Project Description

Many gram-negative bacteria naturally create compounds called endotoxins, which induce pathological symptoms including septic shock in humans. Limulus Amebocyte Lysate (LAL) testing, the gold-standard endotoxin detection test, is used in virtually every area of biomedical product development [4]. The test is derived from horseshoe crab blood, including coagulation Factor C, the primary effector protein. Many horseshoe crabs die each year due to the bleeding process, straining populations and ecosystems along the US Atlantic Coast and in Asia, where it is less sustainable [2]. Moreover, LAL testing is expensive, which creates a barrier to biomedical innovation in low-resource settings [7]. For these reasons, our team sought to synthesize a codon-optimized sequence of Factor C and integrate it into Bacillus subtilis (a gram-positive bacterium) using a pAX01 backbone with a xylose-inducible promoter. In the future, we hope to design a detection mechanism to signal the cleavage of Factor C and the presence of endotoxin.

What Are Horseshoe Crabs?

Horseshoe crabs are large, fourteen-legged marine arthropods that grow to 10 to 20 inches and feed on worms, small mollusks, and algae. They have been around for 450 million years and are often called the “living fossils” [2]. Due to the fact that they show few signs of genotypic evolution, they are incredibly useful and interesting creatures.

Image from: https://www.nwf.org/Educational-Resources/Wildlife-Guide/Invertebrates/Horseshoe-Crab

Atlantic horseshoe crabs ( Limulus polyphemus ) can be commonly found along the Atlantic coast of North America from Nova Scotia to the Yucatan Peninsula near the Gulf of Mexico. Other species like the red knot, ruddy turnstone, and sanderling rely on horseshoe crab eggs for nutrients [1]. Additionally, the horseshoe crab serves as a mini ecosystem, housing algae, worms, and barnacles on their back shell.

Yet, as of today, we find that horseshoe crab populations all over the world are being threatened. In terms of man-made stressors, the common practice of using horseshoe crabs as fertilizer and bait has nearly wiped out horseshoe crab populations along the East Coast and overharvesting is an extreme issue in Asia [8].

When our team spoke with Dr. Thomas Novitsky, the retired President/CEO of Cape Cod Biosystems, about horseshoe crab harvesting in the United States, he explained the effects of the United States’ regulations:

"The horseshoe crab harvest in the US is currently sustainable. Mortality from bleeding is estimated to be about 15% mainly due to handling, i.e. transport, storage conditions, time out of water, etc. Dr. Stanley Watson [President/Owner of Associates of Cape Cod Inc], myself, and Dr. Jack Levin [who discovered LAL] with whom I still collaborate, believe that bleeding in itself does not kill the crab. As I said, the horseshoe crab harvest in the US is sustainable and that includes crabs used for eel and whelk bait, which sadly is 100% lethal. In addition, the majority of crabs harvested are used for bait. Thus, if bait use were eliminated and bleeding practices were improved (and regulated), horseshoe crab populations would most likely increase."

However, as for the other three species of horseshoe crabs, regulations and appropriate environmental protection is sparse and far in between. Thus, in many Asian coastal ecosystems, horseshoe crab harvesting proves immense damages towards the species’ survival but also the overall health of those ecosystems.

As our team discussed with Dr. NG Wai-Chuen of the Hong Kong Agriculture, Fisheries, and Conservation Department:

"Currently, only 4 horseshoe crab species remain in the world and amongst them, all three Asian horseshoe crab species (i.e. Tachypleus tridentatus, Tachypleus gigas & Carcinoscorpius rotundicauda ) had been recorded in Hong Kong in 1990s. However, scientific studies indicated that horseshoe crab populations have dropped dramatically worldwide since the last decades and there was also a declining trend of juvenile horseshoe crabs on local shores. In recent years, only two species ( T. tridentatus & C. rotundicauda ) could be recorded in Hong Kong and they are mainly found on some sandy beaches or mudflats.

While capture for blood extraction is not known to happen here, major threats to local horseshoe crabs include coastal development which causes disturbance or loss of their habitats in particular spawning and nursery grounds; and pollution which may reduce their reproduction and survival rate. Although over-harvesting of the animal for food, fertilizer and fish bait (which is a major threat in other Asian countries) does not seem to be a concern in Hong Kong given they are not a target fishery species, scientific studies indicated that horseshoe crabs still formed a small part of the by-catch from local trawling activities."

Why Are Horseshoe Crabs Bled?

The horseshoe crab has an open circulatory system, which means all internal surfaces and organs are bathed in blood. Since any pathogen introduced to the body can easily access all vital organs, the horseshoe crab has a developed immune system that can quickly clot the toxins and seal the rest of the body from it. The crab’s blood contains amoebocytes that can detect bacterial toxins and isolate them by trapping them through a process called coagulation [9].

Image from: https://www.cnn.com/2014/09/04/health/this-crabs-blood-could-save-your-life/index.html

Due to its unique endotoxin detecting possibility, those amoebocytes are used to create the Limulus Amoebocyte Lysate test, or the LAL assay by the biomedical industry. The biomedical industry is extremely reliant on horseshoe crab blood. The price of a quart of blood is around $15,000, and LAL tests cost from $195 to $250 [6]. About 600,000 crabs are caught every year during the spawning season, drained of blood in the pericardium, and released back to the environment. In the bleeding process, more than 30% of the crab’s blood is taken and due to the large loss of blood, 20-30% of crabs die because of disorientation and changes in behavior and physiology [3]. After harvests, the reproductive abilities of females decline, which means they are less likely to breed. A recent trend shows an increase in horseshoe crab harvest.

What Is LAL and Factor C?

Limulus amebocyte lysate (LAL) is an aqueous mixture of the components making up the horseshoe crab immune cells known as amebocytes. In the horseshoe crab immune system, these amebocytes help defend the organism against pathogens. One of their functions is of particular interest: when lipopolysaccharides (LPS) are detected in the horseshoe crab’s bloodstream, a cascade of protein cleavage results in the coagulation of the infected region and thus isolation of endotoxins [7]. This result can be replicated by breaking down amebocytes with distilled water and then exposing the endotoxins to it.
Factor C is a biosensor which serves as the primer for the protein cascade. It consists of a heavy chain (80 kD) and a light chain (43 kD). Normally, it is contained inside the large granules of the amebocyte and travels to the surface of the cell when a LPS receptor binds with an endotoxin [12]. There, the phenylalanine-isoleucine bond at its light chain gets cleaved and it becomes activated as designated factor C. Designated factor C then activates zymogen factor B. Meanwhile, the serine protease zymogen factor G is activated by a glucan molecule. Both factor B and factor G perform the same task of activating the proclotting enzyme, which releases an activation peptide. The clotting enzyme cleaves two peptide bonds in coagulogen, a fibrinogen molecule specific to arthropods, to form an insoluble coagulin gel [13].

Image from: https://www.cnn.com/2014/09/04/health/this-crabs-blood-could-save-your-life/index.html