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At least 253 records · Page 14Linked to original sources
PubMed1999

Horseshoe crab acetyl group-recognizing lectins involved in innate immunity are structurally related to fibrinogen.

We have characterized and cloned newly isolated lectins from hemolymph plasma of the horseshoe crab Tachypleus tridentatus, which we named tachylectins 5A and 5B (TLs-5). TLs-5 agglutinated all types of human erythrocytes and Gram-positive and Gram-negative bacteria. TLs-5 specifically recognize acetyl group-containing substances including noncarbohydrates; the acetyl group is required and is sufficient for recognition. TLs-5 enhanced the antimicrobial activity of a horseshoe crab-derived big defensin. cDNA sequences of TLs-5 indicated that they consist of a short N-terminal Cys-containing segment and a C-terminal fibrinogen-like domain with the highest sequence identity (51%) to that of mammalian ficolins. TLs-5, however, lack the collagenous domain found in a kind of "bouquet arrangement" of ficolins and collectins. Electron microscopy revealed that TLs-5 form two- to four-bladed propeller structures. The horseshoe crab is equipped with a unique functional homologue of vertebrate fibrinogen, coagulogen, as the target protein of the clotting cascade. Our observations clearly show that the horseshoe crab has fibrinogen-related molecules in hemolymph plasma and that they function as nonself-recognizing lectins. An ancestor of fibrinogen may have functioned as a nonself-recognizing protein.

ABO Blood-Group System↗
PubMed2000

Phylogenetic relationship among horseshoe crab species: effect of substitution models on phylogenetic analyses.

The horseshoe crabs, known as living fossils, have maintained their morphology almost unchanged for the past 150 million years. The little morphological differentiation among horseshoe crab lineages has resulted in substantial controversy concerning the phylogenetic relationship among the extant species of horseshoe crabs, especially among the three species in the Indo-Pacific region. Previous studies suggest that the three species constitute a phylogenetically unresolvable trichotomy, the result of a cladogenetic process leading to the formation of all three Indo-Pacific species in a short geological time. Data from two mitochondrial genes (for 16S ribosomal rRNA and cytochrome oxidase subunit I) and one nuclear gene (for coagulogen) in the four species of horseshoe crabs and outgroup species were used in a phylogenetic analysis with various substitution models. All three genes yield the same tree topology, with Tachypleus-gigas and Carcinoscorpius-rotundicauda grouped together as a monophyletic taxon. This topology is significantly better than all the alternatives when evaluated with the RELL (resampling estimated log-likelihood) method.

Animals↗
PubMed2004

A Toll-like receptor in horseshoe crabs.

Non-self-recognition of invading microbes relies on the pattern-recognition of pathogen-associated molecular patterns (PAMPs) derived from microbial cell-wall components. Insects and mammals conserve a signaling pathway of the innate immune system through cell-surface receptors called Tolls and Toll-like receptors (TLRs). Bacterial lipopolysaccharides (LPSs) are an important trigger of the horseshoe crab's innate immunity to infectious microorganisms. Horseshoe crabs' granular hemocytes respond specifically to LPS stimulation, inducing the secretion of various defense molecules from the granular hemocytes. Here, we show a cDNA which we named tToll, coding for a TLR identified from hemocytes of the horseshoe crab Tachypleus tridentatus. tToll is most closely related to Drosophila Toll in both domain architecture and overall length. Human TLRs have been suggested to contain numerous PAMP-binding insertions located in the leucine-rich repeats (LRRs) of their ectodomains. However, the LRRs of tToll contained no obvious PAMP-binding insertions. Furthermore, tToll was non-specifically expressed in horseshoe crab tissues. These observations suggest that tToll does not function as an LPS receptor on granular hemocytes.

Amino Acid Sequence↗
PubMed2002

Long-term in vitro generation of amoebocytes from the Indian horseshoe crab Tachypleus gigas (Müller).

Amoebocyte is the single type of cell circulating in the horseshoe crab hemolymph, which plays a major role in the defense system of the animal. Granules present in these cells are sensitive to nanogram quantities of bacterial endotoxins, which form the basis of the Limulus amoebocyte lysate (LAL) test. Normally, amoebocytes for the production of the LAL are collected by cardiac puncture; hence, development of the in vitro culture system for amoebocytes will reduce the variability of the lysate and help to conserve the 400 million-yr-old living fossil. In the present investigation we have attempted organ culture of gill flaps that have been shown to be the source of amoebocytes. The gill flaps were cultured at 28 degrees C on a rocker platform in a modified L-15 medium supplemented with 10% v/v horseshoe crab serum. This led to the release of amoebocytes outside the gill flaps for a period of 6-8 wk with a more or less steady number of amoebocytes during the weekly harvest. No significant difference was seen in the yield of amoebocytes from male and female horseshoe crabs. Confocal laser microscopy studies revealed significant difference in the size of amoebocytes released in vitro as compared with those obtained in vivo. Thus, we have optimized the culture conditions for the long-term generation of amoebocytes in vitro from the Indian horseshoe crab Tachypleus gigas by reducing the incidence of contamination, simulating in vivo conditions for the organ culture of gill flaps, and improvising the nutritional status using the modified L-15 medium, providing the desired osmolarity and pH.

Animals↗
PubMed1995

A case of orthologous sequences of hemocyanin subunits for an evolutionary study of horseshoe crabs: amino acid sequence comparison of immunologically identical subunits of Carcinoscorpius rotundicauda and Tachypleus tridentatus.

About 83% of the amino acid sequence of hemocyanin subunit HR6 from the Southeast Asian horseshoe crab, Carcinoscorpius rotundicauda, has been determined. There is a difference of about 43% between HR6 and complete sequences of chelicerate hemocyanin subunits from the American horseshoe crab, Limulus polyphemus, and a tarantula, Eurypelma californicum. However, the immunologically identical subunits HR6 and HT6 from Tachypleus tridentatus (Japanese horseshoe crab) show 2.7% sequence difference. Based on the amino acid sequences of HR6 and HT6, the divergence between C. rotundicauda and T. tridentatus occurred about 9.6 million years ago. In the case of horseshoe crab hemocyanin subunits, it seems that the orthologous homologues in many homologous subunits between species are immunologically detectable.

Amino Acid Sequence↗
PubMed2004

Light intensity appears to be more important than an endogenous seasonal clock for regulating structural rhythms in the lateral eye of the horseshoe crab.

The lateral compound eye of the American horseshoe crab, Limulus polyphemus, is an established model system for studying visual and circadian processes. Previous studies have shown there is an endogenous rhythm in retinal structure and this rhythm is enhanced by light. The purpose of this study was to determine whether changes in natural lighting influence these rhythms independent of season. To test this concept, horseshoe crabs were maintained in an aquarium tank and exposed to natural lighting only. The angle of the sun and foliage outside the room where the horseshoe crabs were maintained were such that more natural lighting reached the aquarium tank during winter days than summer days despite the fact that sunlight is more intense in the summer. Horseshoe crabs were sacrificed 2 hours, 5 hours, and 8 hours following local sunrise on a winter day (January 11th) and a summer day (June 19th), respectively, and the dimensions of several retinal parameters were quantified. Preliminary data indicate that certain structural rhythms were significantly more pronounced on the winter day when natural lighting was more intense. For example, 8 hours following sunrise, apertures were 4.2 times as long and 3.2 times more narrow on the winter day than on the summer day. These data suggest that the intensity of natural lighting is more important for regulating structural rhythms in the lateral eye than an endogenous seasonal clock.

Adaptation, Ocular↗
PubMed1989

[Cultivation of horseshoe crab amebocytes].

The in vitro and in vivo cultivation of horseshoe crab amebocytes is studied. Partial bleeding promotes the proliferation of horseshoe crab amebocytes, and could be a useful method for amebocyte production in vivo. The optimal conditions of in vitro cultivation of amebocytes were incubation at 15 degrees C and pH delta 6.8-7.2. The best medium was the hemolymph of the horseshoe crab. Measuring DNA synthesis of amebocytes by 3H-thymidine uptake showed that no proliferation of amebocytes occurs during in vitro cultivation. Because some protozoa were found in the hemolymph, primaquine, quinine, pyrimethamine and metronidazole were used in the culture medium. Primaquine and metronidazole have a protozoacidal effect on the protozoa parasitising in the horseshoe crab.

Animals↗
PubMed1996

Proctolinlike immunoreactivity and identified neurosecretory cells as putative substrates for modulation of courtship display behavior in the blue crab, Callinectes sapidus.

Pheromonally stimulated courtship display (CD) behavior in male blue crabs (Callinectes sapidus) is characterized by rhythmic waving of the fifth legs. The waving of the fifth legs is modulated by proctolin in freely moving crabs and in reduced preparations. To begin to identify an anatomical substrate for CD behavior we have localized putative proctolinergic cells and described the morphology of neurosecretory neurons known to oscillate during pheromonal stimulation of reduced preparations. Proctolin-induced CD occurs with developmental and seasonal dependence. Male crabs altered hormonally by eyestalk ligation spontaneously produce CD behavior. We have localized proctolinlike immunoreactivity (PIR) in the central nervous system (CNS) and compared this immunoreactivity across sexes, developmental stage, eyestalk ligation, and seasonal conditions to determine whether or not expression of PIR is correlated with CD behavior. PIR was found in most areas of the CNS. Clusters of PIR-positive cells were found in the sinus gland and eyestalk ganglia, olfactory neuropil with associated cell bodies, and in a large cell cluster in the subesophageal region of the ventral nerve cord. Three pairs of cell bodies in different cell body groups in the brains of adult crabs stained positively for PIR but did not stain in the youngest juvenile animals. Comparison of PIR distribution with toluidine blue studies of the ventral nerve cord indicated a high likelihood that cells in the PIR-positive cluster of the subesophageal ganglia were also members of the cluster of neurons identified as oscillatory neurosecretory neurons.

Animals↗
PubMed1996

Neurogenesis in the developing crab brain: postembryonic generation of neurons persists beyond metamorphosis.

A considerable amount of information is available about the structure and function of the central nervous system in adult crustaceans. However, little effort has been directed toward understanding embryonic and larval neurogenesis in these animals. In the present study we recorded neurogenesis in the brain of laboratory-reared larvae of the spider crab Hyas araneus. Proliferating cells were detected immunocytochemically after in vivo labeling with 5-bromo-2'-deoxyuridine. This method has already been used to study the proliferation of neuroblasts in the ventral nerve cord of spider crab larvae. In the brain, a set of mitotically highly active neuroblasts was found in newly hatched zoea 1 larvae. These neuroblasts are individually identifiable due to their position and therefore a schematic map of the cerebral neuroblasts could be established. The number of active neuroblasts is high from hatching throughout the molt to the zoea 2. This proliferative action then decreases dramatically and has ceased at the time of first metamorphosis toward the megalopa larva. However, many ganglion mother cells born by unequal division of neuroblasts then go through their final division throughout the subsequent megalopa stage. In the brain, all mitotic activity has ceased at the time of second metamorphosis with the exception of a cluster of labeled nuclei within the olfactory lobe cells. In this cluster, the generation of neurons persists beyond the second metamorphosis into the crab 1 stage. Meanwhile, the neuropil volume of the olfactory lobes increases 10-fold from hatching to the crab 1. These results are discussed with regard to reports on neuronal proliferation during adult life in insects and rodents.

Animals↗
PubMed1991

Biochemistry of seminal secretions of the crab Scylla serrata with reference to sperm metabolism and storage in the female.

Biochemical studies on the male reproductive tissues and seminal secretions have been made with reference to sperm metabolism and different stages of maturity in the crab Scylla serrata. The results reveal that the seminal plasma and spermatophores are rich in protein, carbohydrate, and lipid. In general, organic components of spermatophores are considerably higher than those of seminal plasma. Enzyme studies show that the succinate dehydrogenase (SDH) activity is very low, whereas fumarate reductase (FR) and lactate dehydrogenase (LDH) exhibit high activity. Electrophoretic studies on LDH show that, in addition to the occurrence of a sperm-specific fraction, LDHx, the M-type subunits are predominant in the mature spermatophores. These results from enzyme studies suggest that sperm metabolism is mainly anaerobic, utilizing the carbohydrates as substrates. The results for maturational changes reveal that the male reproductive tissues and their secretions contain lesser quantity of organic components in the immature crabs; as the maturity proceeds, there is not only concentration of organic substances but also an increase in the size of spermatophores. The concentration of biochemical constituents is highest in the proximal vas deferens (PVD), suggesting that the granular seminal plasma as well as the sperm-agglutinating substance and spermatophoric wall are secreted in this region. The spermatheca of the unmated female crabs are poor in organic constituents. After mating, their contents are enriched by organic substances derived from contributions of the seminal substances. During sperm storage in the spermatheca, only the carbohydrates decline steeply. A low activity of SDH, but a moderate level of LDH and a high level of FR activity, is recorded in the spermathecal content of mated crabs, providing further evidence for anaerobic metabolism of sperm during storage in female. A sharp fall in the stored carbohydrates constitutes further evidence in this regard.

Age Factors↗
PubMed1994

Regeneration and molting effects on a proprioceptor organ in the Dungeness crab, Cancer magister.

Decapoda Crustacea molt in order to grow; some species, such as the Dungeness crab Cancer magister, achieve a very large size. Does sensory neuron hyperplasia in internal proprioceptors accompany this growth? To determine this, neurons in propodite-dactylopodite (PD) chordotonal organs were counted in first walking legs of juvenile (5th through 9th instar) and adult (10th through 13th instar) C. magister. We found that the PD organs of J5 crabs have about 56 neurons; the number increases to about 61 neurons in J6 crabs. Significant hyperplasia now occurs because an average of 79 neurons are found in the PD organs of J7 crabs. Little to no hyperplasia accompanies the several succeeding juvenile and adult molts (ca. 82-86 neurons are present). Because autotomized limbs are regenerated upon molting, we also examined how the number of PD organ neurons in regenerated legs compares with those of pristine legs. Newly regenerated legs (termed 1st stage regenerates) have fewer sensory neurons than do their contralateral pristine partners (65 vs 81); larger regenerated legs which have attained nearly normal size as a result of additional molts (2nd stage regenerates) still have fewer neurons than their pristine partners (69 vs 81). Additionally, in contrast to those of pristine walking legs, the elastic strand of PD organs from 1st stage regenerates in a misshapen sheet containing a cluster of small neurons with no obvious functional organization. Nonetheless, neurophysiological recordings indicate that all the receptor types typical for pristine legs (movement and position cells) are represented. The PD organs of 2nd stage regenerates differentiate to the shape and neuronal organization of pristine legs.

Animals↗
PubMed1998

The freshwater river crab, Potamonautes warreni, as a bioaccumulative indicator of iron and manganese pollution in two aquatic systems.

The aim of this study was to determine the potential use of the freshwater river crab, Potamonautes warreni, as a bioaccumulative indicator of iron and manganese pollution in aquatic ecosystems. Water and sediment analysis of the two study sites (Germiston Lake and Potchefstroom Dam) revealed that while levels of manganese were higher in Germiston Lake, iron concentrations were higher in Potchefstroom Dam. Metal analysis of P. warreni revealed that while the crabs from Potchefstroom Dam contained slightly higher iron levels than those from Germiston Lake, manganese concentrations in P. warreni from the latter site were significantly higher than those in the crabs from the former site. Iron and manganese levels in these organisms were influenced by the size, mass, and sex of the crabs on occasion, but these relationships were not always consistent at both of the sites. The results of this study clearly indicate that the ultimate levels of iron and manganese attained in P. warreni do vary depending on the site from which animals are collected. From this, it is suggested that these crustaceans be incorporated into biomonitoring protocols, particularly in areas that are subjected to elevated metal levels in the environment

Animals↗
PubMed2000

Inhibition of ovarian growth by cadmium in the fiddler crab, Uca pugilator (Decapoda, ocypodidae).

The effect of cadmium chloride (1 mg/L) on oocyte growth of the fiddler crab, Uca pugilator, was studied during the slow vitellogenesis phase of ovarian maturation of this carb. In vivo experiments were done with both intact and eyestalkless crabs. The intact cadmium-exposed crabs exhibited a significantly lower oocyte diameter than the controls at the end of the 2-week exposure period, but no significant differences were detected among the eyestalkless crabs, suggesting that the effect of cadmium could be on the sinus gland in the eyestalks, increasing secretion of the gonad-inhibiting hormone (GIH). To test this hypothesis, in vitro experiments were done, incubating pieces of ovary with and without eyestalk tissue, in the presence of thoracic ganglion, needed for oocyte growing due to the secretion of the gonad-stimulating hormone (GSH). Only when eyestalk tissue was present in the incubation media was oocyte growth inhibited by cadmium compared to the controls. These results strongly suggest that cadmium acts at least in part to increase the secretion of GIH from the sinus gland. GIH could then in turn act by (a) inhibiting secretion of GSH by the thoracic ganglion, (b) directly inhibit the oocytes, or (c) both (a) and (b).

Animals↗
PubMed1998

Tissue-specific patterns and steady-state concentrations of ecdysteroid receptor and retinoid-X-receptor mRNA during the molt cycle of the fiddler crab, Uca pugilator.

In the fiddler crab, Uca pugilator, we have investigated the temporal expression of receptors in various tissues using probes that encode Uca ecdysteroid receptor (UpEcR) and retinoid-X-receptor (UpRXR) gene homologs. During molt stages C4 through D1-4, UpEcR and UpRXR transcripts are expressed in regenerating limb buds, gills, eyestalks, hypodermis, hepatopancreas, muscle from nonregenerating walking legs, and the large cheliped. Some of these tissues have not previously been recognized as ecdysteroid-target tissues. Levels of ecdysteroids in the hemolymph fluctuate significantly during the molt cycle of U. pugilator. The variation in steady-state concentrations of UpEcR transcripts in tissues from C4 to D1-4 implies molt cycle-related differences in the potential of these tissues to respond to changing titers of ecdysteroids in the hemolymph. In singly autotomized crabs, highest concentrations of UpEcR transcript in some tissues did not coincide with the highest levels of circulating ecdysteroids, suggesting that UpEcR expression in these tissues is not dependent on high ecdysteroid titers and may be induced by low or rising concentrations of ecdysteroids. UpEcR and UpRXR genes were expressed simultaneously in tissues, supporting the possibility of heterodimerization for EcR and RXR in vivo. In some tissues, however, levels of transcripts differed, suggesting other possible receptor interactions. Moreover, UpEcR expression in tissues from multiply autotomized crabs differed from the expression patterns in tissues from singly autotomized crabs.

Animals↗
PubMed1995

High-affinity antigen binding by chelating recombinant antibodies (CRAbs).

We have developed a strategy for making antibody fragments with high binding affinities by harnessing the chelate effect. We create a bispecific antibody fragment (Chelating Recombinant Antibody or CRAb) that recognizes adjacent and non-overlapping epitopes of the target antigen, and is flexible enough to bind to both epitopes simultaneously. Here the strategy is illustrated with two antibodies that form complexes of known three-dimensional structure against different epitopes of lysozyme. Computer graphic modelling indicated that two single-chain antibody fragments (scFv) derived from antibodies D1.3 (Ka = 10(8) M-1) and mutant HyHEL-10 (Ka = 10(6) M-1) could be linked together on the surface of lysozyme by a flexible and hydrophilic polypeptide between the C terminus of one fragment and the N terminus of the other. The CRAb gene was assembled and the CRAb expressed by secretion from bacteria. The purified CRAb was shown to have a much higher affinity than either of the scFv fragments, as shown by competition ELISA (Kd > 10(9) M-1), bandshift on gels (Ka > 2 x 10(9) M-1) and fluorescence quench (Ka > 1.3 x 10(10) M-1).

Amino Acid Sequence↗
PubMed1994

Cytoplasmic localization of transcripts of a complex G+C-rich crab satellite DNA.

The primary sequence and higher order structures of a G+C-rich satellite DNA of the Bermuda land crab Gecarcinus lateralis have been described previously. The repeat unit of the satellite is approximately 2.1 kb. In exploring a possible function for this satellite, we asked whether it is transcribed. As a probe for transcripts, we used a segment of DNA amplified from a 368 bp EcoRI fragment from the very highly conserved 3' end of the satellite DNA. During polymerase chain reaction (PCR) amplification, the probe was simultaneously either radiolabeled or biotinylated. Tissue- and stage-specific transcripts were observed when blots of poly(A)+ mRNAs recovered from polysomes isolated from crab tissues [including midgut gland (hepatopancreas), limb bud, and claw muscle] were probed with the satellite DNA fragment. The presence of satellite transcripts in polysomal mRNAs is strong evidence that the transcripts had reached the cytoplasm. To corroborate the presence of transcripts in the cytoplasm, we investigated in situ hybridization of satellite probes with RNAs in tissue sections. Biotinylated satellite DNA probes were applied to sections of midgut gland, limb bud papilla, ovary, or testis of anecdysial crabs. Retention of RNAs in tissue sections was improved by UV-irradiation prior to hybridization. Transcripts were abundant in the cytoplasm of all tissues except testis. Sections of crab midgut gland treated with RNase A prior to hybridization and sections of mouse pancreatic tumor served as controls; neither showed any signals with the probe.

Animals↗
PubMed1993

Potent debriding ability of collagenolytic protease isolated from the hepatopancreas of the king crab Paralithodes camtschatica.

The debriding activity of the protease complex produced from the hepatopancreas of the king crab Paralithodes camtschatica was evaluated in vivo. The results clearly showed that the crab preparation had a high debriding ability. Individual proteases were isolated chromatographically from the crab complex to compare their debriding activities. All the purified proteases were active in vivo. The most active component of the complex was the isozyme C of crab collagenolytic protease, a member of the chymotrypsin-like protease class.

Animals↗
PubMed1986

The innervation of the pyloric region of the crab, Cancer borealis: homologous muscles in decapod species are differently innervated.

The muscles of the pyloric region of the stomach of the crab, Cancer borealis, are innervated by motorneurons found in the stomatogastric ganglion (STG). Electrophysiological recording and stimulating techniques were used to study the detailed pattern of innervation of the pyloric region muscles. Although there are two Pyloric Dilator (PD) motorneurons in lobsters, previous work reported four PD motorneurons in the crab STG (Dando et al. 1974; Hermann 1979a, b). We now find that only two of the crab PD neurons innervate muscles homologous to those innervated by the PD neurons in the lobster, Panulirus interruptus. The remaining two PD neurons innervate muscles that are innervated by pyloric (PY) neurons in P. interruptus. The innervation patterns of the Lateral Pyloric (LP), Ventricular Dilator (VD), Inferior Cardiac (IC), and PY neurons were also determined and compared with those previously reported in lobsters. Responses of the muscles of the pyloric region to the neurotransmitters, acetylcholine (ACh) and glutamate, were determined by application of exogenous cholinergic agonists and glutamate. The effect of the cholinergic antagonist, curare, on the amplitude of the excitatory junctional potentials (EJPs) evoked by stimulation of the pyloric motor nerves was measured. These experiments suggest that the differences in innervation pattern of the pyloric muscles seen in crab and lobsters are also associated with a change in the neurotransmitter active on these muscles. Possible implications of these findings for phylogenetic relations of decapod crustaceans and for the evolution of neural circuits are discussed.

Acetylcholine↗