JoVE   
You do not have subscription access to articles in this section. Learn more about access.

  JoVE Biology

  
You do not have subscription access to articles in this section. Learn more about access.

  JoVE Neuroscience

  
You do not have subscription access to articles in this section. Learn more about access.

  JoVE Immunology and Infection

  
You do not have subscription access to articles in this section. Learn more about access.

  JoVE Clinical and Translational Medicine

  
You do not have subscription access to articles in this section. Learn more about access.

  JoVE Bioengineering

  
You do not have subscription access to articles in this section. Learn more about access.

  JoVE Applied Physics

  
You do not have subscription access to articles in this section. Learn more about access.

  JoVE Chemistry

  
You do not have subscription access to articles in this section. Learn more about access.

  JoVE Behavior

  
You do not have subscription access to articles in this section. Learn more about access.

  JoVE Environment

|   

JoVE Science Education

General Laboratory Techniques

You do not have subscription access to videos in this collection. Learn more about access.

Basic Methods in Cellular and Molecular Biology

You do not have subscription access to videos in this collection. Learn more about access.

Model Organisms I

You do not have subscription access to videos in this collection. Learn more about access.

Model Organisms II

You do not have subscription access to videos in this collection. Learn more about access.

Essentials of
Neuroscience

You do not have subscription access to videos in this collection. Learn more about access.

Essentials of Developmental Biology

You have subscription access to videos in this collection through your user account.

 JoVE Biology

Blood Collection from the American Horseshoe Crab, Limulus Polyphemus

1,2, 3

1Department of Molecular and Cell Biology, University of California, Davis, 2Marine Biological Laboratory - MBL- woods hole, 3Department of Biological Sciences, Hunter College of CUNY

Article
    Downloads Comments Metrics

    You must be subscribed to JoVE to access this content.

    This article is a part of   JoVE Biology. If you think this article would be useful for your research, please recommend JoVE to your institution's librarian.

    Recommend JoVE to Your Librarian

    Current Access Through Your IP Address

    You do not have access to any JoVE content through your current IP address.

    IP: 54.81.123.248, User IP: 54.81.123.248, User IP Hex: 911309816

    Current Access Through Your Registered Email Address

    You aren't signed into JoVE. If your institution subscribes to JoVE, please or create an account with your institutional email address to access this content.

     

    Summary

    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 α2-macroglobulin. Blood is collected from the heart and the blood cells and plasma are separated by centrifugation.

    Date Published: 10/13/2008, Issue 20; doi: 10.3791/958

    Cite this Article

    Armstrong, P., Conrad, M. Blood Collection from the American Horseshoe Crab, Limulus Polyphemus. J. Vis. Exp. (20), e958, doi:10.3791/958 (2008).

    Abstract

    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 α2-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.

    Protocol

    Anatomical features of the horseshoe crab relevant to bleeding (Fig. 1)

    Figure 1

    Figure 1

    1. The three major divisions of the body, from anterior to posterior, are the prosoma (P), the opisthosoma (O) , and the telson (T) 1.
    2. The anterior and lateral free margins of the prosoma is the flange.
    3. The posterior-most indentation of the opisthosoma, where the telson articulates, is the terminal bay.
    4. The joint where the prosoma and the opisthosoma articulate is the hinge (H).
    5. The heart is located along the dorsal midline, just beneath the carapace of prostoma and opisthosoma2 (Fig. 2).


      Figure 2

      Figure 2

    General considerations, sterility and protection from exposure to lipopolysaccharide (endotoxin)

    1. The major enemy of the successful bleed is cell clumping, exocytosis, and formation of the coagulin clot. Cell aggregation and exocytosis are stimulated by lipopolysaccharide (a.k.a., endotoxin, LPS), a product of the Gram-negative bacteria. The threshold concentration of LPS for exocytosis for washed cells is 0.1 - 1 mg/mL but cells suspended in plasma are activated by significantly lower concentrations 3. Endotoxin is not inactivated by simple autoclaving and can be assumed to be present on surfaces and in solutions and reagents that are not certified to be endotoxin-free.
    2. To reduce the chances of clotting during collection of blood, select only perfect, undamaged animals for bleeding. Pre-chill the animals 1-2 h in the 4°C room. Practice sterile technique. Avoid contamination with endotoxin.
    3. LPS-free 3% saline is used in clinical medicine and can be purchased from medical suppliers (see table of specialized reagents and supplies). LPS can be removed from glassware and metal by incubation at 180°C for 4 hours. Sterile single-use syringe needles, Petri-style culture dishes, and screw-cap centrifuge tubes are all LPS-free and can be used without modification so long as proper sterile technique is practiced.
    4. The stability of the blood cells of different animals differs, with the cells of some individual animals undergoing spontaneous degranulation, resulting in the formation of the coagulin clot, even when the animal is bled with the greatest care. The blood cells of other individual horseshoe crabs are significantly more stable and, when proper procedures are followed, remain fully granular during bleeding and separation of cells from plasma.
    5. Several agents have been reported to stabilize the blood cells following bleeding, including caffeine (bleed into 0.25 volumes of 10 mM caffeine in LPS-free 3% NaCl) 4, propranolol (1 mM final concentration) 5, dimethylsulfoxide 6, divalent cation chelation (bleed into an equal volume of 0.1 M dextrose, 0.03 M sodium citrate, 0.026 M citric acid, 10 mM disodium ethylenediaminetetraacetic acid (Na2EDTA), pH 4.6) 7, inhibitors of the G-protein and phospholipase-C pathways (cholera and pertussus toxins, U73122) 8, substitution of chloride with isethionate anion 9, chloride channel blockers 9, cyclic-AMP antagonists 9, sulfhydryl reagents (5 mM N-ethyl maleimide, NEM) 5, and the membrane-active detergent Tween-20 (bleed into 0.1 volume 2% Tween-20 in LPS-free 0.5 M NaCl).

    Supplies and reagents for bleeding the horseshoe crab: collection of large volumes of blood

    1. one or more large, undamaged horseshoe crab
    2. squirt bottle containing 70% ethanol
    3. Kimwipes
    4. 14 gauge needle
    5. sturdy forceps
    6. ice bucket with ice
    7. 50 mL screw-cap plastic disposable centrifuge tubes
    8. rack to hold the 50 mL tubes
    9. counter-top centrifuge
    10. sterile 50 mL serological pipettes
    11. bulb-type pipette filler
    12. disposable sterile filter apparatus
    13. vacuum pump
    14. LPS-free 3% NaCl solution (for collection of blood cells)
    15. Tween-20 (for collection of blood cells)

    Preparations for large-volume bleeding

    1. For bleeding, use large, undamaged animals. Chill the animal for 1 h in a cold room before bleeding.
    2. Establish a comfortable arrangement with a stable support for an ice-bucket that is about 0.3 meters high (the disposable Styrofoam shipping container for mailing cold or frozen reagents is appropriate) and a low chair for the operator. The ice bucket is placed on the support structure and the chair is drawn up to this unit. You should be comfortable sitting for an extended period in the chair with your elbows resting on your thighs, and with the left hand holding a struggling horseshoe crab positioned above the blood collection tubes propped upright in a bed of ice in the ice bucket.
    3. Pre-chill 4 - 6 of the 50 mL disposable plastic centrifuge tubes with screw closures in the ice bath. The tubes are set upright in the ice ranged around the perimeter of the ice bath container. Just before starting the bleeding operation, remove the caps from the tubes and place caps on adjacent table, adhering to sterile technique.
    4. Remove the cap of a 14 ga needle and use the sturdy forceps to loosen the needle in its sleeve, but do not remove needle from the sleeve. MAINTAIN STERILITY. DO NOT TOUCH ANY PART OF THE NEEDLE WITH THE FINGERS. Place sleeve with needle on an adjacent counter with the exposed butt of the needle elevated from contact with any surface to maintain its sterility.
    5. Moisten a Kimwipe with 70% ethanol.

    Bleeding the animal (specifically for the right-handed operator)

    1. Holding the animal for bleeding: grasp animal in the left hand with the four fingers in contact with the anterior flange of the prosoma and the thumb grasping the posterior part of the opisthosma at the base of the tail (the terminal bay). The ventral surface of the animal faces the palm of your hand. The telson lies along the base of the thumb and projects over the wrist. Flex the animal gently so that prosoma and opisthosoma are at right angles to one another (Figure 3a).

      Figure 3

      Figure 3

    2. Clean the exposed hinge joint connecting prosoma and opisthosoma with 70% ethanol applied with an ethanol-moistened Kimwipe (Figure 3b).
    3. Rotate the left hand such that fingers 2 - 4 are uppermost and the dorsal surface of the animal faces up. With the right hand, insert the 14 ga syringe needle, still in its protective sleeve, between fingers 2 and 3 of the left hand. Take the forceps in the right hand and remove the needle from its protective sleeve by grasping the butt with the forceps (Figure 3c). Rotate the left hand so that dorsal surface of the hand faces to your left, thumb and index finger are up, and the animal is upside down, with its dorsal surface facing the right hand. Position the butt of the needle above the first of the 50 mL collection tubes and orient the animal so that the sharp end of the needle is pointed at the hinge joint and so that the needle shaft is parallel to the dorsal midline (Figure 3d). Insert the sharp end of the needle through the hinge joint into the lumen of the heart, keeping the needle positioned parallel to the dorsal midline and inserted at the midline and with the butt of the needle still positioned above the opening of the collection tube. The needle is inserted about 1 - 2 cm deep into the portion of the heart in the prosoma. As soon as the heart is punctured, blood will flow rapidly (Figure 4a, arrow), so it is important to ensure that the delivery end of the syringe needle is positioned over the mouth of the collection tube to collect that initial surge of blood.
    4. Blood will exit the heart initially in a continuous stream that, after about 30 - 50 mL of blood have been collected, will slow to individual drops (Figure 4b). To improve blood flow, it may be good policy to re-orient the tip of the needle slightly. Tilt the needle up or down, left or right; slide its tip deeper into the heart or pull it to closer to the hinge joint to find the position that optimizes blood flow. Do not compress the animal excessively, because this risks damaging the animal and may cause the extravasated blood to initiate clotting.

      Figure 4

      Figure 4

    5. As each collection tube fills with blood, rotate the ice bath to bring the next empty tube beneath the delivery end of the needle.
    6. As bleeding slows, you run the risk of having air flow in a retrograde direction into the needle and into the heart. This can occur when the animal straightens out, thereby reducing the blood pressure. Try to prevent this by maintaining the animal in the flexed position or by allowing straightening to occur only slowly.
    7. When the animal stops giving blood, remove the bleeding needle from the heart. Re-cap the collection tubes and centrifuge immediately in the table-top centrifuge, 1000 RPM, 5 min. The blood cells will form compact pellets at the bottoms of the collection tubes. Because all of the components for blood clotting are contained within secretory vesicles of the blood cells, as soon as plasma is separated from cells, the risk of clotting of the plasma is eliminated.
    8. It is imperative to avoid clotting of the extravasated blood. The following measures reduce the risk of clotting: Pre-chilling the animal and maintaining the collection tubes in an ice bath; gentle handling of the animal; reduction of the rapidity with which the blood flows out of the syringe needle; centrifugation of the blood immediately after collection; avoidance of contamination of needles and collection tubes with endotoxin; maintenance of sterile procedure. As was mentioned above, some individual animals have especially irritable blood cells that initiate exocytosis even when these precautions are followed.
    9. After centrifugation, inspect the tubes for signs of blood clotting - strands of cells and gelatinous clot material attached to the walls of the tube, gelatinous material at the top of the clot, or, worse, large amounts of clot with entrapped blood cells in the fluid column (Figure 4c, arrow). The material from these tubes is serum, not plasma, and contains the secreted products of the blood cells.
    10. Transfer the plasma to sterile tubes with the 50 mL sterile serological pipette Exercise care when pipetting to avoid aspirating any of the cells from the cell pellet in the bottom of the tube. It is best to leave a few mL of plasma above the pellet to avoid disturbing the cell pellet.
    11. The procedure described above is designed to collect large volumes of plasma uncontaminated by the secretory products of the blood cells. To collect large volumes of washed blood cells, collect the blood into 0.1 volumes of 2% Tween-20 dissolved in 3% LPS-free NaCl solution. Centrifuge the cells gently for 10 minutes to establish a loose cell pellet, then wash the cells with several changes of LPS-free 3% NaCl. An extract of the cells that contains the secretory products of the blood cells can be prepared by lysing the washed cells in LPS-free distilled water. This lysate contains a diverse array of proteins and peptides that operate in anti-microbial immune defense 10,11 and also contains coagulogen, the zymogen form of the structural protein of the extracellular blood clot, and the collection of proteases that convert coagulogen to coagulin, the form of the protein that polymerizes into the fibrils of the clot 12. This extract is similar to the commercially available product, Limulus amebocyte lysate or LAL, which is used to assay for the presence of lipopolysaccharide 13. The protease cascade responsible for proteolytic modification of coagulogen to coagulin is activated by LPS 14.
    12. Disposition of animals after bleeding. Bleeding conducted by the methods identified above is tolerated well by the animal. When working at the Marine Biological Laboratory at Woods Hole, I return animals to the ocean after bleeding with the expectation that they will survive. If animals are maintained in aquaria distant from the normal range of horseshoe crabs, they can be bled repeatedly once or twice a month. As is described below, animals maintained in captivity require high quality water and frequent feeding to maintain their health. Euthanasia can be accomplished by destruction of the dorsal ganglion, which is situated in the dorsal midline between the eyes.

    Processing and storage of plasma

    1. Protection from proteases. If the plasma is to be used for protein purification, it should be treated with protease inhibitors immediately after collection. Add phenylmethylsulfonylfluoride (PMSF) to a final concentration of 1 mM. PMSF is kept as a 0.1 M stock solution in DMSO or ethanol. It is quite unstable in water 15, so cannot be relied on for lasting protection, but addition immediately after separation of plasma from blood cells will inactivate the small quantities of proteases that might have been released from the cells.
    2. Sterile filtration: sterilize the plasma by ultrafiltration under vacuum using a disposable medium-filtration device fitted with a 0.22 mm pore filter. These filters have a tendency to clog. It is advisable to centrifuge the plasma before filtering to reduce this problem. If large volumes of plasma are processed, it is advisable to pre-filter the plasma first using a Millipore filter with a 1 mm pore size, then with a 0.45 mm pore size Millipore filter.
    3. Plasma can be protected from bacterial contamination either by adding NaN3 to a final concentration of 0.2 mg/mL or by sterile filtration. Unsterile plasma should never be stored for periods longer than 2 days without one or the other of these anti-bacterial measures being taken.
    4. Blood clotting. Unlike the mammalian clotting system, the Limulus plasma contains none of the elements for blood clotting. Instead, the entire machinery for clot formation, the structural protein of the clot (coagulogen) and the system of proteases that process coagulogen to render it capable of polymerizing into the insoluble clot, is found in the secretory granules of the blood cells 16. So if the blood cells are removed before they have a chance to undergo exocytosis, the plasma is entirely free of the proteins for clotting and will remain fluid indefinitely.
    5. Protein purification. Hemocyanin is present at 40 mg/mL in plasma and is a 48-mer of a 70 kDa subunit. It can be isolated by ultracentrifugation (40,000 X g, 8 h) or gel filtration with a large pore size exclusion resin such as BioGel A5m 17. The C-reactive proteins are present at 1 - 5 mg/mL and can be isolated by affinity isolation on phosphorylethanolamine-Sepharose, with elution with a calcium chelator 18. The broad-spectrum protease inhibitor, a2-macroglogulin, has been purified by gel filtration on Sephacryl S-300 resin after hemocyanin and the C-reactive proteins have been removed 19.

    Microscopic examination of the motile blood cells in culture

    1. Establishment of amebocyte lawns in vitro: LPS-free 3% NaCl solution is added to sterile plastic Petri-style culture dishes (1 mL for a 35 mm dish, 2.5 mL for a 60 mm dish, 6 mL for a 90 mL dish). Blood is then obtained by cardiac puncture using the method described above, but with the modification that a 23 gauge, 1 in sterile syringe needle is used instead of the 14 gauge needle. Blood flows drop-by-drop from the 23 gauge needle and 1 drop is collected into the 35 mm dish, 3 drops into the 60 mm dish, and 9 drops into the 100 mm dish. The blood cells are then mixed uniformly in the 3% NaCl solution by gentle swirling, first in a circular pattern, then back-and-forth. The preparation is incubated for 5 min at room T to allow blood cells to attach to the dish surface, then the initial saline is replaced with a buffer of choice3.
    2. If it is desired to maintain the substratum-attached blood cells in an undegranulated condition, the initial saline-blood plasma mixture is replaced with fresh LPS-free 3% NaCl. Plasma accelerates the spontaneous degranulation of the blood cells even in the absence of LPS, and its removal stabilizes the undegranulated state of the cells.
    3. If the initial saline is replaced with sterile plasma, the blood cells transform from the ovoid shape of the circulating blood cell (Fig. 5) and flatten onto the substratum, and then initiate degranulation and the formation of a layer of the coagulin extracellular blood clot above the lawn of flattened amebocytes 20.

      Figure 5

      Figure 5

    4. Establishment of explant cultures of aggregated amebocytes. When blood is collected under sterile, LPS-free conditions, the blood cells settle out from suspension and aggregate to form a tissue-like mass. A convenient container for this is the glass embryo dish rendered LPS-free by incubation for 4 h at 1800C. Blood collected in this container by cardiac puncture using a 19 gauge needle is incubated for 1 - 2 h at room T, then pieces 1 mm square are cut from the amebocyte aggregate with a sterile 18 gauge syringe needle and are sandwiched between two coverglasses or a coverglass and a slide separated by fragments of coverglasses to act as spacers. After about half an hour, amebocytes begin migrating from the periphery of the explant onto the coverglass (Fig. 6a), where they can be viewed with phase contrast or DIC optics 21. Initially, the migrating cells are compact with hyaline pseudopods (Fig. 6b (m)) that are extended above the migration substratum, and that then are lowered into contact with the surface and forward motion involves the flow of the granular cytoplasm into the pseudopod 22. Later, the cells flatten (Fig. 6b (f)) and initiate degranulation (Fig. 6b (d)) and cease locomotion 23. The culture chamber described here allows the perfusion of different experimental agents into the culture chamber to investigate their effects on cell motility and granule exocytosis 9. An extended methodological review of amebocyte culture for microscopic examination can be found in 24.

      Figure 6

      Figure 6

    Maintenance of adult horseshoe crabs in aquaria

    Horseshoe crabs require high quality sea water and can be maintained in aquaria fitted with a water purification system that provides particle filtration, aeration, and denitrification of the water25. Animals should be fed 2 or 3 times a week on shrimp, lobster, fish, or squid. Feeding is accomplished by removing the animal from the aquarium, placing it on its back, and placing the food on the mouth. If hungry, the animal will soon begin swallowing the food. Depending on the sophistication of the sea water system, the animal should be maintained for an hour or so until it defecates in a separate container of sea water or it can be returned to the aquarium after feeding has begun. For long-term maintenance, the animal should be kept away from light to prevent the growth of green or blue-green algae on the surface of the carapace. Algae erode the carapace and since the adult does not molt, this will cause death to the animal 26. In the wild, animals spend much of the time partially buried in sand, but sand or gravel in the aquarium presents a difficulty in maintaining the level of cleanliness important for long-term maintenance.

    Subscription Required. Please recommend JoVE to your librarian.

    Discussion

    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 eel and conch fisheries29, the American horseshoe crab is still reasonably plentiful and allows the non-destructive collection of 50 mL of blood from a small adult and as much as 400 mL from a large female. Collection of even this much blood can be completed in less than 10 minutes. I know of no other readily available invertebrate that affords so much blood with so little effort.

    The importance of the LAL test for endotoxin, an indicator for the presence of Gram-negative bacteria, has stimulated interest in the immune system of the horseshoe crab13. The LAL test depends on the activation of the initiating protease of the blood clotting system by LPS14, with a read-out of clot formation or of a colorimetric assay for protease activity. The two elements of immunity that have received the greatest attention have been the proteins of the plasma and the several proteins and peptides of the secretory granules of the blood cells. The result has been the elevation of the horseshoe crab to the status of having the best-described immune system for any long-lived invertebrate11

    Subscription Required. Please recommend JoVE to your librarian.

    Disclosures

    Acknowledgements

    Original research described above was supported by NSF grant 0344630.

    References

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

    Comments

    13 Comments

    Thank you for this great video! How long can you keep the amebocytes in culture? Can they be frozen for long-term storage? Best wishes, Christine
    Reply

    Posted by: Christine B.October 21, 2008, 6:34 PM

    The amebocytes will survive for several days when cultured in sterile Limulus plasma.  The cells that initially migrate rapidly over the culture surface remain motile for only a short period - minutes to an hour or so, then flatten on the culture surface (microscope slide, Petri dish surface) and degranulate.  These highly flattened cells remain alive for an extended period but are no longer migratory and do not alter their shape.
    I have not tried freezing Limulus amebocytes for long-term storage.  Because the cells are so prone to degranulate when removed from the animal, I suspect that they might degranulate during processing with the methods that are standard for the freezing of vertebrate tissue culture cells.  It is possible that the combination of one or more of the agents that retard cellular degranulation (see item #5 in the section, “General considerations...” of the written article) with the cryoprotectants used to freeze-preserve vertebrate culture cells might be successful for freeze-preserving the Limulus amebocyte.
    Reply

    Posted by: AnonymousApril 23, 2009, 8:39 PM

    thank you for the great answer!
    Reply

    Posted by: Andy W.March 5, 2014, 7:44 AM

    Thanks very much for posting this interesting and informative article! What percentage of horseshΠcrabs survive the experience of blood donating? Also, since it's so beneficial to us, is there some way to enhance the experience, for the horseshΠcrab, to make it nicer, and maybe also increase the survival rate? Thanks very much for any further information you can give, Pat
    Reply

    Posted by: AnonymousDecember 29, 2008, 11:08 AM

    The mortality of horseshŒ crabs that have been subjected to the bleeding methods depicted in the video is very low.  We found that animals tolerated multiple episodes of large-volume bleeding over a period of two months without any deaths.  This indicates that animals that are bled only once, then returned promptly to the ocean are at a very low risk of dying.
    I have seen no evidence that horseshŒ crabs experience noticeable discomfort during the bleeding process.  The hinge joint, which is the site of insertion of the blood collection needle, appears to have little in the way of nervous innervation or pain receptors.  Probably the most important concern is in the conditions of maintenance of animals in aquaria - clean aquarium conditions, clean water, appropriate feeding, etc.  It is important for long-term maintenance in aquaria that the animals be maintained at low light levels to reduce the possibility for the growth of green and blue-green algae on the surface of the carapace because this leads to erosion of the carapace and death of the animal.
    Reply

    Posted by: AnonymousApril 23, 2009, 6:52 PM

    I hope my post went through.                            Pat
    Reply

    Posted by: AnonymousDecember 29, 2008, 11:09 AM

    A very thorough description, but I do not personally have access to live horseshΠcrabs. How might I go about obtaining a sample of horseshΠcrab blood?
    Reply

    Posted by: AnonymousJune 22, 2009, 12:34 PM

    June ²5, ²009
    I do not know of a supplier of Limulus hemolmph. Pel-Freeze Biologicals [http://www.pelfreez-bio.com/Home.asp] used to sell this but they no longer stock this product. It is possible to purchase live horseshŒ crabs (Marine Biological Laboratory, Woods Hole, 508-²89-7375, [specimens@MBL.EDU]), and then to follow the instructions in the video to obtain blood from the animal. If blood preparation is a one-time action, then you would be stuck with a live horseshŒ crab to house and feed but with no further use for the animal. If that is an unappealing prospect , then probably the best solution to your problem would be to attempt to establish a collaboration with some investigator bleeds horseshŒ crabs and who could provide you with a sample of hemolymph.
    Reply

    Posted by: AnonymousJune 25, 2009, 4:20 PM

    Dear Pete,

    Thank you for a very informative video, i enjoyed watching it very much! I am interested in carrying out research on horseshΠcrabs. Please can you advise me as to how old the horseshΠcrab has to be before it can be bled? thank you very much for your time.

    Mo
    Reply

    Posted by: Mo h.April 9, 2010, 2:16 PM

    January 6, ²011
    Dear Mo,
    It is possible to bleed even juvenile horseshŒ crabs. I use short, #²7 or #²5 needles and aspirate the blood slowly into a syringe. It is possible to collect only small volumes of blood from the juvenile animal, so for experiments requiring large volumes, the adult is the best choice.
    Peter Armstrong
    Reply

    Posted by: AnonymousJanuary 6, 2011, 4:44 PM

    This is a very good video for handling horseshΠcrab bleeding. I really enjoyed to watch it!
    Do the flatten cells have the same quality as well as active cells do?
    Can you fix your PDF file because it cannot be downloaded?
    Thank you.
    James
    Reply

    Posted by: AnonymousMay 3, 2011, 1:56 AM

    isolation of blood from horseshΠcrabs
    Reply

    Posted by: AnonymousNovember 1, 2011, 2:29 PM

    What would be the feasibility of more companies opening up to the harvesting of horseshΠcrab blood? As it seems this process could be duplicated with relative ease, what is the likelihood of more corporations cropping up with the intent of bleeding horseshΠcrabs?
    Thank you for your time.

    -William
    Reply

    Posted by: William S.December 9, 2011, 1:30 PM

    Post a Question / Comment / Request

    You must be signed in to post a comment. Please or create an account.

    Metrics

    Waiting
    simple hit counter