages for the majority of sites, while Co, Mn, and Ni only did for a few sites. Zinc also showed an increase across sites from embryo to larval stage, however was the only one to show a decrease in concentration from egg to embryo stage at all sites. Interestingly, Mn at Manhasset Bay presented embryo and larval stages to be 50 fold greater than all other sites while the egg stage showed similar values to other sites; this high degree of uptake could be due to a high concentration in the overlying water. All essential trace elements can be accumulated from the environment but greater concentrations may be influenced by abiotic factors and the predominant uptake route (aqueous versus diet) at each life stage. Future laboratory experiments are required to investigate factors that influence essential trace element accumulation and loss in horseshoe crab early life stages.

Responses of growth and hemolymph quality in juvenile Chinese horseshoe crab Tachypleus tridentatus (Xiphosura) to sublethal tributyltin and cadmium.

Responses of growth endpoints and hemolymph constituents in juvenile Chinese horseshoe crab Tachypleus tridentatus under treatments of 0.01 and 0.1 mg/l tributyltin (TBT) and 0.1 and 1 mg/l cadmium (Cd) were examined in a 12-week experiment. A significant decline in final body weight, final prosomal width, percentage of individuals molted and mean molting time was detected under TBT exposures. While morphological abnormalities of the juveniles between TBT treatments and the control were statistically indistinguishable, significantly higher occurrences of carapace erosion and appendage loss were noted under 0.1 and 1 mg/l Cd exposures. Various hemolymph quality indicators, including hemolymph plasma protein level, amebocyte viability and percentage of granular-spherical state of amebocytes of the juveniles exposed to TBT or Cd were significantly lower than the control. Such a decrease in hemolymph quality suggested deleterious effects of metal contaminant-induced stressors on the health status of the juveniles even at low exposure levels (i.e., 0.01 mg/l TBT and 0.1 mg/l Cd). Changes of hemolymph parameters in juvenile horseshoe crabs were more sensitive than growth performance as well as morphological abnormalities in response to metal stressors, and can be used as an indicator to reflect habitat conditions and contaminant levels.

Limulus amebocyte lysate testing: adapting it for determination of bacterial endotoxin in 99mTc-labeled radiopharmaceuticals at a hospital radiopharmacy.

UNLABELLED: A bacterial endotoxin test (BET) is required to detect or quantify bacterial endotoxin that may be present in radiopharmaceutical preparations. The test uses Limulus amebocyte lysate, which, in the presence of bacterial endotoxin and divalent calcium ions, causes the formation of a coagulin gel. (99m)Tc-labeled radiopharmaceuticals have chelating ligands such as diethylene triamine pentaacetic acid (DTPA), ethylene dicysteine (EC), L,L-ethyl cysteinate dimer (ECD), N-[2,4,6-trimethyl-3 bromoacetanilid] iminodiacetic acid (mebrofenin), dimercapto succinic acid-III (DMSA-III), dimercapto succinic acid-V (DMSA-V), and several others, which form a coordination complex with Na-(99m)Tc-O4 in the presence of reducing agents. During BET by the gel-clot method, the free sulfhydryl (-SH) and carboxyl (-COOH) in some of the chelating agents in the final (99m)Tc-labeled radiopharmaceuticals decrease the free divalent calcium ion concentration, which in turn inhibits coagulin gel formation. This study was designed using the premise that addition of calcium chloride solution to the reaction mixture would nullify this effect. METHODS: We present here the data obtained from BET assay analysis of (99m)Tc-labeled radiopharmaceuticals and the cold kits from which they are made (EC, ECD, methoxyisobutylisonitrile, DTPA, mebrofenin, methylene diphosphonic acid [MDP], DMSA-III, and DMSA-V) using 2 different dilutions, maximum valid dilution (MVD) and half maximum valid dilution (MVD/2), with and without the addition of calcium chloride at a final concentration of 300 µM. RESULTS: It was observed that at MVD and MVD/2 all of the (99m)Tc-labeled kits exhibited interference in coagulin gel formation with the exception of (99m)Tc-methoxyisobutylisonitrile, (99m)Tc-MDP, (99m)Tc-mebrofenin, and (99m)Tc-ECD. However, only the cold kits of methoxyisobutylisonitrile and MDP did not show inhibition. An addition of calcium chloride solution nullified this interference at both MVD and MVD/2 in all of the (99m)Tc-labeled radiopharmaceuticals in which interference was observed. CONCLUSION: In practice, Limulus amoebocyte lysate testing is not a method of choice for (99m)Tc-labeled radiopharmaceuticals because these radiopharmaceuticals exhibit interference. However, our study proves the hypothesis that the addition of calcium chloride can circumvent this problem. The addition of calcium chloride provides an enhanced biologic quality control testing option for the final formulation of (99m)Tc-labeled radiopharmaceuticals at the hospital radiopharmacy end.

Biophysical mechanisms of endotoxin neutralization by cationic amphiphilic peptides.

Bacterial endotoxins (lipopolysaccharides (LPS)) are strong elicitors of the human immune system by interacting with serum and membrane proteins such as lipopolysaccharide-binding protein (LBP) and CD14 with high specificity. At LPS concentrations as low as 0.3 ng/ml, such interactions may lead to severe pathophysiological effects, including sepsis and septic shock. One approach to inhibit an uncontrolled inflammatory reaction is the use of appropriate polycationic and amphiphilic antimicrobial peptides, here called synthetic anti-LPS peptides (SALPs). We designed various SALP structures and investigated their ability to inhibit LPS-induced cytokine secretion in vitro, their protective effect in a mouse model of sepsis, and their cytotoxicity in physiological human cells. Using a variety of biophysical techniques, we investigated selected SALPs with considerable differences in their biological responses to characterize and understand the mechanism of LPS inactivation by SALPs. Our investigations show that neutralization of LPS by peptides is associated with a fluidization of the LPS acyl chains, a strong exothermic Coulomb interaction between the two compounds, and a drastic change of the LPS aggregate type from cubic into multilamellar, with an increase in the aggregate sizes, inhibiting the binding of LBP and other mammalian proteins to the endotoxin. At the same time, peptide binding to phospholipids of human origin (e.g., phosphatidylcholine) does not cause essential structural changes, such as changes in membrane fluidity and bilayer structure. The absence of cytotoxicity is explained by the high specificity of the interaction of the peptides with LPS.

Visual efference in Limulus: in vitro temperature-dependent neuromodulation of photoreceptor potential timing by octopamine and substance P.

Efferents from the brain of Limulus course toward its lateral eye and release octopamine and substance P into it. These neurotransmitters have previously been found to act as neuromodulators in this visual system by altering the size of its responses to light. We report here that both also modulate the timing of the receptor potentials (RPs) evoked by brief light flashes and that these timing effects are temperature dependent. Specifically: We extend our previous report that octopamine prolongs ambient RPs in a categorical fashion and here demonstrate that it does the same at colder temperatures. Categorical means that a given RP is either clearly prolonged in a dramatic fashion or its duration is otherwise unremarkable. Octopamine also accelerates the onsets of RPs when they are evoked by weak flashes under cold temperatures. Contrariwise, substance P accelerates RPs at all temperatures and this acceleration dramatically reduces the sluggishness that is otherwise typically present at low temperatures. Quantitative analysis of intensity-response functions also demonstrated that light sensitivity under substance P is significantly augmented. The plain temporal antagonism between these two modulators demonstrates that the visual system of Limulus possesses a well-poised mechanism which could be used to adjust the timing of its neural processing to interface well with the temporal characteristics of those visual stimuli that are currently present.

C-reactive protein collaborates with plasma lectins to boost immune response against bacteria.

Although human C-reactive protein (CRP) becomes upregulated during septicemia, its role remains unclear, since purified CRP showed no binding to many common pathogens. Contrary to previous findings, we show that purified human CRP (hCRP) binds to Salmonella enterica, and that binding is enhanced in the presence of plasma factors. In the horseshoe crab, Carcinoscorpius rotundicauda, CRP is a major hemolymph protein. Incubation of hemolymph with a range of bacteria resulted in CRP binding to all the bacteria tested. Lipopolysaccharide-affinity chromatography of the hemolymph co-purified CRP, galactose-binding protein (GBP) and carcinolectin-5 (CL5). Yeast two-hybrid and pull-down assays suggested that these pattern recognition receptors (PRRs) form pathogen recognition complexes. We show the conservation of PRR crosstalk in humans, whereby hCRP interacts with ficolin (CL5 homologue). This interaction stabilizes CRP binding to bacteria and activates the lectin-mediated complement pathway. We propose that CRP does not act alone but collaborates with other plasma PRRs to form stable pathogen recognition complexes when targeting a wide range of bacteria for destruction.

Response of the blood clotting system of the American horseshoe crab, Limulus polyphemus, to a novel form of lipopolysaccharide from a green alga.

Lipopolysaccharide (LPS, endotoxin) is a component of Gram-negative bacteria and is the principal indicator to the innate immune systems of higher animals of a Gram-negative bacterial invasion. LPS activates the blood clotting system of the American horseshoe crab, Limulus polyphemus. By stimulating blood cell degranulation, LPS triggers the release of the proteins of the clotting system from the cells, and by activating a protease cascade that converts coagulogen, a soluble zymogen, to coagulin, the structural protein of the clot, LPS triggers the production of the fibrillar coagulin blood clot. Although originally thought to be restricted to the Gram-negative bacteria and the cyanobacteria, LPS, or a very similar molecule, has recently been described from a eukaryotic green alga, Chlorella. Here we show that, like LPS from Gram-negative bacteria, the algal molecule stimulates exocytosis of the Limulus blood cell and the clotting of coagulin. The coagulin clot efficiently entraps the cells of Chlorella in a network of fibrils. Invasion and erosion of the carapace by green algae is an important cause of mortality of Limulus, and it is suggested that the cellular response to aLPS may contribute to defense against this pathogen.

Protein kinase C-induced disorganization and endocytosis of photosensitive membrane in Limulus ventral photoreceptors.

Protein kinase C (PKC) desensitizes the light response in photoreceptors from the ventral optic nerve of the horseshoe crab Limulus. Photoisomerization of Limulus rhodopsin leads to phosphoinositide hydrolysis, resulting in the production of inositol trisphosphate and diacylglycerol (DAG). Inositol trisphosphate mobilizes intracellular stores of Ca(2+), resulting in photoreceptor excitation in Limulus, while DAG may activate PKC. We investigated whether PKC-mediated desensitization of the photoresponse is accompanied by ultrastructural changes in the rhodopsin-bearing photosensitive membrane (rhabdom) in Limulus ventral photoreceptors. PKC activation by (-)-indolactam V in darkness induces disorganization and swelling of the rhodopsin-containing microvilli and endocytosis of rhabdomeral membrane. The effects of (-)-indolactam V on dark-adapted photoreceptor ultrastructure are reversible, are stereospecific, are blocked by coapplication of PKC inhibitors, and closely match those induced by continuous, bright light. Rhabdom disorganization and endocytosis via PKC activation may, therefore, contribute to desensitization of the light-adapted photoreceptor.

Categorical and prolonged potentials are evoked when brief, intermediate-intensity flashes stimulate horseshoe crab lateral eye photoreceptors during octopamine neuromodulation.

Octopamine, a major efferent neurotransmitter in the lateral eye of the horseshoe crab (Limulus polyphemus), has previously been shown to modulate photoreceptor responses evoked by long flashes. Quantification of these data indicates that this modulation produced a genuine increase in sensitivity to light which cannot be entirely due to an increase in optical efficiency consequent on an anatomical alteration. Other previous studies demonstrated that extrinsic current can modulate Limulus lateral eye photoreceptor cells by inducing a bistable membrane potential with two distinct states. The present study was therefore undertaken to find out if octopamine could modulate visual responses by inducing prolonged and bistable polarization shifts similar to those demonstrated in several other neural systems. Intracellular microelectrodes were used to execute an electrophysiological study of the receptor potentials evoked in the lateral eye of Limulus when brief (20-ms) flashes were delivered while 50 microM octopamine perfused dark-adapted photoreceptors. The combined chemical and optical stimuli prolonged photoreceptor responses to light to the degree that they often exceeded the duration of the brief stimulus by hundreds of milliseconds. Moreover, these prolonged potentials were clearly bistable because they were categorical--either a prolongation was perceptually clear-cut and present or it was not, with no intermediate patterns being observed. During seawater control perfusions, such prolongations were absent. This appears to be the first demonstration of such categorical and prolonged potentials in a photoreceptor neuron. This finding particularly suggests that efferent-driven neuromodulation can enable the development of a persisting short-term representation of a brief stimulus, with this representation being retained at the most distal possible neural site.

Facilitation of the responses to injections of inositol 1,4,5-trisphosphate analogs in Limulus ventral photoreceptors.

Injection of inositol 1,4,5-trisphosphate and its metabolically resistant analogs InsP3S3 and L-chiro-2,3,5-InsP3 into the ventral photoreceptors of Limulus results in the release of calcium from internal stores and in a current flow into the cells. We show here that the dependence of the current response on the amount of analog injected is supralinear. The injections also facilitate the responses to subsequent injections. We analyze the kinetics of the responses either by very slow application of the analogs directly into the lobe that is sensitive to InsP3 and light or by delivering a pulse into the nonsensitive lobe of the cell, in both cases creating a ramp of rising concentration in the sensitive region. Typically, a long latent period was followed by a strong brief inward current. The ratio between the latency and the duration of the response, defined as twice the time from half-amplitude to the peak of the response, reaches values greater than 10. Our analysis shows that this value cannot be attained within realistic models whose only nonlinearity is the cooperative binding of the ligand to its receptor. The observed ratio, however, can be achieved with a positive feedback model. Treatments that lead to partial depletion of calcium stores reversibly increase the latency of the response. We conclude that the mechanisms of the response of Limulus ventral eye to the metabolically resistant analogs of InsP3 probably involves a positive feedback mechanism and that the carrier of the feedback is likely to be Ca2+.

Excitatory actions of FMRFamide-related peptides (FaRPs) on the neurogenic Limulus heart.

The actions of FMRFamide-related peptides (FaRPs) on the neurogenic heart of the horseshoe crab, Limulus polyphemus, were investigated. Excitatory chronotropic effects were produced by application of TNRNFLRFamide, SDRNFLRFamide, GYNRS-FLRFamide, or pQDPFLRFamide to the intact heart preparation. Effects were dose-dependent with a threshold of 10(-9) M or less. TNRNFLRFamide and SDRNFLRFamide increased the burst rate of the isolated Limulus cardiac ganglion. Synthetic FaRPs produced inotropic excitation of the heartbeat as well. GYNRSFLRFamide, TNRNFLRFamide, SDRNFLRFamide, and pQDPFLRFamide increased heart contraction strength at a threshold dose of approximately 10(-8) M. TNRNFLRFamide and SDRNFLRFamide enhanced electrically evoked contractions of the Limulus myocardium, elicited contracture in some preparations, and increased the excitability of cardiac muscle fibers. The presence of cardioactive FaRPs in the Limulus central nervous system was suggested by reverse phase HPLC of acidified methanol extracts of Limulus nervous tissue. Four peaks of FaRP-like bioactivity were detected with the Busycon radula protractor muscle bioassay. These peaks also contained FaRP-like immunoreactivity. Two of these partially purified peaks produced excitatory chronotropic effects on the intact Limulus heart preparation similar to those produced by synthetic FaRPs.

Fast desensitization of the response to InsP3 in Limulus ventral photoreceptors.

In Limulus ventral photoreceptor cells the time-course of the desensitization of InsP3 response was measured by an injection-pair paradigm. Pressure pulses of InsP3 were delivered into the cell with various interpulse intervals. The desensitization of the response to the second injection of each pair approached totality at 200 ms, which is the duration of the response to a single pressure pulse of InsP3. Lowering extracellular calcium did not affect the time-course of the desensitization. Lowering the temperature slowed down both the time-course of the response to InsP3 and the time-course of the desensitization to the same extent. These findings suggest that the desensitization is powerful enough and its onset fast enough to contribute to the transience of the InsP3 response. The time-course of the desensitization suggests it may influence light adaptation.