BSc Thesis, 2019 | Chairgroup of Marine Animal Ecology, Wageningen University & Research 1 Defining Optimal Husbandry conditions for the Atlantic Horseshoe Crab, Limulus polyphemus A comparative study on literature and current culture practices in public aquaria Hilmar N. S. Derksen, Tinka A. J. Murk, Max Janse ABSTRACT The Atlantic horseshoe crab, Limulus polyphemus L. (Xiphosurida: Lumilidae) plays a vital role in coastal ecosystems and is an important species in physiological studies. Over the past three decades, horseshoe crab populations have rapidly declined due to wild capture for fishing industries and biomedical industries, stressing the necessity for conservation efforts and raising public awareness. Public zoos and aquaria largely contribute to this cause by keeping horseshoe crabs in captivity. However, a complete and detailed set of optimal rearing conditions was still lacking. This study provides first evaluation of current husbandry practises among public aquaria and comparisons with natural and culture conditions suggested in literature. As a result we could determine that optimal water conditions are; 19-21°C, salinity 29ppt, 7.9pH, Dissolved Oxygen (DO) 7.1mg/L, Alkalinity 2.7mEq/L for juveniles and ±18°C, salinity 20-28ppt, ±7.7pH, DO 8.5-9.3mg/L for adults. Flow- through tanks and sufficient filtration including trickling- or sand filters in combination with UV or ozonation should secure a viable habitat for the animals. Toxic substances as ammonia and nitrates should be kept as low as possible, the maximum levels of these, however, are not yet known. Important for good health is to supplement horseshoe crab diet with plant-based food sources and to limit the food quantities to their daily energy and protein requirements. KEY WORDS: Limulus polyphemus, horseshoe crab, husbandry, aquaculture, captive, rearing INTRODUCTION Horseshoe crabs, commonly referred to as living fossils, have survived virtually unchanged for over 200 million years and oldest fossils of ancestral species date back even further, to approximately 445 million years ago (Rudkin & Young, 2009; Rudkin, Young, & Nowlan, 2008) As a group of chelicerate, aquatic arthropods, horseshoe crabs ‘Leach, 1819’ (Xiphosurida; Limulidae) are more closely related to modern-day scorpions and spiders than to true crustaceans. In fact, recent genetic analysis revealed that horseshoe crabs might actually represent a group of aquatic arachnids (Ballesteros & Sharma, 2019). Limulus polyphemus is the most studied of the four extant horseshoe crab species found worldwide and is endemic to the Atlantic coast of North America ranging from Maine to the Gulf of Mexico. The other 3 species; Carcinoscorpius rotundicauda, Tachypleus gigas and Tachypleus tridentatus, can be found in coastal areas between India and Japan (Smith & Berkson, 2005; Walls, Berkson, & Smith, 2002). The Atlantic horseshoe crab is an important species used as a laboratory model animal in prominent research regarding vision & circadian rhythms, embryology of marine invertebrates and their unique endocrine system (Berkson & Shuster, 1999; Liu & Passaglia, 2009; Rudloe, 1979; Zaldívar-Rae, Sapién-Silva, Rosales-Raya, & Brockmann, 2009). Additionally, Limulus polyphemus is a key species in coastal ecosystems and benthic food chains, where the eggs provide an essential food source to supplement the diet of more than a dozen species of migratory shorebirds (Clark, Niles, & Burger, 1993; Gillings et al., 2007; Graham, Botton, Hata, Loveland, & Murphy, 2009). Since 1980 horseshoe crab populations have been rapidly declining with large numbers of animals captured in the wild for their use in fertilizer, cattle feed and as bait in commercial fishing industries (Berkson & Shuster, 1999; Botton, 1984). It has been estimated that a 2.5 million crabs are harvested annually for eel- and whelk fishing alone, grossing an economical value of approximately $13 - $17 million (Manion, West, & Unsworth, 2008). In recent years L. polyphemus has become most notable for the collection of its copper-based blue blood, which contains an important endotoxin indicator ‘Limulus Amoebocyte Lysate’ (LAL). The underlying mechanism involves a cascade of serine proteases capable of detecting miniscule quantities of pyrogenic endotoxins produced by Gram-negative bacteria (Ding & Ho, 2010). Therefore, horseshoe crab blood has been widely used in testing the sterility of medical devices, implants and injectable drugs over the past three decades (Ding & Ho, 2010; Smith & Berkson, 2005). An estimated 250,000 horseshoe crabs are captured, transported and bled annually. These the animals are put under considerable stress and it has been shown that post-bleeding mortality rates increased with 7.5% for L. polyphemus kept in captivity and up to 15% for animals released back into the wild (M. Thompson, 1999; Walls et al., 2002). In early 2000s, researchers succeeded in mapping the genes underlying the production of Limulus Amoebocyte Lysate and identified the primary component (Factor C) of the coagulation cascade (Ding & Ho, 2001; Ding, Navas, & Ho, 1993). Using recombinant technology and genetic engineering, they have accomplished the production of a recombinant Factor C (rFC), which is now incorporated into the PyroGene kit, an alternative test for LAL (Ding & Ho, 2010). Although the recombinant Factor C has been commercially available for almost 15 years and has been found to be equivalent to the LAL test, its use in pharmaceutical industries worldwide is still lacking. This is presumably due to historical single-source supplier concerns, ongoing validation procedures and compendial standards, whilst the horseshoe crab populations are still being diminished by fishing and blood-harvesting industries (Bolden & Smith, 2017; Williams, 2018). Due to its ecological value and the importance of L. polyphemus in the physiological research and biomedical field, there is high interest in restoring the horseshoe crab populations (Mishra, 2009). Culturing the animals in the lab has been suggested to reduce the necessity for wild-dcaptured horseshoe crabs and ensure a year around source of biological research material (Kropach, 1979). Additionally, horseshoe crabs kept in public zoos and aquaria could largely contribute to education and raising awareness on the conservation status of this species. Carmichael & Brush (2012) have reviewed multiple studies to RESEARCH ARTICLE 1 Department of Marine Animal Ecology, Wageningen University & Research, Droevendaalsesteeg 4, Wageningen, The Netherlands; 2 Burgers’ Zoo, Anton van Hooffplein 1, Arnhem, The Netherlands; * Author for correspondence (post@hilmarderksen.nl)