The American horseshoe crab Limulus polyphemus is vitally important for its role in the biomedical industry. Amoebocytes in the hemolymph, or blood, of horseshoe crabs isolate foreign bacteria by detecting and quickly clotting around endotoxins. Because of their remarkable clotting abilities, these amoebocytes are extracted from the blood to create Limulus Amoebocyte Lysate (LAL), which is used to test for the presence of toxins in medical devices and injectables.
Understanding the factors that influence amoebocyte density, metabolic stress, and mortality in horseshoe crabs has important management implications. Selective harvesting of horseshoe crabs with the highest amoebocyte levels may allow biomedical bleeding facilities to generate similar levels of LAL while using fewer crabs. This would be more efficient and cost-effective for those facilities while reducing the number of horseshoe crabs that die due to harvesting. The main goal of the proposal is to determine factors (e.g. age, holding pond water temperature) that influence blood chemistry (e.g., amoebocyte density), metabolic stress, and mortality in horseshoe crabs harvested for the biomedical industry.
Project Findings
Adult male horseshoe crabs were collected from April to June in 2022 and 2023. All crabs were brought to environmentally controlled tanks for further study. To study age-related impacts, the water in the tanks was kept at 23 °C and 30 ppt salinity. To study the impacts of temperature, horseshoe crabs were randomly assigned and acclimated to tanks of 30 ppt salinity with one of three water temperature treatments: 18 °C, 23 °C, or 28 °C, for one week. A standard metabolic rate was measured and then horseshoe crabs were either mock-bled or had one-third of their blood volume collected. After the one-week acclimation period, metabolic rate was measured using an intermittent flow-through respirometer that quantified the amount of oxygen consumed over a period of 15 minutes. Crabs were then randomly binned into bled or mock-bled treatment groups.
Collected hemolymph was used for histochemistry (the practice of differentiating cellular structures through staining) and nuclear magnetic resonance-based metabolomics (which characterizes metabolites within an organism’s tissues and biofluids). An organism’s metabolome changes in response to external stressors, such as environmental factors.
Researchers found that horseshoe crabs at the highest temperature had a significantly higher pre-bleed standard metabolic rate than those at lower temperatures. There was little evidence that age or water temperature over the course of one week affected horseshoe crab blood histochemistry or mortality in bled crabs. However, both age and water temperature affected metabolomic profiles. 17 metabolites were significantly different between young and old horseshoe crabs, while 17 known and 2 unknown metabolites were significantly different across temperature treatments.
Analyses showed that older horseshoe crabs and horseshoe crabs held at higher water temperatures had higher levels of lactate, among other markers, indicating increased oxidative stress in these crabs. These results suggest that stress responses in horseshoe crabs that may not be immediately visible at the cellular level (i.e., through blood histochemistry) are measurable at the metabolomic level.
Resulting Publications
Litzenberg, Kerryanne. (2023). The Physiological Effects of Age and Temperature on Blood Chemistry, Metabolism, and Mortality of Harvested Horseshoe Crabs (Limulus polyphemus). College of Charleston.