Tales of the Horseshoe Crab
Microbial Solutions
Regina Kelder

Tales of the Horseshoe Crab

Two scientists share their insights on this 444-million-year-old invertebrate.

The American horseshoe crab is uniquely valuable in vaccine contamination control. The crab's blue, copper-rich blood clots when it encounters endotoxins, highly toxic, heat-stable lipopolysaccharides that comprise the outer cell membrane of gram-negative species of bacteria like Escherichia coli, Neisseria, Salmonella, Shigella and Pseudomonas. Since the early 1970s, the horseshoe crab's bacterial endotoxin clotting response has been the basis of the highly effective Limulus Amebocyte Lysate (LAL) assay used throughout the biopharmaceutical industry to detect the presence of endotoxins in products and instruments such as injectable drugs, vaccines and surgical implants. (see Eureka blog). Approximately 70 million LAL/TAL tests are performed each year.

But the horseshoe crab is not resistant to outside forces. "Living Fossils and Blue Blood: The Story of the Horseshoe Crab and Human Health,"  a complimentary lecture held Aug. 13 at the New England Aquarium's Simons IMAX® Theatre, highlighted some of those challenges. One major concern in certain geographic regions is the widespread harvesting of horseshoe crabs for eel and whelk bait. This practice is illegal in South Carolina, but still allowed, though with some restrictions, along most of the Mid-Atlantic and New England seaboard. (New Jersey, which has moratorium on all horseshoe crab harvesting, is the exception.) But the development of artificial bait could help reduce the pressure on the adult horseshoe crab population, says Kathryn Tuxbury, Associate Veterinarian at the New England Aquarium, who kicked off the lecture last Thursday.

Tuxbury referenced Nancy Targett, a University of Delaware researcher, who has developed a bait product that combines compounds found in brown seaweeds and kelp, with a small amount of coarsely ground horseshoe crab and food-grade chemicals including baking soda and citric acid. The material forms a gelatinous material that keeps up to four days. Tuxbury said researchers have also substituted invasive Asian crab in their artificial bait, further reducing reliance on the horseshoe crab.

The artificial bait is currently undergoing a large study in Massachusetts to see if the eel and whelk like it, so time will tell if it has any lasting impact on conservation efforts. Studies conducted by Tuxbury 15 years ago, and more current surveys, suggest that horseshoe crab populations in New York and New England are declining, unlike in South Carolina, where harvesting of crabs is restricted to biomedical manufacturing, or in Maryland and Delaware, where fisherman are prohibited from harvesting female horseshoe crabs. Data suggest the populations in the Southeast and Mid-Atlantic are stable or rising, according to the Atlantic States Marine Fisheries Commission.

Here are a few other highlights of the lecture, which included John Dubczak, General Manager of Charles River's Endotoxin and Microbial Detection Division.

Fungal Infections and Nutritional Deficiencies.

Horseshoe crabs face a variety of challenges in the wilderness. But one threat that seems largely confined to captive populations, such as those residing in aquariums, are fungal infections. During an internship at the National Aquarium in Baltimore Tuxbury and others characterized a fungus Fusarium solani that causes the gills to thicken abnormally and discolor.  It can become systemic and cause widespread damage in crabs, and they eventually succumb to it.  The researchers found Fusarium solani in eight out of 10 tissue samples that they cultured; they published the study last year. Tuxbury said one can treat the fungus, but it's long-term and quite expensive.  Diet can also affect the well-being of these arthropods. Because captive horseshoe crabs can't feast on the worms, clams, detritus, dead fish and algae that their wild sisters and brothers are able to get to, they run the risk of developing protein deficiencies. To counter these multiple challenges, the New England Aquarium tries to minimize the stress on horseshoe crabs as much as possible, says Tuxbury. They make sure the animals are submerged in water almost all of the time and that they are not over-used for onsite and offsite demonstrations. The crabs are examined every three months and aquarium staff monitors the water quality and temperature regularly.

The Big Bang.

It was Dr. Jack Levin, a hematology researcher at Johns Hopkins, who determined that the amebocytes in horseshoe crab blood were responsible for the clotting, and that endotoxins from the bacteria were the trigger. But it was Dr. Frederik Bang, a pathologist at Johns Hopkins interested in diseases of marine animals, who actually discovered LAL in the blood of American horseshoe crabs in the 1950s. While studying the crabs' response to bacterial injections Bang found that the blood coagulated in the presence of the contamination, and that the product of the blood cells produced a gel when exposed to gram-negative bacteria.  A decade later, Bang and Levin began collaborating on studies looking at the mechanism by which endotoxins mediate the coagulation of LAL, says Dubczak.  They were also interested in the development of a clinical test, which led them to another Johns Hopkins scientist, James Cooper (and the founder of CRL's Endosafe division). Cooper was studying radiopharamceuticals, whose short lives made the rabbit pyrogen test used then for endotoxin detection impractical. Cooper applied the LAL test, found it worked even better than the rabbit test. The rest, as they say, is history.

How to cite:

McEnery, Regina. Tales of the Horseshoe Crab. Eureka blog. August 17, 2015. Available: https://eureka.criver.com/tales-of-the-horseshoe-crab/