Kepley BioSystems Horseshoe Crab Publications

Krisfalusi-Gannon J, Ali W, Dellinger K, Robertson L, Brady TE, Goddard MKM, Tinker-Kulberg R, Kepley CL and Dellinger AL (2018) The Role of Horseshoe Crabs in the Biomedical Industry and Recent Trends Impacting Species Sustainability. Frontiers in Marine Science. 5:185. doi: 10.3389/fmars.2018.00185

Abstract: Every year the Atlantic horseshoe crab (Limulus polyphemus) arrives on shore to spawn, a sight once taken for granted. However, in addition to the gradual climate changes impacting all ecosystems, commercial demand from the widespread application of Atlantic horseshoe crab blood in industrial endotoxin testing and steady use as eel and whelk bait has brought the future of this enduring species into question. In response, regulations have been adopted to enhance the traceability and record keeping of horseshoe crab harvest, which has historically been difficult to track. However, these regulations do not restrict or limit LAL harvest in any significant manner. Still, sometimes-lethal biomedical bleeding process and associated behavioral changes pose a risk to horseshoe crab viability after bleeding and once returned to the waters. As a result, regulators and environmentalists are concerned that current trends and overfishing of this marine arthropod will significantly impact the surrounding ecosystem. This review examines their role and recent trends in the biomedical industry that are impacting these ancient creatures and the derivative effect on shorebirds, while considering emerging alternatives where feasible, as well as ways to ensure sustainable and pragmatic harvesting strategies. Ultimately, healthy populations of horseshoe crabs are vital to restoring and maintaining ecosystems while balancing the need for medical and research applications entirely dependent on these unique creatures.

Published: 05 June 2018.

Tinker-Kulberg R, Dellinger K, Brady TE, Robertson L, Levy JH, Abood SK, LaDuca FM, Kepley CL and Dellinger AL (2020) Horseshoe Crab Aquaculture as a Sustainable Endotoxin Testing Source. Frontiers in Marine Science. doi: 10.3389/fmars.2020.00153

Abstract: Horseshoe crab (HSC) hemolymph is the source of Limulus amebocyte lysate (LAL), a critical component in sterility testing that ensures drug and medical device safety for millions of patients every year. Wild HSC populations have been declining as a result of its use as whelk and eel bait, environmental changes, and its capture and bleeding for hemolymph by the biomedical industry, thus posing significant risks to species viability and the LAL raw material supply chain. We designed a controlled aquaculture habitat to husband HSCs and evaluated the effects of captivity on health markers (e.g., amebocyte density, hemocyanin levels, and LAL activity). We found HSC aquaculture to be practicable, with routine hemolymph harvesting resulting in high LAL quality, while safeguarding animal well-being with 100% HSC survival. Further, low-impact hemolymph harvesting via an indwelling catheter revealed rapid amebocyte rebound kinetics after consecutive 10% hemolymph extractions. Sustainable supplies of LAL could also be adapted to address daunting trends in septicemia and antimicrobial resistance. LAL is uniquely sensitive and specific for gram-negative bacteria, which represent 70–80% of pathogens that typically lead to sepsis. However, erratic results associated with interfering substances plagued efforts to adapt LAL for clinical use in the past. We report the development of a new LAL-based assay that can detect gram-negative bacteria and endotoxins in human blood without interference using aquaculture-derived LAL. Based on this research, sustainable LAL production from aquaculture could potentially satisfy industry needs with a fraction of one year’s current capture via year-round harvesting from a finite cohort of HSCs and expand raw materials supplies for potential future clinical applications.

Published: 01 April 2020.

Tinker-Kulberg R, Dellinger AL, Gentit LC, Fluech BA, Wilder CA, Spratling IL, Stasek DJ, Kepley CL, Robertson L, Goddard MK, Brady TE, Tōland L and Dellinger K (2020) Evaluation of Indoor and Outdoor Aquaculture Systems as Alternatives to Harvesting Hemolymph From Random Wild Capture of Horseshoe Crabs. Frontiers in  Marine Sciences. 7:568628. doi: 10.3389/fmars.2020.568628

Abstract: This study evaluated two approaches to the aquaculture of Limulus polyphemus with the ultimate goal of harvesting Limulus amebocyte lysate (LAL) at an industrial scale. To monitor Horseshoe crabs (HSCs), a combination of physical, biochemical and cellular components were examined for HSC cohorts in an indoor recirculating aquaculture system (RAS) and an outdoor on-bottom pen culture system (PCS) over a 6-month period. The metrics included body weight, hemocyanin (Hc) concentration, amebocyte density, and LAL reactivity. In addition, a simulated biomedical bleeding event (extracting 30% of the total hemolymph volume) was performed to assess the impact on physiochemical properties of the hemolymph and amebocytes. Overall, the HSCs fared better in the RAS compared to the PCS, with higher rebound kinetics with respect to Hc, amebocyte density, LAL reactivity, and with 100% survival in the RAS cohort. Further, hemolymph reinfusion (after amebocyte removal) was shown to improve HSC recovery time. In summary, outcomes of this research show that a RAS, coupled with adequate nutrition and monitoring can provide HSCs with a suitable environment for sustainable hemolymph extraction and year-round LAL production.

Published: 22 October 2020.

Tinker-Kulberg R, Dellinger A, Brady TE, Robertson L, Goddard MKM, Bowzer J, Abood SK, Kepley C and Dellinger K (2020) Effects of Diet on the Biochemical Properties of Limulus Amebocyte Lysate From Horseshoe Crabs in an Aquaculture Setting. Frontiers in Marine Science. 7:541604. doi: 10.3389/fmars.2020.54160

Abstract: The Limulus amebocyte lysate (LAL) isolated from cells in the horseshoe crab (HSC) hemolymph is a critical resource for global biomedical and pharmaceutical quality control and sterility testing. Given the necessity of and limitations associated with wild capture, a conservational approach to LAL harvesting would benefit the medical community that relies on the raw material while helping ensure species viability. We posited that aquaculture and year-round collection represented a sustainable alternative for the production of LAL from a finite HSC cohort, thereby averting the impact of current practices on wild populations. Given the specter of captivity diseases linked to diet, such as panhypoproteinemia, this work, at the outset, focused on optimizing a feed formulation to ensure animal vitality. In turn, each preparation required evaluation with respect to effects on LAL as well as vital HSC health markers so as to meet or exceed industry requirements and establish a new supply chain paradigm. In this controlled husbandry study, we conducted three 8-week feeding trials and demonstrated a ~7-fold LAL reactivity range among the HSC feed groups. Relative protein abundance patterns of HSC amebocyte clotting factors (i.e. Factor C, Factor B, and proclotting enzyme) were influenced by diet in particular, and the up-regulation of specific LAL factors correlated with enhanced activity. These results also cite the discovery that coagulation Factor C, the LPS-sensitive serine protease proenzyme, may be a phosphoprotein.

Published: 29 October 2020.