Effects of Diet on the Biochemical Properties of Limulus Amebocyte Lysate From Horseshoe Crabs in an Aquaculture Setting

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 reactivity. These results also cite the discovery that coagulation Factor C, the LPS-sensitive serine protease proenzyme, may be a phosphoprotein.

Transcriptomic Signature of Horseshoe Crab Carcinoscorpius rotundicauda Hemocytes’ Response to Lipopolysaccharides

“…This may suggest that different genetic characteristics and habitat could be associated with differences in the immune response. Factors such as species, microbiome composition, diet, ocean acidification, and geographical ubication, among other factors, have been associated with differences in the genome, transcriptome, and immune parameters of HSCs [ 51 , 52 , 53 , 54 , 55 ].…”

Evaluation of Indoor and Outdoor Aquaculture Systems as Alternatives to Harvesting Hemolymph From Random Wild Capture of Horseshoe Crabs

“…The effects and optimization of diet and different bleeding volumes and regimens, coupled with optimal recovery times, on HSC health and LAL production from HSCs raised in an indoor RAS have been recently reported (Tinker-Kulberg et al, 2020a). The use of intravascular catheters surgically implanted in the respective pericardial membranes of the HSCs, which allowed for routine hemolymph collection without repetitive membrane punctures, has also been recently investigated (Tinker-Kulberg et al, 2020b).…”

“…In the indoor system, HSCs (n = 24) were provided a natural feed (e.g., glass minnows and krill; Aylesworth's Fish and Bait, St. Petersburg, FL, United States) ad libitum and maintained as described previously (Tinker-Kulberg et al, 2020a). A health assessment was recorded for each HSC upon intake (i.e., sex, weight, carapace length/width, as well as an appendage, carapace, eye, and mouth evaluation).…”

Aquatic and Terrestrial Invertebrate Welfare

“…For L. polyphemus, the majority of the diet consists of a wide variety of small invertebrate prey [114]. Despite this finding, most diets in human care consist of a relatively small variety of large invertebrates (e.g., decapods, cephalopods) and teleost fishes [92,115]. This difference in diets may be a factor in the hypoproteinemia frequently found in adult L. polyphemus, and possibly in the general poor survival rate of the species in aquaculture [106,115].…”