Development of methodology and reference intervals for the analysis of the free-ranging Atlantic horseshoe crab Limulus polyphemus hemolymph

Jill E. Arnold, Catherine A. Hadfield, Leigh A. Clayton, Carolyn Cray, Darbi Jones, Mark Payton

Research output: Contribution to journalArticlepeer-review

1 Scopus citations


Background: The Atlantic horseshoe crab, Limulus polyphemus, is valuable to fishing and biomedical industries, for education in public aquaria, and of ecological importance because the eggs provide a critical food source for migratory shorebirds. While hematologic and biochemical analyses are fundamental tools for health assessment, reports are limited for the four extant species of horseshoe crabs; L polyphemus (East Coast North America), Carcinoscorpius rotundicauda (mangrove horseshoe crab Southeast Asia), Tachypleus gigas, and Tachypleus tridentatus (Southeast and East Asia). Objectives: This prospective study measured hemolymph values to determine RIs from free-ranging horseshoe crabs during the annual breeding aggregation in Delaware Bay, USA. Methods: Near-shore animals were briefly manually restrained for hemolymph collection from the cardiac sinus. Hematologic samples (25 males, 25 females) were collected for hemocytometer counts using the Natt and Herrick method. A second set of 50 animals (25 males, 25 females) was sampled for biochemistry measurands, including copper concentration by spectrophotometer, osmolality by vapor pressure osmometer, and protein electrophoresis. Results: Total hemocyte counts varied widely (RI 6600-44 300/µL). Electrolytes were consistent with osmoconformers living in seawater. There was a marked difference in sexes, with females showing much lower protein, glucose, and copper levels. Eleven females had multiple outliers, and all data were excluded from RI calculations. Conclusions: Further studies of this species outside of spawning season are needed. Expanding our knowledge of horseshoe crab health is important to decreasing morbidity and mortality in research applications and maximizing longevity and overall health in display settings.

Original languageEnglish (US)
Pages (from-to)259-272
Number of pages14
JournalVeterinary Clinical Pathology
Issue number2
StatePublished - Jun 2021


  • cell count
  • hemocytes
  • hemocytometer
  • hemolymph
  • Limulus polyphemus

ASJC Scopus subject areas

  • veterinary(all)


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