Name: Blood Collection from the American Horseshoe Crab, Limulus Polyphemus (Video) | JoVE Journal
Uploaded: 2008-10-13T00:00:00.000+00:00
Duration: 12 min 48 s
Description: Learn blood collection from the American horseshoe crab, Limulus polyphemus, for biomedical research. This method demonstrates cardiac puncture to obtain amoebocytes for bacterial detection assays.

ai_agent_IP

ai_agent

maintenance

instantTrialDurationMinutes

isBiopharma

enableNewMyJoveSectionDesign

educationFeatures

seoMetaApi

ai_copilot_institution_blacklist

ai_chat_institution_blacklist

freeAccessWindow

ssoRoleCapture

scheduledMaintenanceBanner

joveLabsFeatures

disable_autoplay_for_bots

extended_trial_duration

5 minutes

playerTrialDesktop

Original research described above was supported by NSF grant 0344630.

<ol><li>Lochhead, J. H. Selected Invertebrate Types. Brown, F. A. , John Wiley and Sons, Inc. New York, NY. 360-381 (1950).</li>
<li>Shuster, C. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+circulatory+system+and+blood+of+the+horseshoe+crab+Limulus+polyphemus+L.%3A+a+review%5BTitle%5D)+AND+%22US+Dept+Energy%2C+Fed+Regul.+Comm%22%5BJournal%5D)">The circulatory system and blood of the horseshoe crab Limulus polyphemus L.: a review.</a> US Dept Energy, Fed Regul. Comm. , Washington. (1978).</li>
<li>Conrad, M. L., Pardy, R. L., Wainwright, N., Child, A., Armstrong, P. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Response+of+the+blood+clotting+system+of+the+American+horseshoe+crab%2C+Limulus+polyphemus%2C+to+a+novel+form+of+lipopolysaccharide+from+a+green+alga%5BTitle%5D)+AND+%22Comp+Biochem.+Physiol+A+Mol.+Integr.+Physiol%22%5BJournal%5D)">Response of the blood clotting system of the American horseshoe crab, Limulus polyphemus, to a novel form of lipopolysaccharide from a green alga.</a> Comp Biochem. Physiol A Mol. Integr. Physiol. , 144-423 (2006).</li>
<li>Nakamura, T., Morita, T., Iwanaga, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Intracellular+proclotting+enzyme+in+limulus+%28Tachypleus+tridentatus%29+hemocytes%3A+its+purification+and+properties%5BTitle%5D)+AND+%22J.+Biochem.+%28Tokyo%22%5BJournal%5D)">Intracellular proclotting enzyme in limulus (Tachypleus tridentatus) hemocytes: its purification and properties.</a> J. Biochem. (Tokyo. 97, 1561-1574 (1985).</li>
<li>Levin, J., Ornberg, R. L. Blood Cells of Marine Invertebrates: Experimental Systems in Cell Biology and Comparative Physiology. Cohen, W. D. , Alan R. Liss, Inc.. New York, NY. 259-260 (1985).</li>
<li>Liang, S. M., Liu, T. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Studies+on+the+Limulus+coagulation+system%3A+inhibition+of+activation+of+the+proclotting+enzyme%2C+by+dimethyl+sulfoxide%5BTitle%5D)+AND+%22Biochem.+Biophys.+Res.+Commun%22%5BJournal%5D)">Studies on the Limulus coagulation system: inhibition of activation of the proclotting enzyme, by dimethyl sulfoxide.</a> Biochem. Biophys. Res. Commun. 105, 553-559 (1982).</li>
<li>Soderhall, K., Smith, V. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Separation+of+the+haemocyte+populations+of+Carcinus+maenas+and+other+marine+decapods%2C+and+prophenoloxidase+distribution%5BTitle%5D)+AND+%22Dev.+Comp+Immunol%22%5BJournal%5D)">Separation of the haemocyte populations of Carcinus maenas and other marine decapods, and prophenoloxidase distribution.</a> Dev. Comp Immunol. 7, 229-239 (1983).</li>
<li>Solon, E., Gupta, A. P., Gaugler, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Signal+transduction+during+exocytosis+in+Limulus+polyphemus+granulocytes%5BTitle%5D)+AND+%22Dev.+Comp.+Immunol%22%5BJournal%5D)">Signal transduction during exocytosis in Limulus polyphemus granulocytes.</a> Dev. Comp. Immunol. 20, 307-321 (1996).</li>
<li>Armstrong, P. B., Rickles, F. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Endotoxin-induced+degranulation+of+the+Limulus+amebocyte%5BTitle%5D)+AND+%22Exp.+Cell+Res%22%5BJournal%5D)">Endotoxin-induced degranulation of the Limulus amebocyte.</a> Exp. Cell Res. 140, 15-24 (1982).</li>
<li>Iwanaga, S., Kawabata, S. Evolution and phylogeny of defense molecules associated with innate immunity in horseshoe crab. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aIwanaga%2C+S+%22Front+Biosci%22">Front Biosci</a>. Shuster, C. N., Barlow, R. B., Berkman, L., Brockmann, H. J. 3, (1998).</li>
<li>Armstrong, P. B. The American Horseshoe Crab. , Harvard University Press. Cambridge, MA. 288-309 (2003).</li>
<li>Iwanaga, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+limulus+clotting+reaction%5BTitle%5D)+AND+%22Curr.+Opin.+Immunol%22%5BJournal%5D)">The limulus clotting reaction.</a> Curr. Opin. Immunol. 5, 74-82 (1993).</li>
<li>Levin, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+Limulus+amebocyte+lysate+test%3A+perspectives+and+problems%5BTitle%5D)+AND+%22Prog.+Clin.+Biol.+Res%22%5BJournal%5D)">The Limulus amebocyte lysate test: perspectives and problems.</a> Prog. Clin. Biol. Res. 231, 1-23 (1987).</li>
<li>Koshiba, T., Hashii, T., Kawabata, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+structural+perspective+on+the+interaction+between+lipopolysaccharide+and+factor+C%2C+a+receptor+involved+in+recognition+of+Gram-negative+bacteria%5BTitle%5D)+AND+%22J.+Biol.+Chem%22%5BJournal%5D)">A structural perspective on the interaction between lipopolysaccharide and factor C, a receptor involved in recognition of Gram-negative bacteria.</a> J. Biol. Chem. 282, 3962-3967 (2007).</li>
<li>James, G. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Inactivation+of+the+protease+inhibitor+phenylmethylsulfonyl+fluoride+in+buffers%5BTitle%5D)+AND+%22Anal.+Biochem%22%5BJournal%5D)">Inactivation of the protease inhibitor phenylmethylsulfonyl fluoride in buffers.</a> Anal. Biochem. 86, 574-579 (1978).</li>
<li>Murer, E. H., Levin, J., Holme, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Isolation+and+studies+of+the+granules+of+the+amebocytes+of+Limulus+polyphemus%2C+the+horseshoe+crab%5BTitle%5D)+AND+%22J.+Cell+Physiol%22%5BJournal%5D)">Isolation and studies of the granules of the amebocytes of Limulus polyphemus, the horseshoe crab.</a> J. Cell Physiol. 86, 533-542 (1975).</li>
<li>Decker, H., Ryan, M., Jaenicke, E., Terwilliger, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((SDS+induced+phenoloxidase+activity+of+hemocyanins+from+Limulus+polyphemus%2C+Eurypelma+californicum+and+cancer%5BTitle%5D)+AND+%22%22%5BJournal%5D)">SDS induced phenoloxidase activity of hemocyanins from Limulus polyphemus, Eurypelma californicum and cancer.</a> , 276-17796 (2001).</li>
<li>Armstrong, P. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+cytolytic+function+for+a+sialic+acid-binding+lectin+that+is+a+member+of+the+pentraxin+family+of+proteins%5BTitle%5D)+AND+%22J.+Biol.+Chem%22%5BJournal%5D)">A cytolytic function for a sialic acid-binding lectin that is a member of the pentraxin family of proteins.</a> J. Biol. Chem. 271, 14717-14721 (1996).</li>
<li>Armstrong, P. B., Melchior, R., Quigley, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Humoral+immunity+in+long-lived+arthropods%5BTitle%5D)+AND+%22J.+Insect+Physiol%22%5BJournal%5D)">Humoral immunity in long-lived arthropods.</a> J. Insect Physiol. 42, 53-64 (1996).</li>
<li>Armstrong, P. B., Armstrong, M. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+decorated+clot%3A+Binding+of+agents+of+the+innate+immune+system+to+the+fibrils+of+the+limulus+blood+clot%5BTitle%5D)+AND+%22Biol.+Bull%22%5BJournal%5D)">The decorated clot: Binding of agents of the innate immune system to the fibrils of the limulus blood clot.</a> Biol. Bull. 205, 201-203 (2003).</li>
<li>Armstrong, P. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Interaction+of+the+motile+blood+cells+of+the+horseshoe+crab%2C+Limulus.+Studies+on+contact+paralysis+of+pseudopodial+activity+and+cellular+overlapping+in+vitro%5BTitle%5D)+AND+%22Exp.+Cell+Res%22%5BJournal%5D)">Interaction of the motile blood cells of the horseshoe crab, Limulus. Studies on contact paralysis of pseudopodial activity and cellular overlapping in vitro.</a> Exp. Cell Res. 107, 127-138 (1977).</li>
<li>Armstrong, P. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Motility+of+the+Limulus+blood+cell%5BTitle%5D)+AND+%22J.+Cell+Sci%22%5BJournal%5D)">Motility of the Limulus blood cell.</a> J. Cell Sci. 37, 169-180 (1979).</li>
<li>Armstrong, P. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Adhesion+and+spreading+of+Limulus+blood+cells+on+artificial+surfaces%5BTitle%5D)+AND+%22J.+Cell+Sci%22%5BJournal%5D)">Adhesion and spreading of Limulus blood cells on artificial surfaces.</a> J. Cell Sci. 44, 243-262 (1980).</li>
<li>Armstrong, P. B. Blood Cells of Marine Invertebrates. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aArmstrong%2C+PB+%22Lab.+Animal%22">Lab. Animal</a>. Cohen, W. D. , A.R. Liss. New York, NY. 253-258 (1985).</li>
<li>Smith, S. A., Berkson, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Laboratory+culture+and+maintenance+of+the+horseshoe+crab+%28Limulus+polyphemus%29%5BTitle%5D)+AND+%22Lab.+Animal%22%5BJournal%5D)">Laboratory culture and maintenance of the horseshoe crab (Limulus polyphemus).</a> Lab. Animal. 34, 27-34 (1980).</li>
<li>Leibovitz, L., Lewbart, G. A. The American Horseshoe Crab. Shuster, C. N., Barlow, R. B., Brockmann, H. J. , Harvard University Press. Cambridge, MA. 245-275 (2003).</li>
<li>Rudkin, D. M., Young, G. A., Nowlan, G. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+oldest+horseshoe+crab%3A+a+new+xiphosurid+from+Late+Ordovician+Konservat-Lagerstatten+deposits%5BTitle%5D)+AND+%22Palaeontology%22%5BJournal%5D)">The oldest horseshoe crab: a new xiphosurid from Late Ordovician Konservat-Lagerstatten deposits.</a> Palaeontology. 51, Manitoba, Canada. 1-9 (2008).</li>
<li>Shuster, C. N., Sekiguchi, K. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aShuster%2C+CN+%22The+American+Horseshoe+Crab%22">The American Horseshoe Crab</a>. Shuster, C. N., Barlow, R. B., Brockmann, J. J. , Harvard University Press. Cambridge, MA. 103-132 (2003).</li>
<li>Walls, E. A., Berkam, J., Smith, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+horseshoe+crab%2C+Limulus+polyphemus%2C+200+million+years+of+existence%2C+100+years+of+study%5BTitle%5D)+AND+%22Rev.+Fisheries+Sci%22%5BJournal%5D)">The horseshoe crab, Limulus polyphemus, 200 million years of existence, 100 years of study.</a> Rev. Fisheries Sci. 10, 39-73 Forthcoming.</li>
<li>Patten, W. The Evolution of the Vertebrates and Their Kin. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aPatten%2C+W+%22P.+Blakiston%27s+Son+and+Co%22">P. Blakiston's Son and Co</a>. , Philadelphia, PA. 195-209 (1912).</li>
</ol>

There are four species of horseshoe crab, Limulus polyphemus from the east coast of North America and three species that range from Japan to the Bay of Bengal. The animal is identified as a "living fossil" because anatomically similar forms have been found as fossils from 445 million years ago 27. The horseshoe crab represents a long-lived animal, requiring 10 - 13 years to reach maturity and living at least 20 years 28. Although it has been subjected to extensive harvesting as bait for the...

blood collection from the american horseshoe crab, limulus polyphemus

Blood Collection from the American Horseshoe Crab, Limulus Polyphemus

The horseshoe crab has the best-characterized immune system of any long-lived invertebrate. The study of immunity in horseshoe crabs has been facilitated by the ease in collecting large volumes of blood and from the simplicity of the blood. Horseshoe crabs show only a single cell type in the general circulation, the granular amebocyte. The plasma has the salt content of sea water and only three abundant proteins, hemocyanin, the respiratory protein, the C-reactive proteins, which function in the cytolytic destruction of foreign cells, including bacterial cells, and &alpha;2-macroglobulin, which inhibits the proteases of invading pathogens. Blood is collected by direct cardiac puncture under conditions that minimize contamination by lipopolysaccharide (a.k.a., endotoxin, LPS), a product of the Gram-negative bacteria. A large animal can yield 200 - 400 mL of blood. For the study of the plasma, blood cells are immediately removed from the plasma by centrifugation and the plasma can then be fractionated into its constituent proteins. The blood cells are conveniently studied microscopically by collecting small volumes of blood into LPS-free isotonic saline (0.5 M NaCl) under conditions that permit direct microscopic examination by placing one of more LPS-free coverglasses on the culture dish surface, then mounting those coverglasses in simple observation chambers following cell attachment. A second preparation for direct observation is to collect 3 - 5 mL of blood in a LPS-free embryo dish and then explanting fragments of aggregated amebocytes to a chamber that sandwiches the tissue between a slide and a coverglass. In this preparation, the motile amebocytes migrate onto the coverglass surface, where they can readily be observed. The blood clotting system involves aggregation of amebocytes and the formation of an extracellular clot of a protein, coagulin, which is released from the secretory granules of the blood cells. Biochemical analysis of washed blood cells requires that aggregation and degranulation does not occur, which can be accomplished by collecting blood into 0.1 volumes of 2% Tween-20, 0.5 M LPS-free NaCl, followed by centrifugation of the cells and washing with 0.5 M NaCl.

The American horseshoe crab, Limulus polyphemus, is arguably the most convenient source for large quantities of blood of any invertebrate. The blood is simple in composition, with only one cell-type in the general circulation, the granular amebocyte, and only three abundant proteins in the plasma, hemocyanin, the C-reactive proteins, and &alpha;2-macroglobulin. Blood is collected from the heart and the blood cells and plasma are separated by centrifugation.

The horseshoe crab has the best-characterized immune system of any long-lived invertebrate.  The study of immunity in horseshoe crabs has been facilitated ...

In order to bleed the American Horseshoe Crab Limulus PolyPhen, one must hold the crab in a flexed position and insert a syringe needle into the hinge to puncture its heart. Once its circulatory system is accessed, an adult crab can be drained of nearly all of its blood. Why bleed?The horseshoe crab Its blood contains a highly motile type called an amoeba site that secretes specialized clotting factors which react violently in the presence of gram-negative bacteria. A fact which is led to the adoption of the Limulus amoeba Cyte lysate assay or gram-negative test by the FDA. Hi, I'm Peter Armstrong from the Department of Molecular and Cellular Biology at the University of California Davis.Over today I'm reporting from the Marine Biological Laboratory in Woods Hole, Massachusetts, where I conduct research during the summer. Today we will show you how to collect blood from the American horseshoe crab. Limitless polyphemus ar arguably the best source for large volumes of blood for any invertebrate.So let's get started. Horseshoe crabs can be kept in captivity. They require high quality sea water and can be maintained in Marina Aquar that provide aeration and denitrification of the wate

blood-collection-from-the-american-horseshoe-crab-limulus-polyphemus

Research

JoVE Journal

Biology

In this video we perform electroretinogram recording, optic nerve recording, and intraretinal recording with the American horseshoe crab, Limulus Polyphemus. These electrophysiological paradigms can be used for investigating the neural basis of vision in a research or teaching lab.

Using the Horseshoe Crab, Limulus Polyphemus, in Vision Research

This video will demonstrate how to obtain hemocytes (blood cells) from the Hawaiian bobtail squid, Euprymna scolopes for use in cell biological and bacterial adhesion assays. Hemocytes will be stained with a fluorescent dye and exposed to GFP-labeled bacteria.

Obtaining Hemocytes from the Hawaiian Bobtail Squid Euprymna scolopes and Observing their Adherence to Symbiotic and Non-Symbiotic Bacteria

Insect hemocytes carry out many important functions, both immune and non-immune, throughout all stages of insect development. Our present knowledge of hemocyte types and function comes from studies on insect genetic models. Here, we present a method for extracting, quantifying and visualizing hemocytes from wild caterpillars.

A Simple Protocol for Extracting Hemocytes from Wild Caterpillars

The Drosophila hemolymph serves as both blood and interstitial fluid, circulating throughout the body during the larva and adult stages. This video describes a protocol to collect larval hemolymph in order to quantify the circulating cells.

Cuticle Disruption: A Method to Collect Hemolymph from Drosophila Larvae

This video demonstrates the hemocyte disturbance assay, evaluating hemocyte re-adhesion in Drosophila larvae's hematopoietic pockets. Mechanical disturbance releases resident hemocytes, elevating circulating hemocyte counts. Post-recovery, re-adherence of resident hemocytes is confirmed, illustrating reversibility.

A Hemocyte Disturbance Assay to Assess Hemocyte Re-Adhesion to Hematopoietic Pockets

Fishery-induced changes to exploited crustacean fisheries, such as the American lobster fishery, could potentially influence their reproductive dynamics, leading to a reduction in mating success. This study's goal was to develop a noninvasive method for ascertaining the mating success of female lobsters that may be physiologically or functionally mature.

A Noninvasive Method For In situ Determination of Mating Success in Female American Lobsters (Homarus americanus)

Blood samples are useful for assessing biomarkers of physiological states or disease in vivo. Here we describe the methodology to sample blood from the lateral tail vein in the rat. This method provides rapid samples with minimal pain and invasiveness.

Sampling Blood from the Lateral Tail Vein of the Rat

We describe a method to collect quantifiable hemolymph efficiently from small arthropods for subsequent analysis.

A Precise and Quantifiable Method for Collecting Hemolymph from Small Arthropods

Here we present a method to collect a large volume of blood from swine using percutaneous intracardiac cannulation connected to a vacuum pump, as a terminal procedure.

Large Volume Blood Collection from Swine for In Vitro Applications: Use of Intracardiac Cannulation and a Vacuum Pump as a Terminal Procedure

Here, we present a protocol for an optimized blood collection technique in murine subjects through facial vein venipuncture using a lancet. We use this technique alongside flow cytometric assessment to characterize cellular phenotypes of transgenic, congenic, and reporter strains. It is also suitable for serum cytokine analysis and diabetes studies.

Facial Vein Venipuncture for Murine Blood Collection

Learn blood collection from the American horseshoe crab, Limulus polyphemus, for biomedical research. This method demonstrates cardiac puncture to obtain amoebocytes for bacterial detection assays.

Blood Collection from the American Horseshoe Crab, Limulus Polyphemus

Explore More Videos