Improving the Bleeding Protocol for Horseshoe Crabs With the Development of a Partially- Underwater Bleeding Apparatus

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Authors: Rohan Arora & Evan London

Peer Reviewer: Janice Rateshwar

Professional Reviewer: Dr. Shawn Stickler


Abstract

The horseshoe crab population has been on the rapid decline due to the exploitation of horseshoe crab blood. Horseshoe crab blood has important components such as hemocyanin that are vital for the isolation of Limulus amebocyte lysate (LAL). In the scientific industry, LAL is used in bacterial endotoxin testing, which it helpful in pharmaceutical development. Recognizing that the horseshoe crab population has been quickly declining, scientists have attributed this to the severe hypoxic conditions they face during the industry-standard bleeding process. The current industry-standard bleeding process is unsustainable considering the decline in horseshoe crab population; therefore, the aim of this research is to develop and test an apparatus that is partially submerged in an effort to reduce the degree of hypoxic conditions suffered by horseshoe crabs during the bleeding process and to increase the survival rates of these crabs post-bleeding. In order to achieve the objectives outlined for the study, the researchers set up the lab equipment through an extensive process that took into account ideal aquaculture for the horseshoe crabs. In addition to this, they planned and constructed a prototype of the developed apparatus. Through an extensive testing process, the research suggests the developed prototype was effective in increasing the survival rates of the horseshoe crabs within the post-bleeding critical period in comparison to the industry-standard bleeding protocol. The research also suggests the dire need to update the current bleeding protocol that exists in order to promote sustainability. In addition to this, the implications of this study are significant; scientists that build upon this study have the potential of expanding this prototype to more industrial settings and directly testing for reductions in hypoxic conditions. 


Introduction

With the advent of biotechnology in the past century, several terrestrial and marine organisms have been found to be extremely useful in the medical and pharmaceutical industry. One of the most influential of these organisms is the Atlantic horseshoe crab, scientifically known as Limulus polyphemus. For the past several decades, horseshoe crabs have been exploited due to the high hemocyanin concentration in their blood. Hemocyanin is used for the isolation of Limulus amebocyte lysate (LAL). LAL is used for bacterial endotoxin testing, which helps detect and quantify endotoxins from Gram-negative bacteria, and the main significance for this testing is to ensure that pharmaceutical products that are currently being developed do not contain dangerous pyrogenic substances, such as the bacterial endotoxin, which is known to cause fever in mammals (“Bacterial Endotoxins Test”). 

 Although LAL is especially useful for industry bacterial endotoxin testing, the current practice of bleeding horseshoe crabs is unsustainable, as supported by the high mortality rate of horseshoe crabs (Van Holde, 1995). The current protocol for the horseshoe crab bleeding process exposes horseshoe crabs to hypoxic conditions because the crabs are not submerged in water for the majority of the bleeding process (Walls & Berkson, 2003). In order to address the unsustainability of the current protocol, the researchers hope to develop a better protocol for the bleeding process that incorporates a new apparatus that avoids exposure to hypoxic conditions by bleeding the crabs with the bulk of their body, including their gills, entirely underwater. By developing a system in which these hypoxic conditions are avoided, the researchers will be able to benefit biomedical companies and horseshoe crab populations. After the development of the system, the researchers will test the newly developed protocol by bleeding the control group with the industry-standard protocol and bleeding the treatment group with the newly developed apparatus. The treatment group would have lesser exposure to hypoxic conditions, and the researchers would be able to analyze the effectiveness of the developed system in terms of mortality rate. 

As introduced earlier, horseshoe crab bleeding is a common practice in the biomedical industry; however, horseshoe crab populations are declining at a rapid rate. The main justification for the researchers’ proposal to improve the protocol with the development of a new apparatus is the high mortality rate of horseshoe crabs caused by exposure to hypoxic conditions during the industry-standard bleeding protocol. The root cause for this hypoxic condition is the fact that they are not submerged in water for the majority of the practice. The crabs are also exposed to hypoxic conditions during the shipping and handling procedures in their capturing and bleeding process. According to a study conducted by Elizabeth A. Walls and Jim Berkson at Virginia Tech in 2003, unbled crabs showed an extremely low mortality rate, while bled crabs during a two-week post-bleeding window showed a much higher rate. After analyzing the extensive data collected, Walls and Berkson found that unbled crabs have a mortality rate of roughly 0.5% while bled crabs have an average mortality rate of 8%, a figure that likely increased after publication of the 2003 study.

By addressing this issue head-on, the researchers for this study will be able to develop a system that makes the bleeding process more sustainable and efficient for biomedical companies and horseshoe populations. The innovative system the researchers developed and its respective scientific study will yield additions to existing protocol and bleeding apparatus that can be used by these same biomedical companies to enhance the existing industry standard for horseshoe crab bleeding. In the past, studies have analyzed hypoxic conditions in horseshoe crabs in various conditions. The majority of studies measure oxygen levels in horseshoe crabs by taking hemolymph samples. Hemolymph contains components that fulfill those blood and lymph-based functions. For instance, hemolymph contains hemocyanin, a protein that binds oxygen similar to hemoglobin. 

Hemolymph samples are extremely useful in determining oxygen levels of the crabs’ blood, and many important discoveries have been made from such samples. A study conducted by Allender et al (2010) obtained the hemolymph sample from the crabs’ cardiac sinus. Then, they measured oxygen content by inserting the sample into a specific CG4+ cartridge and then placed it into an Abbot Point of Care i-stat blood analyzer. After analyzing the data from the blood analyzer, they found that evolutionarily horseshoe crabs are able to maximize oxygen uptake in long-term ‘out of water’ conditions; however, they suffer severe hypoxia from short-term removal from the water, a common practice in the horseshoe crab bleeding industry.  The principal justification for this study lies in the fact that the scientific community needs to increase the sustainability of the horseshoe crab bleeding process and help these declining horseshoe populations from the factors that are most linked to their high mortality rate. For that reason, it is vital that scientists look into the development of a submerged bleeding process to prevent hypoxic conditions in crabs.


Methods 


Lab Setup

In order to achieve the objective effectively, the research team divided the study into two phases consisting of both development and testing of the partially-submerged bleeding apparatus. The researchers began the study by ordering horseshoe crabs from KP Aquatics and setting up tanks for the horseshoe crabs used for the testing phase of the study. On December 11th, 2018, the researchers acquired a utility rack next to the lab bench for the horseshoe crab tanks. The researchers then placed twelve empty ten-gallon tanks onto the utility rack, labeling them by number from 1 to 12. Using zip-ties, they attached two extension cords, which were connected to the existing cord reel above the utility rack, to the middle beam connectors in the utility rack. After placing the tanks and attaching extension cords to the beam connectors, they  proceeded to construct filters for the tanks using 20-gallon AquaClear tank filters with CycleGuard and BioMax. The protocol for the construction of these tank filters was outlined in the instructions provided in the box. After testing the twelve filters, the researchers assigned each filter to a specific tank and placed it on the ridge of each tank. Shortly after the tanks started to successfully filter water, they filled the tanks with saltwater. For the next several weeks, they continued to monitor the salinity of the tanks using the Instant Ocean hydrometer in the lab. The researchers aimed for a salinity between 20-35 parts per thousand and recorded the salinity in the tanks regularly to ensure it was between that range. When the salinity was too low, they refilled the evaporated water with saltwater; when the salinity was too high, they emptied some of the water out and refilled it with freshwater. Every week, the researchers refilled the tanks with freshwater due to water lost to evaporation. They also added API Stress Coat Aquarium Conditioner fluid with the instructed dosage. In summary, the researchers set up twelve tanks with the proper aquaculture and environment for horseshoe crabs. 


Introduction of Horseshoe Crabs

After receiving the shipment of horseshoe crabs, the researchers opened the box and observed that the crab supplier provided the team with three extra specimens. All the crabs appeared to be male and had an approximate diameter of one inch. The researchers introduced two crabs each to tanks 1-4 and 8-12, and three each to tanks 5-7 to accomodate for the extras. They fed the crabs biweekly with API Sinking Shrimp Pellets Bottom Feeder Fish Food and continued to monitor salinity and temperature of the water to ensure it was within the range allotted. They separated the tanks on the data tables into different groups with the crabs in tanks 1-6 being the treatment group and those in 7-12 as the control groups. 


Construction of Apparatus

In order to develop the apparatus, the researchers first began to sketch out the general outline of what they wanted it to look like. Recognizing that the study had a tight budget, the researchers attempted to use cheaper alternatives to what researchers in the industry would use to construct an industrial-grade apparatus. After further refining the plan and identifying relevant parts, they developed a sketch of the prototype and purchased the necessary materials.

Figure 1: Final sketch of the prototype apparatus.


Below is a list of the materials used to construct the apparatus:

Materials

  • Two ? in. wing nuts
  • 24 half-inch flat washers
  • Two ½ in. galvanized steel hex bolts
  • Six large paper clips
  • One 10-inch mini bungee cord
  • Hot glue gun
  • Watertight plumbing epoxy
  • Hand drill with bit
  • Scrap wood
  • Two Sterilite storage containers
  • White corrugated plastic sheet
  • Scissors
  • X-Acto stainless steel single-blade knife

After obtaining the necessary materials, they cleared a lab bench to construct the apparatus. They began by first cutting out a 4.5 in. by 10 in. panel out of the larger sheet using an X-Acto knife. After this, the researchers put the sheet aside and began to make the paper clip connectors that they would fasten onto the bolt and attach the panel with. In order to shape the paperclip, the researchers threaded the paperclip by wrapping it around one of the half-inch bolts. They made six of these connectors and ensured the connectors would fit snugly in the bolt. 

Arora & London, Figure 1: Photograph showing how connectors were made by wrapping one side around the threads of the bolt.
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Figure 1: Photograph showing how connectors were made by wrapping one side around the threads of the bolt.
Arora & London, Figure 2: Photograph of one of the completed connectors.
Figure 2: Photograph of one of the completed connectors.

Using a hand drill with a ? inch drill bit, the researchers drilled a hole through the PVC pipe. Using the X-Acto knife, they cleaned up the residue left by the drill and made the hole slightly bigger to snugly accommodate the ½ in bolt. After whittling the hole, the researchers then fastened the pipe to a clamp and forced the bolt to thread the now-larger pipe to ensure it fit snugly. 

Arora & London, Figure 3: Photograph of PVC pipe post-drilling with bolt threaded through hole.

Figure 3: Photograph of PVC pipe post-drilling with bolt threaded through hole.

After removing the bolt from the PVC pipe, the researchers proceeded to add three washers, then one of the connectors, followed by two more iterations of this pattern of construction. Then, they screwed the bolt back into the PVC pipe, ensuring that half of the bolt was visible on both sides of the PVC pipe. The researchers then continued with the standard construction pattern for three more iterations. Finally, they added three extra washers at the end of the last connector and fastened down the end of the bolt with a wing nut. They tightened the wing nut a precise amount to allow only the necessary rotational movement of the washer/connector system, since lateral movement along the bolt would compromise the stability of the apparatus.

Arora & London,Figure 4: Photograph of connectors on PVC pipe bolt.
Figure 4: Photograph of connectors on PVC pipe bolt.

Using the hand drill with the ? inch drill bit, the researchers drilled a hole into the bottom of the Sterilite storage container. Next, the researchers drilled two additional smaller holes separated by an inch between them on the shorter side of the container above the bottom hole. Using a blade, they enlarged the bottom hole to a diameter within which a second bolt would fit snugly when threaded.

Arora & London, Figure 5: Image showing the hole with the second bolt through it, and the two smaller holes above.
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Figure 5: Image showing the hole with the second bolt through it, and the two smaller holes above.

The researchers then secured the bolt to the storage container by applying watertight plumbing epoxy between the bolt and the storage container’s exterior side. On the interior, they secured the bolt to the inside by hot-gluing the bolt at its base while positioning the bolt relatively upright.

Arora & London, Figure 6: Bottom of the container with the protruding bolt head secured by epoxy.
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Figure 6: Bottom of the container with the protruding bolt head secured by epoxy.

After completing the apparatus’ base, the researchers cut out four identical wood blocks using a band saw and used them as pegs to elevate the apparatus, using hot glue to attach the pegs to the four corners of the base. Next, they slid the PVC pipe with the washer/connector system onto the epoxy-secured base bolt. They attached the PVC pipe with a mini bungee cord so the pipe would be easily removable, running the cord through the two holes they drilled on the edge of the smaller side of the storage container and crossing it around the PVC pipe itself.

Arora & London, Figure 7: PVC pipe secured with bungee prior to completion of the washer/connector system.
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Figure 7: PVC pipe secured with bungee prior to completion of the washer/connector system.

The researchers then secured the ends of the paperclip connectors to the plastic panel that they cut out earlier by sliding the wire into the corrugations in the plastic panel and hot gluing them together. To secure the horseshoe crabs to the base, they acquired four rubber bands and punched holes through the plastic sheet. They stretched the rubber bands through the holes, creating two different sizes to account for the size range of the crabs and secured them in a taut position with hot glue.


Testing 

After constructing the apparatus, the researchers proceeded to the testing phase of the project to see if the apparatus was effective in increasing survival rates. After the three month growth and acclimation period of the crabs, the researchers were able to safely test on them. Before testing, the researchers held the crabs in dry containers for 24 hours and gently patted the crabs dry. This was in an effort to replicate the industry standard in which prior to bleeding, the crabs are left in a dry environment for 24-72 hours. It is important to note that the researchers had separate dry containers for each tank, and only the crabs from that respective tank were placed in these dry containers. After the 24-hour period, the researchers massed each of the crabs using an electronic balance and recorded these values. These values were used when calculating the total blood volume of each of the crabs. In order to calculate the blood volume, the researchers applied the linear relationship of blood volume to weight that a study Lenka Hurton, Jim Berkson, and Stephen Smith published in 2005. This study was able to describe the linear relationship between blood volume to weight with an equation:


H=-5.7+92s+257.2w-41ws


where H =hemolymph volume (mL), s =sex (males s =0, females s =1), and w =wet body weight (kg) (Hurton et al.). 

The blood volume calculations for the crabs are attached below with wet body weight (g), gender, formula-calculated blood volume, and extraction amount in the appendix labeled as Table 1. For this study, the extraction amount was 35% of total hemolymph volume. 

After calculating the total hemolymph volume and extraction amount for the horseshoe crabs, the researchers proceeded to chill all the crabs for an hour prior to beginning the bleeding process. After the chilling process, they placed each crab on the white platform of the apparatus and fastened them with their cardiac sinus facing upwards. For the experimental group, the researchers lowered the platform such that the gills of the horseshoe crab were submerged while the cardiac sinus itself was not submerged in the water. For the control group, the researchers did not lower the platform in an effort to replicate the dry bleeding industry standard. Using a one-milliliter BD syringe and needle, the researchers bled the crabs by inserting the needle into the cardiac sinus and then extracting the appropriate volume(refer to Table 1) . After bleeding each crab, the researchers left them in the dry container for 30 minutes and then put them back into their respective tanks. They then monitored the survival of the crabs for a 24-hour period and recorded the crabs that had died. After it was discovered that a crab died, the researchers removed it from the tank and placed it into a dry container for that respective tank. By the end of the 24-hour period, they were able to analyze the degree of survival of the horseshoe crabs with statistical analysis. 


Results

The study examined the effect the partially-submerged bleeding apparatus would have on survival rates of horseshoe crabs. Over the course of the entire year, the researchers were able to prepare the lab, construct and test the apparatus, and derive statistical significance. The null hypothesis for the study stated that the type of bleeding apparatus used will not make a statistical difference on survival rates of the crabs within their 24-hour critical period. In addition, the alternative hypothesis predicted that the partially-submerged bleeding apparatus would yield higher survival outcomes for the horseshoe crabs than the existing industry standard of dry bleeding within the 24-hour post-bleeding critical period. Before running statistical analysis, the researchers first compiled the raw data they collected regarding each crab’s survival and compiled it with respect to which group the crab belonged to. The compiled data is attached below. 

TankSample SizeSurviving Crabs(counted)
Tanks 1-6 (Experimental)148
Tanks 7-12 (Control)133
Table 2: Number of horseshoe crabs that survived post-bleeding 24-hour critical period.

After compiling the data, the researchers ran a 2-proportion z-test to determine if the data was statistically significant. The statistical test provided a strong p-value of  .0359, at the 0.05 statistical significance level. This significantly low p-value showed sufficient evidence against the null hypothesis and suggested that the apparatus did indeed influence survival rates of the horseshoe crabs. The small blue area in Figure 8 represents the p value and implies that there is a slim chance the differences in survival outcomes occurred by random chance. 

Table 1. Wet measured body weight, calculations for hemolymph volume, and calculations for extraction volume for each crab in their respective tanks.
p1=proportion of control crabs that survived=.5714p2=proportion of treatment crabs who survived=.2308
Significance Level=.05Z(control)=-1.64; z=-1.8 ; p-value=.0359
Thus, the researchers were able to REJECT the null hypothesis in favor of the alternative hypothesis.
Table 3: Table shows results of 2-proportion z-test with null hypothesis and proportions used in 2-proportion z-test.
Arora & London, Figure 8: Graph showing p-value and results of z-test for two proportions.
Figure 8: Graph showing p-value and results of z-test for two proportions. 



Discussion

The purpose of the study was to see if there was a correlation between the horseshoe crabs’ gills being submerged in water during the bleeding process and survival rates of these said horseshoe crabs. The researchers hypothesized that the developed apparatus, which allowed for the horseshoe crab gills’ to be submerged, would yield higher survival outcomes within the tested window. They found that the data was statistically significant, and thus the researchers were able to reject the null hypothesis, implying that the type of apparatus used, more specifically the submersion of crab gills, is statistically significant. Although the results of the z-test showed the data was significant, the low sample size of the groups prevents the researchers from gaining much additional significance from the study(Table 2). In addition to this, for future studies that aim to replicate this study, the researchers hope the sample size is far higher than that of this study, as a study with a higher sample size would help confirm these findings.

Another major area the study tried to tackle was the issue of dry bleeding horseshoe crabs, which is currently the industry standard. After the bleeding process, the researchers noticed that the dry bleeding process places the horseshoe crabs under immense stress, so much so that even after extracting a miniscule amount of blood, the shells of the crabs became much softer than the original. All in all, it is clear that dry bleeding as a normal practice is extremely detrimental to horseshoe crabs and has contributed to their spike in mortality rates within the past several decades. 

The researchers’ idea to construct an apparatus had three major objectives. The first goal was for the new bleeding system to ensure that the gills are submerged in water, which they expected would reduce the levels of hypoxia. In addition to this, another major goal of the study was for the apparatus to both be easy-to-use and easy-to-build, allowing ease in replication by other scientists. Rather than using complex materials, the researchers relied on a budget of roughly twenty US dollars to construct the apparatus, with most of the materials commonly found at home improvement stores like The Home Depot or Lowes. When reflecting on whether they were able to achieve these three major goals, the researchers realize that the developed apparatus has opened the door for future scientists to build more industrial-grade apparatuses that are based on the researchers’ finalized sketch. The researchers were able to accomplish most of the goals for this project; the gills were able to be submerged for the entire bleeding process, the apparatus was easy to build for the layman, and it was adjustable with respect to size. The only major issue they ran into was the rubber bands that were on the corrugated plastic platform had more tensile strength that a lot of the crabs could handle, and this increased tension caused a few of the crabs’ shells to become damaged while they were fastened into the apparatus. The researchers believe the way to solve this in the future would be to use some kind of string rather than a rubber band or have a way to adjust the rubber bands for each crab. 

Although the experimental design was well-thought out, the researchers did discover some potential improvements to the design. The most significant improvements were to increase the sample size and also the size of the crabs themselves. An increased sample size would allow for increased power of the study to draw conclusions. To the point of the size of the crabs, when the horseshoe crabs were delivered to the research team by the vendor, they were roughly half an inch in diameter and a bulk of the study was spent allowing the crabs to molt and grow into a bigger size. Even after the three month acclimation and growth period the researchers provided the crabs, most of the crabs only ranged from half an inch to two inches. This extremely low size makes the bleeding process much more difficult to conduct and makes the post-bleeding recovery for the crabs far more challenging. The extraction volumes the researchers calculated were so small that precision became an issue when extracting. In addition to this, an increased size of the horseshoe crabs would allow for tagging of the crabs’ shells, which would allow further data analysis to occur as more detailed information can be gathered about each crab.

 In addition to this, the researchers recommend future scientists using a direct measurement method for hypoxia. In this study, the researchers’ goal was to make an apparatus that would reduce the hypoxic conditions that horseshoe crabs face in the bleeding process, yet they depended only on survival outcomes to indirectly measure hypoxia levels. Although hypoxia is the leading cause of the increased mortality rate in horseshoe crabs during the bleeding process, it would be extremely valuable to quantitatively describe the impact of the apparatus on hypoxia levels. By doing so, it would open the door to future studies that would improve on the researchers’ design to minimize hypoxia levels. Many studies in the past have used equipment such as CG4+ cartridges and I-stat point analyzers for direct hypoxia testing, and the researchers recommend future studies apply these types of equipment when improving the design from this study. 

Lastly, a major issue the researchers had when testing was using the syringe to extract blood from the crab. Due to the small size of the crabs, the extraction volume was extremely small, thus there was a large degree of human error while using the syringe. To combat this, the researchers recommend future studies using auto-extractors or semi-auto extractors for the blood extraction process as these devices would be more accurate when extracting extremely small volumes of blood. Looking forward, they hope that future scientists and manufacturers can make an industry-grade version of this prototype to test in a mass-bleeding setting and improve their protocol based on the results.


Acknowledgements

This paper was written within the research program at Thomas Jefferson High School for Science and Technology in the Oceanography and Geophysical Systems laboratory. The co-authors are most grateful towards the support displayed by faculty for their guidance throughout this study, especially Dr. Stickler for overseeing the study. In addition, the co-authors greatly appreciate the equipment and funding provided by the TJ Partnership Fund towards the research program because the funding has made projects like this possible. 

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